The tendency of population aging is continuously increasing, which is directly correlated with a significative number of associated pathologies. Several metabolic bone diseases such as osteoporosis or chronic kidney disease–mineral and bone disorders involve a high risk of fractures. Due to the specific fragility, bones will not self-heal and supportive treatments are necessary. Implantable bone substitutes, a component of bone tissue engineering (BTE) strategy, proved to be an efficient solution for this issue. The aim of this study was to develop composites beads (CBs) with application in the complex field of BTE, by assembling the features of both biomaterials’ classes: biopolymers  and ceramics , in a combination described for the first time in the literature. The CBs prepared by double crosslinking  showed adequate physico-chemical characteristics and capabilities  for bone tissue repair. Moreover, preliminary in vitro studies on cell cultures highlighted that the CBs were free of cytotoxicity and did not affect the morphology and density of cells. The results indicated that the beads based on a higher concentration of guar gum have superior properties than those with carboxymetilated guar, especially in terms of mechanical properties and behaviour in simulated body fluids. Metabolic bone diseases (MBDs) include a significant spectrum of clinically related disorders, caused by calcium, phosphorus, magnesium or vitamin D abnormalities. The most common triggers for MBDs are hereditary hypophosphatemia, hyperparathyroidism or a deficiency of vitamin D. Osteoporosis, osteomalacia  and fluorosis are the most frequently diagnosed, while Paget’s disease, chronic kidney disease-mineral bone disorder, fibrous dysplasia and osteogenesis imperfecta are rarely met ,2.Usually, the patients with MBDs do not experience symptoms, evidence of illness or abnormality, until they develop disastrous musculoskeletal complications, such as fractures . FracturBone tissue engineering (BTE) is an intensely studied area that offers promising solutions for treating bone disorders, especially for the reconstruction of serious bone defects, such as those caused by fractures . ImplantNaturally occurring polymers are among the most used biomaterials in bone substitutes manufacturing. Polysaccharides dominate the top-level position in this application, mainly due to their easy availability, eco-friendliness, and non-toxicity. Guar gum (GG) and alginate (ALG) are two polymers widespread in nature, obtained from cheap naturally available and affordable sources .Due to its glycopolysaccharide structure, GG, isolated from ground endosperm of Cyamopsis tetragonolobus or Cyamopsis psoraloides, interacts and binds with proteins in a physiological environment . Carboxy2+) it generates a stable gel [ALG, another vegetal polysaccharide, isolated from brown algae, is frequently chosen in tissue engineering in general, and in bone tissue engineering, based on its remarkable properties: biocompatibility, biodegradability, injectability and capacity to support cell growth. Moreover, in the presence of divalent cations 2(PO4)6—approximate, Alfa Aesar, Kandel, Germany); alginic acid, sodium salt ; guar gum  or carboxymethyl guar gum .For the preparation of the beads, the following were used: calcium phosphate tribasic, 34–40% Ca ; CaCl2 The reagents used to study in vitro interaction with simulated body fluids were: a simulated body fluid (SBF) prepared following a protocol described in a previous article ); physioIn vitro cell behaviour assays were performed using the same DMEM and HBSS mentioned above; HBSS with calcium and magnesium, no phenol red; fetal bovine serum (FBS); antibiotics mixture ; Thiazolyl Blue Tetrazolium Bromide (MTT); Dimethyl sulfoxide (DMSO) and Calcein-AM solution, 4 mM in DMSO, min. 90% (HPLC). Apart from HBSS, which was purchased from Biological Industries, Beit-Haemek, Israel, all the other reagents were purchased from Sigma-Aldrich, Steinheim am Albuch, Germany.® Mini Ultrapure Water Systems .All the solutions used in this study were prepared with type 1 ultrapure water from Arium2, in the presence of HA. Inspired by the composition of the human bone [2 bath (0.4 M) and left to crosslink for 2 h at room temperature. After repeated washing procedures with ultrapure water, the beads were dried using a halogen moisture analyzer , at a drying temperature of 105 °C.The composite beads (CBs) were obtained by ionic crosslinking of ALG, GG and CMGG with CaClman bone , the CBsThe microstructural features of the beads and their compositions were examined using the Hitachi SU-70 Field Emission Scanning Electron Microscope (FE-SEM) with Oxford Energy-dispersive X-ray Spectrometer (EDX) . Before examination, each bead was cross-sectioned and covered with a thin platinum layer. The working parameters for FE-SEM-EDX were: 20 kV accelerating voltage, working distance between 16.8 and 17.6 mm, 30° elevation and 20 keV energy level.−1, at a 4 cm−1 resolution.CBs’ chemical structure was analysed using Fourier-transform infrared spectroscopy with attenuated total reflection module (ATR–FTIR), on Nicolet Summit Pro FTIR Spectrometer with Everest ATR accessory, from Thermo ScientificTM, Waltham, MA, USA. The spectra were collected at the following parameters: average of 16 scans between 4000 to 400 cmCBs’ mechanical features were studied by an axial compression test, performed on a texture analyzer (TA) device—TA-XT2 Plus , equipped with a 5 kg load cell.Before each analysis, the bead diameter was measured with a digital caliper (mean diameter was 1.7 ± 0.1 mm), and then placed in the center of the TA-XT2 sample platform. To better understand the interaction between organic and inorganic compounds, from a mechanical point of view, pure HA was also analysed. For this, HA discs  were prepared using CrushIR™ digital hydraulic press  by applying a force of 7.6 tons.TA-XT2 is connected to a computer, the commands and the results being given/displayed using a specific software (Exponent). For each analysis, the following parameters were used: 1 mm/second pre-test speed, 2 mm/second compression speed, and 0.2% deformation (ε). The device displays a force-time curve at a specific ε, expressed in g (1 g = 0.0098 N). This force, beads/HA discs diameter and ε were used to determine the compressive strength—σ (MPa) and Young’s Modulus—E (MPa), using Hooke’s law. Four aliquots from each CBs and four HA disks were tested, the results being expressed as mean ± standard deviation, ® ThermoMixer C with ThermoTop lid, used to prevent condensation on tube lid and tube wall , without shaking. In all the cases, the physiological medium was refreshed after each measurement (daily for the first 4 days and then weekly).Four different acellular aqueous media were chosen to study CBs’ in vitro interaction: DMEM (the same used for cell culture assays), HBSS, SBF and physiological sodium chloride solution 0.9%. Beads with an initial weight of 3–4 mg were immersed in 1 mL of each of the four mentioned solutions, in a 2 mL Eppendorf microcentrifuge tube and placed at 37 °C, in EppendorfSD) of the beads based on wo  and we :The gravimetric method was used to determine the swelling degree , in complete DMEM enriched with 10% FBS and 1% P/S/N, in plates of 48 wells (10 × 103 cells/well). After a proper sterilization , the beads were incubated with the already attached cells (direct contact). Considering the size of the beads, and their interaction with DMEM and HBSS, 48 well plates were used and the MTT test was accomplished for four aliquots  at 24, 48 and 72 h.The MTT assay was performed separately on two different cell lines: MG63 human osteosarcoma cells  and A431 human epidermoid carcinoma cells , after the approval of the Ethical Committee of Grigore T. Popa University of Medicine and Pharmacy of Iasi, Romania (no. 141 from 26 January 2022). A standard protocol, already described by our group in another published paper, was used . In brieThe Calcein-AM assay was carried out after 72 h of direct contact of the beads with MG63 cells, as CBs’ main application is bone tissue engineering. In brief, after removing the beads and the medium from the wells, the cells were washed two times with HBSS with calcium and magnesium. A volume of Calcein-AM solution prepared in HBSS (2:1000) was added in each well to completely cover the cell layer and was maintained for 40 min in incubator, at specific conditions for cell cultures. Inverted Phase Contrast Microscope , 10× objective, was used to study cells morphology and density.CBs based on ALG, GG/and HA were prepared by ionic crosslinking, known as a simple, common and cost-effective gelation method to form alginate-based materials . The pre2 crosslinking and high drying temperature, were chosen in order to obtain beads with adequate mechanical strength and suitable handling properties, which can sustain fractures repair and regeneration. To the best of our knowledge, using a halogen moisture analyser to design composite beads has not yet been reported in the literature. Four different CBs in terms of biopolymers content were obtained, their codification and composition being detailed in These two concepts, CaClOn a 1 mm scale, the FESEM micrographs exhibited beads with micro spherical shape, a compact structure, and rough surface, with no evidence of cracks. The roughness of the CB surface is useful because it improves cells’ attachment, proliferation and differentiation .As the degree of magnification increased (between ×350 and ×1100), it was observed that the HA microparticles are uniformly distributed in the polysaccharide’s matrix, attributed to the ultrasonic homogenization used in the preparation process. Also, the inorganic components of the beads were well embedded and densely packed in the organic phase . The HA At a magnification between ×13,000 and ×25,000, some cracks were observed in the beads structure that frequently appear in the drying process of the crosslinked alginate-based materials  and is a® , whose structure can be associated with that of CMGG beads. As an important conclusion of the study, the authors highlighted the fact that both porous structures sustained bone ingrowth on their surfaces and inside them after their implantation in rat calvaria [Regarding the porosity of the CBs, calvaria . Moreovecalvaria .These differences between GG beads and CMGG beads, in terms of porosities and ceramic distribution in the organic part, are explained by the results of a study published by Dalei et al. , where GEDX is a valuable microanalysis used in various biomedical fields; with regard to the specimens containing calcium phosphates, it is considered a semi-quantitative method, since it allows the determination of Ca/P molar ratio, indicating the nature of the calcium phosphates located close to the matrix surface . The eleIn the case of calcium phosphate bioceramics for bone reconstruction, due to natural bone composition biomimetics, the Ca/P ratio significantly influenced CBs in vitro interaction with osteoblast. For instance, it was reported that a Ca/P ratio value higher than 2 decreased osteoblast viability; nevertheless, high osteoblast alkaline phosphatase activity was identified for the bioceramics with the Ca/P ratio between 0.5–2.5 . Moreove10(OH)2(PO4)6, the molecular weight of 1004.67 g/mol, which is known to have a theoretical Ca/P ratio of 1.67 [2+ ions and the biopolymers functional groups and a high crosslinking degree. Moreover, CB4 showed an irregular porous surface structure with particles aggregated between them. Since EDX offers the results from the surface of the analysed sample rather than from inside of it, the high value of Ca/P can also be related to these aspects.According to the producer data, the HA incorporated in the beads structure have the estimated chemical formula Ca of 1.67 . General of 1.67 ,50. Ca/PBy FTIR spectroscopy, the functional groups of the four CBs and the interactions between components were identified. The wavenumber values for each functional group identified in the polymers, HA and the four CBs, can be seen in −1 and 1420 cm−1 are specific to the biopolymers: in the case of ALG representing COO− asymmetric and symmetric stretching [−1 can be assigned to the vibrational modes of ν3 PO43–, whereas those at about 600 cm−1 and 560 cm−1 can be attributed to vibrational modes of ν4 PO34– [On the ATR–FTIR spectra of the four CBs  the absoretching , while fretching . Regardiν4 PO34– .−1 from all the spectra are specific to the biopolymers (C–C stretching) that overlap those of the HA (ν3 PO43−) [2+ ions to the guluronic acids [The high intense peaks identified near 1020 cm3 PO43−) . Also, tic acids , suggest− symmetric stretch peak  and 1.66 MPa (CB2), comparable with those of human trabecular bone, placed between 0.1 and 16 MPa, according to the comprehensive review of Gerhardt and Boccaccini . MoreoveThis observation is relevant for our study, because as EDX data showed, the only bead with a Ca/P molar ratio smaller than 1.67 (1.625) is CB2 which is the sample with the highest σ. Regarding the compressive or elasticity modulus, the smallest value obtained was 6.18 MPa (CB1), while the highest was 8.32 MPa (CB2). E of CB2 is comparable to that found by Ji et al. (9.5–12.4 MPa) for other bone tissue engineering scaffolds [2+ ions [The suitable values of σ and E obtained at the mechanical assays, comparable with those of human trabecular bone and other ceramics, are assigned to the intermolecular crosslinking . As alre2+ ions , thus re2+ ions  preparedEven if is generally believed that a high degree of crosslinking involves superior mechanical properties, Montoya-Ospina et al.  recentlyRegarding pure HA discs, the following values were determined: σ = 0.0913 ± 0.00117 (MPa) and E = 0.4566 ± 0.0058 (MPa) , which aBased on their similarities in terms of composition with natural bone, ceramic/polymer composites are considered superior materials, being part of the third-generation orthopedic biomaterials. They combine the key features of the two classes, resulting in superior materials with great bioactivity, biodegradability, mechanically strong yet at the same time flexible .These properties are governed by the interfacial interactions between organic and inorganic components ,75. ThesComparing the beads with GG and those with CMGG, in terms of mechanical features, the results indicated that a high concentration of GG generated the most resistant bead, attributed to the high molecular weight of the polymer.The first step in the in vitro and in vivo characterization of any material intended to be used in direct contact with the human body, is their immersion in different acellular aqueous environments. Simulated body fluid (SBF), developed by Kokubo et al. , was iniIn order to get information about how the beads will behave in cell cultures assays, their swelling degree was studied for 14 days, denoted as the mean time for these assays. The obtained results are displayed in The values attained for SD in DMEM were relatively constant, especially for CB1 and CB2. After seven days of immersion, a slight decrease was noticed for CB2. CB3, and CB4 graphs showed an inconstant behaviour.2, as during the preparation process an egg-box model of crosslinking is created, which is in fact a chelate structure that generated very stable junctions [2+ ions. The swelling process starts with the diffusion of the water in the bead structure that induces the dissociation of the temporary junctions and the release of Ca2+ ions. The polymeric network expands, resulting in an increase of SD, until a critical value is reached; then, the stable chelate junctions are dissociated, and the degradation of the material starts [Regarding the values obtained for SD in HBSS, CB2, CB3, and CB4 swelled quickly in the first 48 h, followed by an equilibrium phase until day seven, then by a sudden decrease in the value in the 2nd week. The obtained results are specific for the alginate-based materials that are crosslinked with CaClunctions . Moreovel starts .The second part of the extensive study of beads performance in simulated body fluids was their immersion in physiological serum and SBF for 28 days, with the aim to obtain data concerning their behaviour after implantations in a bone defect, as in vivo experiments request much longer times.In Another important aspect related to CB4, the bead with the highest Ca/P ratio, was the fact that the smallest values for SD among all four aqueous media  were obtOn the other hand, the highest values of SD were obtained for CB1, the bead with the most modest compressive strength and Young’s Modulus, so is the least resistant from a mechanical point of view. A high degree of swelling is mainly associated with a porous structure, which is directly correlated with low mechanical strength, the balance between an adequate porous structure and superior mechanical properties being a serious challenge for bone tissue engineering materials .In summary, GG formed more stable interactions and junctions in the preparation process compared with CMGG, but future studies are necessary to better understand the encountered mechanism. The bioactivity of the beads after immersion in SBF will be studied in the near feature, as it represents one of the main subjects of another study, where in vitro and in vivo behaviours of CBs will be analysed in detail.Developed first by Mosmann et al.  40 yearsAs mentioned in To further confirm this feature, the beads were tested in the same conditions in direct contact with A431. Even if the CBs will be used as bone substitutes, their interaction with this tumoral line was studied, mainly as A431 are acknowledged to be highly angiogenic, exhibiting an increased expression of epidermal growth factor receptors and an ability to significantly produce vascular endothelial growth factor, well known for promoting neovascularization and neo-angiogenesis .The interaction of CBs with A431 , right rCalcein-AM is an easy-to-handle assay that provides useful data on the morphology and density of cells after incubation with compounds/materials intended for biomedical applications. It is a single-staining method based on the enzymatic action of Calcein-AM dye .The results of the assay are presented in Considering the results obtained from the two preliminary biological in vitro assays, more complex cell culture studies can be safely performed, such as the capacity of the beads to sustain mineralization  and finally be considered for implantation into animal models.2+ ions and the biopolymers functional groups (2.35 and 2.7). The use of high drying temperature in the bead preparation, different molar weight polymers, and intermolecular crosslinking determined adequate mechanical strength and suitable handling features that can sustain fractures repair and regeneration. Concentrations of ALG or GG/CMGG and different polymer ratios did not significantly affect in vitro interaction with simulated body fluids and cytocompatibility on the tested cell lines. Thus, the developed HA-ALG-GG/CMGG based beads could be used as a potential bone substitute for bone tissue repair and regeneration. In light of the encouraging results, several perspectives could be adopted, in order to explore an automatic method to obtain optimized beads in terms of weight and dimensions, drug loading, and release studies, as well as complex cell culture assays and in vivo examinations on bone defects.Four types of organic-inorganic composite beads were fabricated for the first time by incorporating HA into the ALG-GG/CMGG-based polymer matrix with different contents. The HA microparticles were uniformly embedded and densely packed into the polysaccharide phase by ionic gelation and a dehydrothermal process, forming beads with spherical shapes, compact structures, and rough surfaces. The Ca/P molar ratio determined by EDX was fitted into the amorphous calcium phosphate type (1.2–2.2), calcium deficient hydroxyapatite (1.5–1.67), strong interaction between the Ca


Slc36a2 and Slc36a3 were all highest in brown adipose tissue. We then generated global Slc36a2 and Slc36a3 knockout mice to investigate their functions in metabolism. Neither loss of Slc36a2 nor Slc36a3 affected the body weight and body composition of the mice. Slc36a2 knockout mice exhibited increased oxygen consumption during the daytime. After cold treatment, inhibition of Slc36a2 significantly decreased the mass of brown adipose tissue compared to wildtype mice, while it lowered the expression level of Cpt1a. Moreover, the serum lipid levels and liver mass were also decreased in Slc36a2 knockout mice after cold treatment. On the contrary, Slc36a3 knockout impaired glucose tolerance and up-regulated serum LDL-cholesterol concentration. Thus, SLC36A2 and SLC36A3 play central and different roles in the energy metabolism of the mice.Brown adipocytes mainly utilize glucose and fatty acids to produce energy, which play key roles in thermogenesis. Furthermore, brown adipocytes also utilize other substrates, such as amino acids, for energy expenditure in various conditions. Here, we report the new physiological roles of proton-coupled amino acid transporters, SLC36A2 and SLC36A3, on global energy metabolism. The relative mRNA expression levels of both  Adipose tissue (AT) is a highly dynamic organ which accounts for 4–40% of the total body weight in adult humans and plays a key role in systemic energy homeostasis and many other biological processes. The major cell type in the AT, known as adipocytes, are composed of three subtypes: white, beige and brown adipocytes based on morphology, gene expression and function. White adipocytes (WAs) are primarily found within visceral and subcutaneous ATs, where their main function is energy storage in the form of triacylglycerol (TAG) within lipid droplets (LDs). One the otgher hand, brown adipocytes (BAs) reside in brown adipose tissues (BAT) in mammals and have abundantly smaller LDs and dissipate energy through non-shivering thermogenesis . FunctioDespite glucose and fatty acids, BAs also use amino acids (AAs) as major nutrients during thermogenesis . It has +/AA symporters that differ from the other mammalian AA transporters (mostly known as Na+/AA symporters or exchangers) [Slc36a2 inhibit acidification of lysosome and S6K re-phosphorylation under the starvation condition [Slc36a2 and Slc36a3 global knockout mouse models to explore the potential functions SLC36A2 and SLC36A3 in metabolism.Proton-coupled amino acid transporters  belong to the transmembrane amino acid symporter family with different expression patterns and substrate selectivity . SLC36A1hangers) . SLC36A1hangers) . SLC36A1hangers) ,21. SLC3hangers) ,23. Comphangers) . Previouondition ,26. Howeondition ,28. In tMice used in this research were all from a C57BL/6N background and were bred and housed in the animal facility of CAM-SU  with free access to acidified water and standard rodent chow food (radiated and autoclaved). Mouse maintenance and experimental use were all under the guidance and supervision based on animal protocols (ZJ-2021-1) approved by the institutional Animal Care & Use Committee of CAM-SU on 24 December 2021.Slc36a2 and Slc36a3 global knockout mice (Knockout First) were made through blastocyst injection of mutant mouse ES cells (Wellcome Trust Sanger Institute). Chimeric mice were then sequenced and the positive insertion ones were bred with wild type C57BL/6N mice to obtain the Slc36a2/Slc36a3 Tm1a mice (abbreviated as Slc36a2/Slc36a3 KO).−1 collagenase type I at 37 °C for 40 min. The digestion was terminated with addition of the same volume of DMEM containing 20% FBS, and centrifuged to remove undigested tissues. Cells were then centrifuged at 1700 rpm for 5 min, and the supernatant was removed to obtain SVF preadipocytes in the pellet. The freshly isolated SVF cells were seeded and cultured in growth medium containing DMEM, 20% FBS, 1% penicillin/streptomycin (P/S) at 37 °C with 5% CO2 before reaching a confluence of 100%. For adipogenic differentiation, growth medium was replaced by induction medium  for 4 days and then differentiated in differentiation medium .Primary WAT SVF preadipocytes were isolated using collagenase digestion and followed by density separation. Briefly, the inguinal white adipose tissue (iWAT) was minced and digested in 1.25 mg mLBody composition  in live animals without anesthesia were recorded using small animal MRI equipment . Each animal was placed in a plastic holder designed for mice without sedation or anesthesia. Subsequently, the holder was planted into the measuring space on the side of the MRI system. To guarantee the accuracy of the results, mice were forced to not move in the holder. Each scan took about 2 min.2) and carbon dioxide production (VCO2) of the mice. The system was placed in the animal facility of CAM-SU under a strictly controlled temperature (24 °C) and light-dark cycle . Each mouse was housed in a chamber with free access to water and food. The whole experiment was performed for 3 days as the first day was for the mice to adapt the chamber. The data presented were all corrected for energy expenditure levels to the body weight of each mouse. Average day (8 a.m.–8 p.m.) and night (8 p.m.–8 a.m.) energy expenditures were the average mean value of all measured data points.An indirect calorimetry system  was used to measure day and night oxygen consumption  according to the manufacturer’s protocols.Total TG from liver samples of −ΔΔCT method normalized to mouse β-Actin as the internal control.Total RNA was extracted from adipose tissues using Trizol Reagent (Invitrogen) following the standard protocol from the manufacturer. Absorption ratios of 260/280 nm (~2.0) were measured using a Nanodrop 3000 spectrophotometer. cDNA was then made through reverse transcription using 3 μg of RNA with random primers and M-MLV reverse transcriptase. Real-time PCR was then carried out using the SYBR Green method on a Roche Light-cycler 480 PCR System. Sequences of the gene-specific paired primers were retrieved from PrimerBank and they are listed in p values of <0.05, <0.01 or <0.001.The two-tailed and unpaired Student’s t test was conducted to calculate the significance of all analyses as presented by mean ± SEM. Statistically significant results were shown by Slc36a1 mRNA levels were highest in the kidney, with lower expression levels from the brain and liver (Slc36a2 and Slc36a3 shared similar expression patterns with an adipocyte maker gene Fabp4 (fatty acid binding protein 4). Their mRNAs were all highest in BAT, followed by lower expression levels in inguinal WAT (iWAT) and epididymal WAT (eWAT)  preadipocytes isolated from iWAT at D8 compared with undifferentiated cells  A. The mRocnemius A. Slc36aed cells B. FurtheSlc36a2 genome region to obtain Slc36a2 knockout mice. Homozygous alleles (Slc36a2 KO) carried the cassette led to early translational termination that generated a truncated peptide, which lost the key transmembrane domains of SLC36A2 , carbon dioxide production (VCO2) and respiration exchange rate (RER) independently of treadmill speed , LDL-cholesterol, HDL-cholesterol and TG levels were all significantly decreased in Slc36a2 KO mice   D,E. More WT mice F. TherefSlc36a2 and Slc36a3 were highly expressed in BAT and that the Slc36a2 level was increased after adipogenic differentiation in vitro. Loss of either Slc36a2 or Slc36a3 has no effect on body weight and body composition. Slc36a2 knockout increases oxygen consumption of mice during the daytime while Slc36a3 knockout does not affect the oxygen consumption. Intriguingly, Slc36a3 KO mice have significantly impaired glucose tolerance which is not observed from Slc36a2 KO mice. Loss of Slc36a2 decreases BAT and liver mass, liver TG concentration and blood lipid levels only after cold-treatment, while Slc36a3 KO increases serum LDL-cholesterol concentration. Further analysis reveals that Slc36a2 loss of function up-regulates the expression level of Cpt1a, which is involved in β-oxidation [Our study demonstrates previously unrevealed roles for SLC36A2 in cold-induced thermogenesis and SLC36A3 in glucose and lipid metabolism. We found that both xidation . Thus, SSlc36a2 KO mouse model to access the potential physiological role of SLC36A2 in vivo, especially in BAs after cold treatment. The results indicated that loss of Slc36a2 improves systemic metabolism, especially lipid metabolism after cold treatment. L-α-amino acids with small aliphatic side chains, such as proline and glycine, are preferred substrates for SLC36A2 [Slc36a2 inhibition may promote mitochondrial respiration through activation of PRODH. Consistent with our result, knockdown of Slc36a2 in brown preadipocyte also increases UCP1 level [+ leak mediated by UCP1 across the inner mitochondrial membrane to generate heat [+-independent and electrogenic AA transporter [+-stimulated and Na+-independent manner [Slc36a2 may affect the H+ leak and modulates UCP1 activity in BA. Indeed, both Slc36a2 overexpression and inhibition have been shown to affect lysosomal acidification [Slc36a2 KO mice was specifically caused by elevated energy expenditure of BA this remains to be investigated by using a BA-specific knockout mouse model. Unfortunately, the third loxP site of the inserted cassette was lost that made it impossible to generate a conditional allele.SLC36As mediate AA transport and are important for the pharmacokinetic profiles of AA-based drugs by mediating their intestinal and kidney transportation. However, their physiological roles in other metabolic organs in vivo have not been investigated. In particular, SLC36A2 is located on the membrane of BA and may serve as an amino acid sensor to orchestrate the functions of the intracellular organelle ,26. Here SLC36A2 ,32. It h SLC36A2 ,34, whic SLC36A2 ,36. Slc3ate heat ,38,39. Snsporter . In cultt manner . Thus, iThermogenesis in BAT is involved in the activation of both the sympathetic nervous system and adrenergic signaling from the central nervous system ,43. GammSlc36a3 is primarily thought to be an orphan transporter and is only found to be expressed in the testes [Slc36a3 in vivo and in vitro have not yet been accessed. In the present study, we found its expression was highest in BAT and explored the role of SLC36A3 in vivo by loss of function using a mouse model. The results showed that Slc36a3 played a role in systemic glucose and lipid metabolism. However, owing to the lack of a specific antibody for SLC36A3, we could not verify the protein localization of SLC36A3 in different tissues. Thus, the tissue specific function of SLC36A3 has not been explored. However, expression patterns of Slc36a2 and Slc36a3 mimicked Fabp4 in vivo, which indicated that SLC36A2 and SLC36A3 may both play roles in AT. Considering the opposite phenotype of the two KO mouse models, we could at least conclude that SLC36A2 and SLC36A3 were indispensable for energy metabolism and there were no compensatory effects in either Slc36a2 or Slc36a3 KO mice.e testes ,47. The Slc36a2 and Slc36a3 assessed the role of SLC36A2 and SLC36A3 by using genetic knockout mouse models, especially in the aspect of metabolism. However, this first in vivo study charactering SLC36As’ function was still limited, especially in terms of technical limitations. First, the current study is mainly based on the results from global KO mice, no conditional alleles have been made to access the tissue-specific roles of SLC36A2 and SLC36A3. Thus, it is impossible now to conclude whether the phenotypes came from BAT. Second, due to the lack of specific antibodies for SLC36A2 and SLC36A3, the endogenous protein levels, distribution and dynamics in various conditions were unable to be characterized. Last, we used KO mice to perform in vivo loss-of-function studies of SLC36A2 and SLC36A3. However, the gain-of-function study through generating overexpression models, especially tissue specific overexpression by transgenic mice or virus injection are still required [This first in vivo functional characterization of required ,49. ThusIn conclusion, our results demonstrate that AA transport through various SLC36As is indispensable for BAT and the systemic energy metabolism.


Shewanella marisflavi BBL25, an electricity-generating microorganism, uses various carbon sources and shows broader sugar utilization than the better-known S. oneidensis MR-1. To determine the sugar-utilizing genes and electricity production and transfer system in S. marisflavi BBL25, we performed an in-depth analysis using whole-genome sequencing. We identified various genes associated with carbon source utilization and the electron transfer system, similar to those of S. oneidensis MR-1. In addition, we identified genes related to hydrogen production systems in S. marisflavi BBL25, which were different from those in S. oneidensis MR-1. When we cultured S. marisflavi BBL25 under anaerobic conditions, the strain produced 427.58 ± 5.85 μl of biohydrogen from pyruvate and 877.43 ± 28.53 μl from xylose. As S. oneidensis MR-1 could not utilize glucose well, we introduced the glk gene from S. marisflavi BBL25 into S. oneidensis MR-1, resulting in a 117.35% increase in growth and a 17.64% increase in glucose consumption. The results of S. marisflavi BBL25 genome sequencing aided in the understanding of sugar utilization, electron transfer systems, and hydrogen production systems in other Shewanella species.Identification of novel, electricity-producing bacteria has garnered remarkable interest because of the various applications of electricigens in microbial fuel cell and bioelectrochemical systems.  Shewanella species are widely studied owing to their metal-reducing activity that reduces solid metal oxides . The cultured cells were stored in 20% glycerol at −80°C as stock solutions for further use. A single colony from the LB agar plate was precultured in 5 ml of LB broth [2, 10% CO2, and 5% H2. Oxygen was removed from the media by flushing the serum bottles with N2 gas. The N2 gas was blown through the media for 10 min to maintain an anaerobic environment, and rubber stoppers were used to prevent biogas escape. All bottles were sealed with rubber stoppers and removed from the anaerobic chamber. Biohydrogen was produced at 30°C and 200 rpm in a shaking incubator for 48 h.All chemicals used for media preparation in this study were purchased from BD Difco Laboratories . LB broth . The preLB broth . These a2 as the carrier gas. The temperatures of the inlet, oven, and GC system detector were set at 150, 80, and 150°C, respectively. The analysis time was 2 min, and a hydrogen peak was identified at approximately 0.8 min. The hydrogen concentration in all experiments was quantified in comparison with 0.2 ml of standard hydrogen gas  [g for 10 min [2SO4), and the experiment was performed at a flow rate of 0.6 ml/min for 40 min [Reagents for GC and high-performance liquid chromatography (HPLC) were obtained from Sigma-Aldrich (USA). To measure biohydrogen production, biogas analysis was conducted using gas chromatography, which is described as follows: the volume of biogas released by each strain under each parameter was measured using a glass syringe (Sigma-Aldrich) and compared. In addition, 0.2 ml of biogas was collected from the headspace and analyzed to validate the biohydrogen content in the total biogas via gas chromatography  using a thermal conductivity detector with a 3.66 m × 3.18 mm × 2 mm Porapak N packed column  and Nic, USA) . To deter 10 min . To measr 10 min . The resr 40 min .S. marisflavi BBL25 produces electricity from lignocellulosic biomass and various carbon sources [S. oneidensis MR-1, it is more efficient in utilizing carbon sources. In addition, S. marisflavi BBL25 produced polyhydroxybutyrate (PHB) using pLW487 containing PHB synthetic genes, suggesting it could potentially be used as a host for biochemical production [S. marisflavi BBL25, genome sequencing was performed. Whole-genome sequencing data of S. marisflavi BBL25 indicated that its genome was 4,364,798 bp long, with a 49.9% guanine-cytosine content and 3,845 open reading frames (ORFs)  , Table 2ome size -39. Howe species . Functioclusters . A totalobserved .S. marisflavi BBL25 electron transport system are shown in mtrA and mtrB and one type of cymA and mrtC genes in S. marisflavi BBL25. Two types of mtrA and mtrB genes suggest the possibility of higher electricity generation [mtrA gene is used by MR-1 to reduce iron (III) and flavins [mtrB gene is known to be associated with the Mtr respiratory pathway [mtrC gene has been purified and demonstrated to exchange electrons with each other and various forms of iron (III), flavins, and electrodes [cymA gene is a cytoplasmic membrane-anchored tetraheme c-type cytochrome that is capable of receiving electrons from the menaquinone pool [The genes related to the neration . The pri flavins . The mtr pathway . The proectrodes . The cymone pool .S. marisflavi BBL25  [Based on genome information and a deep search of metabolic genes, we assigned the metabolic pathway of xylose to pyruvate . We alsovi BBL25 . Formatesis MR-1 . Based ovi BBL25 . Shewanes (KEGG) .Shewanella species have been recognized as potential biohydrogen producers owing to their ability to produce hydrogen from pyruvate under anaerobic stationary-phase conditions in the absence of an external electron acceptor [S. oneidensis MR-1 can oxidize lactate, pyruvate, formate, hydrogen, and some amino acids for respiration and may form a productive partnership with fermentative microorganisms [S. marisflavi BBL25 can produce hydrogen under the same conditions as those of S. oneidensis MR-1, we assumed that S. marisflavi BBL25 produces biohydrogen by utilizing pyruvate and grows under anaerobic conditions in the presence of external electron acceptor, fumarate. S. marisflavi BBL25 could produce biohydrogen and grow under the aforementioned conditions. The highest optical density (OD) (0.1890 ± 0.003) was recorded at 600 nm after 12 h of culture , followed by glycerol and galactose  and the highest cumulative hydrogen volume (877.43 ± 28.53 μl at 600 nm) after 42 h of culture. The results of growth and hydrogen production from xylose are shown in S. marisflavi BBL25 can produce more hydrogen from xylose than glucose and can use multiple carbon sources.The time-dependent production of biohydrogen by  culture . The hig culture . With xyS. marisflavi BBL25 can grow using various carbon sources and generate electricity, especially from C6 biosugars [S. marisflavi BBL25. Shewanella marisflavi BBL25 contains D-glucose PTS permease , IIA (orf_02257), glucokinase (orf_02722), glucose-6-phosphate isomerase (orf_03084), fructose-bisphosphatase (orf_03413), aldolase (orf_00197), phosphoglycerate kinase (orf_00196), and pyruvate kinase (orf_02062)  in the genome. In addition, the metabolic pathway of glucose utilization of S. marisflavi BBL25 is well established due to whole-genome sequencing. Hence, there is a possibility that genes related to the glucose utilization by S. marisflavi BBL25 can be used for the same purpose in other strains that cannot utilize glucose. For xylose utilization, we identified a xylose-utilizing system in S. marisflavi BBL25. It comprised xylose reductase (orf_00894), xylitol dehydrogenase (orf_03529), xylulokinase (orf_02525), and transketolase (orf_00194)  and xylulokinase (xylB) were not found [N-acetylglucosamine uptake system in S. marisflavi BBL25, a well-known carbon source for S. oneidensis MR-1 [S. marisflavi BBL25 comprises an N-acetylglucosamine symporter (orf_03052), N-acetylglucosamine permease (orf_02284), N-acetylglucosamine kinase (orf_02722), N-acetylglucosamine-6-phosphate deacetylase (orf_03054), and glucosamine-6-phosphate deaminase (orf_03370). Overall, Shewanella oneidensis MR-1 cannot catabolize glucose because the glucokinase-encoding gene (glk) is not present in its genome. Moreover, xylose cannot be oxidized to xylulose because xylitol dehydrogenase (XDH) encoded by the gene xyl2 is absent [iosugars . We analf_02062) . Unlike f_00194) . In the ot found . Moreovesis MR-1 , was ides absent , 52.S. marisflavi BBL25, suggesting that glucose and xylose utilization in S. marisflavi BBL25 may be more valuable than in S. oneidensis MR-1  was introduced into S. oneidensis MR-1. We then compared the growth and glucose consumption of this strain and found that the engineered S. oneidensis MR-1 had a higher OD of 0.639 ± 0.01 at 600 nm and consumed 18.3%more glucose than the wild type. In contrast, wild-type MR-1 showed a lower OD of 0.29 ± 0.01 at 600 nm, and carbon source consumption was not observed (Genes related to glucose and xylose utilization were found in sis MR-1 . Althoug glucose , 54. To observed .S. marisflavi BBL25 can utilize various carbon sources and produce electricity. However, the mechanism involved remained unclear. Using genome sequencing, we demonstrated the existence of genes related to carbon source uptake, utilization systems, and electron transfer systems. We also identified the pathways for the biohydrogen-producing system in S. marisflavi BBL25. After whole-genome sequencing of this strain, we attempted to identify and confirm its potential as a hydrogen producer. In comparison to hydrogen production in S. oneidensis MR-1, S. marisflavi BBL25 had more formate dehydrogenase, which indicated that BBL25 had greater potential as a hydrogen producer. This was confirmed by hydrogen production experiments under anaerobic conditions. Moreover, by introducing the glk gene from S. marisflavi BBL25 into S. oneidensis MR-1, we observed glucose utilization and growth using glucose in S. oneidensis MR-1. Based on these findings, we verified that the glucose utilization system of S. marisflavi BBL25 worked, and the possibility of improving S. oneidensis MR-1 using a carbon source-utilizing gene from S. marisflavi BBL25 was confirmed. Thus, our study demonstrated various carbon sources and biohydrogen production systems, which offset the shortcomings of the existing representative strains. Furthermore, the whole-genome data generated in this study indicated the possibility of engineering this strain for higher electricity generation, hydrogen production, and carbon source utilization in other Shewanella species.In this study, we previously found that http://jmb.or.kr.Supplementary data for this paper are available on-line only at


The presence of pleural effusions and ascites in patients is often considered a marker of illness severity and a poor prognostic indicator. This study aims to compare inpatient and outpatient costs of alternative invasive treatments for ascites and pleural effusions.The retrospective single-institution study included inpatient cases treated for pleural effusion (J90 and J91) or ascites (R18) at the University Hospital Cologne (UHC) in Germany between January 01, 2020, and December 31, 2021. Costs for punctures and indwelling catheter systems (ICSs) as well as pleurodesis were analyzed in different comparator treatment pathways. Real-world data from the UHC tertiary care center were based on diagnosis-related group fees from 2020 to 2021. A simulation of outpatient expenses was carried out to compare inpatient and outpatient costs for each pathway from a payer perspective.A total of 4323 cases  were analyzed. For ascites, inpatient implantation with home care drainage was found to be the most expensive option, with total costs of €1,918.58 per procedure, whereas outpatient puncture was the least expensive option at €60.02. For pleural effusions, the most expensive treatment pathway was pleurodesis at €8,867.84 compared with the least costly option of outpatient puncture resulting in total costs per procedure of €70.03. A break-even analysis showed that outpatient puncture remains the most inexpensive treatment option, and the ICS comprises a cost-saving potential. Longevity of several months with the use of ICSs results in both enhanced quality of life for patients and increased cost savings. In particular, the presence of pleural effusions and ascites is considered a marker of illness severity and a poor prognostic indicator.2–4 Moreover, one study suggests that patients with liver disease were twofold more likely to die in an institution with 15% higher costs, compared with those without liver disease.5Ascites is the pathological accumulation of fluid in the free peritoneal space. In general, it is triggered by fluid leakage from blood vessels into the abdominal cavity. Symptoms include abdominal pain, increased abdominal girth, nausea and vomiting, shortness of breath, and fatigue. Ascites is most often associated with cirrhosis of the liver, followed by heart failure, kidney failure, infection, or cancer. Malignant ascites account for ∼10% of all ascites cases and life expectancy is ∼20 weeks from its diagnosis.6 Approximately every second cancer patient develops malignant pleural effusion.7 The most common symptoms are shortness of breath, irritable cough, and chest pain; however, the disease can also be asymptomatic. Median life expectancy ranges from 3 to 12 months for patients with malignant pleural effusion following a palliative treatment strategy alleviating secondary symptoms.8 For nonmalignant pleural effusions, patients have a longer median life expectancy of multiple years.9A pleural effusion is the pathological increase of fluid in the pleural space, and the effusion can be either benign or malignant. Congestive heart failure, pleural infection, and malignancy are the leading causes of pleural effusions.10 Current treatment options aim to prevent recurrences involving diuresis, punctures/paracentesis, shunts, local chemotherapy, and permanent drainage or indwelling catheter systems (ICSs). For ascites, treatment options using an abdominal water pump or transjugular intrahepatic portosystemic shunt are also relevant.So far, an effective antitumor therapy for malignant pleural effusion and malignant ascites due to peritoneal carcinomatosis does not exist. Instead, treatment is most often limited to palliation of symptoms, including dyspnea, nausea, chest, abdominal pain, and anorexia.11–13 Following an outpatient therapy strategy, the ICS enables patients to stay in their home environment.Additional treatment options for pleural effusion include chemical pleurodesis and pleurectomy. However, recent studies show that ICS is more effective and more appropriate for symptom control and improvement of quality of life compared with pleurodesis.14 Additionally, patients with pleural effusions have an increased chance of spontaneous pleurodesis.16These procedures are often initialized under local anesthesia as minimally invasive procedures resulting in less pain compared with other forms of therapy, for example, chemical pleurodesis. Furthermore, these procedures also minimize the risk of injury to internal organs and subsequently lower the infection rate.17 The cost-effectiveness is also shown to depend on various factors such as complications, duration, and severity of the underlying diseases, as well as survival. For malignant ascites, the National Institute for Health and Care Excellence (NICE) anticipated in their Medical Technologies Evaluation Programme (MTEP) savings to the National Health Service (NHS) of the United Kingdom when using peritoneal drainage compared with repeated large-volume paracenteses in an inpatient setting.18Although numerous studies have evaluated potential benefits for patients, there is a lack of health economic studies analyzing the implications of current treatment options from an insurer's perspective.19 In addition, Wu et al. identified tunneled peritoneal catheters to be cost-effective in comparison with large-volume paracentesis due to the reduced risk of infection, emergency department visits, and length of stay (LOS).20The study by Bohn and Ray similarly suggested that a peritoneal catheter will be economically beneficial if patients require large-volume paracentesis more than nine times or if patients drain less than 5 L of fluid per procedure.21 Compared with pleurodesis, the cost of the ICS was significantly lower only when patient survival was less than 14 weeks or when no home care was required.A systematic literature review by Botana-Rial et al. found that the cost of the indwelling pleural system ranges from €1,100 (for patients surviving less than six weeks) to more than €5,000 in case of complications.The purpose of this study was to assess and compare the costs of treatment alternatives for pleural effusion and ascites patients from a payer perspective in Germany. A primary focus lies on the health economic effects of ICS in terms of cost savings.This study is a retrospective single-institution analysis of inpatient cases treated at the University Hospital Cologne (UHC) in Germany between January 01, 2020, and December 31, 2021. According to the International Classification of Diseases, 10th revision, German Modification, (ICD-10-GM), the underlying cases were precoded with either pleural effusion (J90 and J91) or ascites (R18) as the main or secondary diagnosis.20 For pleural effusions, this applies to pleurodesis as well as punctures and ICSs.21As described previously, there are several therapy options for both diseases. For ascites, punctures and ICSs are most commonly described in the literature.For ascites, five different comparator treatment pathways have been defined, that is, A1a, A1b, A2, B1, and B2 compare . Along eFor ascites patients, the ICS implantation can be conducted in an in- or outpatient setting (pathway A1 vs. A2). For the A1 (implantation inpatient) pathway, the implantation can, on the one hand, be the main (primary) cause of hospitalization (A1b). On the other hand, ICS only reflects a secondary treatment during their hospital stay if, for example, another surgery  is the primary cause of hospitalization and ICS is implemented as a result (A1a). Regarding patients who receive a puncture, two groups can be differentiated: outpatient (B1) or inpatient puncture (B2).Looking at the comparator pathways for pleural effusion, the groups are analogous to the ascites comparator pathways  and 2. OThe objective of our analysis is to quantify the costs for both treatment pathways  from a payer perspective.OPS), LOS, and nursing degrees.For inpatient costs, our cost assessment is based on real-world data (RWD) of the UHC. Patients under the age of 20 were excluded. The database includes information on patient characteristics, main and secondary diagnoses, German diagnosis-related group (DRG) allocation and revenue, German procedure classification (Operationen- und Prozedurenschlüssel or for pathway A1 (inpatient ICS implantation),○ LOS: 1–2 days and○ OPS codes▪ 5–549.2 or 8–148.0 for ascites drainage and▪ 8–144.0, 8–144.1, or 8–144.2 for pleurodesis drainage.for pathway B1 (inpatient puncture),○ LOS: 1 day andOPS codes:○ ▪ 1–853.2 and 8–153 for ascites puncture and▪ 1–844 and 8–152.1 for pleural effusion puncture.for pathway C1 (pleurodesis),○ LOS: any LOS andOPS codes: 5–345.1 - 5–345.6.○ The aim was to identify patients who represent the  inpatient treatment pathways. Therefore, we searched the database for the following OPS codes and LOS values:22 In case the cross-check resulted in a DRG with a significantly lower fee, we analyzed and calculated costs for all cases with this respective DRG in the RWD.Based on the median DRG fees of each subgroup, the costs for the comparator pathways were calculated. To validate the median DRG costs resulting from our RWD analysis, we cross-checked the resulting DRG using a web-based grouper tool.Costs that occur in the outpatient sector have been modeled, that is, forpathway A2 (outpatient implantation in the ascites group) andpathway B2 .EBM codes for each procedure performed. In contrast to inpatient OPS codes (which indirectly lead to a certain DRG), the EBM codes are directly associated with a fee.Costing data are taken from the so-called German Uniform Assessment Standard (einheitlicher Bewertungsmaßstab or EBM), which is the basis for remuneration of outpatient services. Outpatient treatments are documented and reimbursed based on (Lauer-Taxe) of the ASEPT ICS. ASEPT is an example of a permanent drainage system, which was approved by the FDA in 2015.23In addition, costs for nursing services were taken from the German schedule of fees for care services. Material costs for an ICS are given by the list price The analysis was conducted from a payer perspective, that is, the German statutory health insurance (SHI). For the inpatient sector, costs of all types are included in the DRG fees. One exception, at least for the German DRG system, is the remuneration for care services that are reimbursed separately. Costs incurred within the inpatient pathways are therefore included using the cost data from the UHC, which are based on DRGs.Based on the RWD, costs incurred by patients with versus without neoplasms have been calculated to assess whether these subgroups incur substantially diverging costs.EBM codes. This included the following components: quarterly fees (lump sum), EBM positions for treatments and ambulatory surgeries, diagnostics, and material costs. Costs for the ICS as well as home care services were added as they are not represented by the EBM.Outpatient cost components, in contrast, are not merged into one single fee, but consist of a variety of different positions that are summed up. We therefore modeled the inpatient pathways by identifying all procedures undertaken for the respective treatment as well as the associated When comparing the home care treatment pathways (including the ICS) with those pathways consisting of regular physician visits (in- or outpatient), the latter options incur not only medical costs but also transportation costs, in particular, for patients with high care needs. Therefore, we aimed to approximate this additional cost component by making the following assumptions: all patients who are dependent on care services  will need and be eligible to receive dedicated patient transport. We determined the share of patients who fall into one of these categories based on our RWD from the UHC.To assess the costs occurring per pathway, each cost component was analyzed regarding frequency. Finally, after analyzing and simulating the total costs per pathway, we compared each pathway in a break-even analysis, emphasizing the costs per treatment duration in weeks. Therefore, we set up the following cost functions:All costs are given in euros (€). We calculated median costs. The RWD from UHC are based on DRG fees from 2020 and 2021. All other costs reflect the 2021 prices.All descriptive statistics have been assessed using Microsoft Excel (MS 365 Business Standard).In total, 4323 ascites and pleural effusion cases have been treated in the UHC from 2020 to 2021. The majority are pleural effusion cases (3396), whereas ascites accounts for 1302 cases. Three hundred seventy-five cases were based on both diagnoses. Across all 4323 cases, the 3 most common main diagnoses can be assigned to the following ICD-10 chapters: (IX.) “Diseases of the circulatory system” (37%), (II) “Neoplasms” (23%), and (XI.) “Diseases of the digestive system” (11%).Because one patient may have more than one case , this reflects 3690 individual patients, of which 3494 were eligible . Of all eligible patients, 63% were male. Most patients (29%) were between 70 and 79 years old, followed by those between 60 and 69 years (25%). Cost calculation for ascites comprises a breakdown into relevant cost positions per pathway compare .Starting with pathway A1a, costs for the inpatient implantation have been determined to be €0 since the procedure was captured as part of a wider reimbursement code. From the German SHI perspective, cases assigned to A1a incur the same costs, independent of treating a patient with ICS or not because the particular case mix calculation is not sensitive to the addition of the “ICS-OPS” (as is often the case with more expensive DRGs).n = 18) and obtained median costs of €1,820.67, which we used in the following analysis. In both pathways, patients proceed with home care drainage, which has been quantified by summing up costs for the ICS drainage material and costs for ambulatory care service. In total, this accounts for €97.91, leading to total costs of €97.91 and €1,918.58 for A1a and A1b, respectively.In pathway A1b , 21 cases fulfilled the criteria. Their median DRG costs amounted to €3,286.19, but after validation using the web grouper, a less costly DRG (Z01C) was identified. Thus, we analyzed all cases with this DRG in our RWD (EBM position 5–549.2 (implantation of an ICS in the abdomen: for ascites drainage), followed by frequent drainage at home.Pathway A2 (outpatient implantation for ascites followed by home care drainage) accounted for €670.98 for the initial treatment procedure. It includes material costs for the ICS and costs for the outpatient surgery reflected by the The subgroup “inpatient puncture” (B1) consisted of 116 patients with median costs per case of €721.54. Our web grouper validation supported this result.Cost components that have been quantified for pathway B2 (outpatient puncture) comprise treatment, examination, and material costs, as well as the quarterly lump sum. The total costs amount to €60.02.The resulting costs for pleural effusion have been determined analogously to the calculation of ascites costs and are summarized in n = 46) and obtained median costs of €2,149.67, which we used in the following analysis.Thirty-three RWD cases fulfilled the criteria for subgroup A1b (inpatient implantation). Their median DRG costs amounted to €3,263.75, but after validation using the web grouper, a less costly DRG (E73B) was identified. Thus, we analyzed all cases with this DRG in our RWD (Implantation costs for a secondary treatment (A1a), again, amounted to €0 (A1a). The subsequent home care drainage cost was €97.91 in pathway A1a as well as in pathway A1b, resulting in total costs of €97.91 and €2,247.58, respectively.n = 22), which could be validated using the web grouper.The RWD analysis of respective pleural effusion cases who were treated with puncture (B1) indicated median DRG costs per case of €833.70 (For an outpatient puncture (B2), the SHI reimburses costs amounting to €70.03, consisting of costs for the puncture itself, the quarterly lump sum, sonography, and materials.n = 79). The median DRG remuneration was €8,867.84. As pleurodeses are ideally only performed once and not regularly, this pathway only incurs nonrecurring costs.Based on the RWD of the UHC, patients who received pleurodesis (C1) have been identified , treatment costs for all pathways can be ranked (lowest to highest costs) as follows:(1).(B2) Outpatient puncture(2).(A1a) Inpatient ICS implantation (secondary reason)(3).(B1) Inpatient puncture(4).(A2) Outpatient ICS implantation (only for ascites cases)(5).(A1b) Inpatient ICS implantation (primary reason)(6).(C1) Pleurodesis The relationships change when analyzing costs over the course of time. Outpatient punctures, predictably, remain the most inexpensive option. Whereas costs for inpatient punctures are relatively low in the short term, costs for all pathways incurring high one-off costs at the beginning will amortize after a period of time, turning them into economically favorable options in the medium and long term. The exact point of time when relationships change depends on the examined comparator treatments.Inpatient treatment with ICS , for example, will save costs compared with inpatient punctures when used for more than 8.2 weeks (ascites) and 7.1 weeks . The share of cases with care service of stages 3 to 5 according to the German classification system amounted to 12.17% of ascites and 11.27% of pleural effusion patients. Based on common market fees, we assumed that a patient's transport to and from the physician's office results in costs of €300 in total. The SHI covers the costs, except for a copayment of €10 per drive. Thus, from a payer perspective, costs of €280 are incurred per patient entitled to patient transport.Combining costs and the percentage of applicable ascites and pleurodesis patients, average costs amounted to €34.08 and €31.54, respectively. Thus, the total costs would increase depending on the frequency of necessary transportation per pathway, which is especially high for outpatient puncture treatment pathways.24 Regarding ascites, outpatient puncture has been identified as the least costly per procedure, annual quarter, and year. On the other hand, ICS when implemented as the primary reason for hospital admission was the most expensive per procedure, while inpatient punctures were the most expensive per annual quarter and year.The results of this analysis demonstrate that there are diverging cost-effective management strategies for recurrent ascites and pleural effusion. Both diseases are associated with high symptom burden for patients and poor quality of life.20Similarly, for pleural effusions, outpatient punctures were found to be the least expensive per procedure, annual quarter, and year. Pleurodesis was the most expensive per procedure and annual quarter, while inpatient puncture was the costliest per year. Similar to the literature, we found that ICS for ascites brings a cost advantage in comparison with inpatient large-volume paracentesis for patients after 2.6 weeks when implemented in an outpatient setting and after 8.2 weeks when implemented in an inpatient setting.21 Several studies indicate that life expectancy in patients with malignant pleural effusion can range from 3 to 12 months.26 In patients with end-stage liver disease with refractory ascites, life expectancy is, on average, six months.27Regarding pleural effusion, we identified that the cost of pleurodesis was higher compared with ICS until 30.2 weeks  and 22.9 weeks  of treatment. Our results are in line with results derived in the study by Botana-Rial et al. who identified a slightly shorter time interval.The present study compared the costs of different management therapies for ascites and pleurodesis. If either disease occurs, an occurrence is more likely toward the end of life, which is an indicator of poor outcomes. Pleural effusion affects the quality of life and is a significant cause of dyspnea and patient suffering. Puncture and drainage provide symptomatic relief. Pleurodesis and ICS share a similar success rate for controlling malignant pleural effusion in terms of survival and patient-reported dyspnea.29 Furthermore, patients with malignant pleural effusions receiving an ICS show fewer hospital readmissions and require fewer pleural procedures in comparison with patients with pleurodesis.29–33 Especially malignant and paramalignant effusions into the pleural space are associated with poor outcomes.34 These patients therefore may be better suited for symptomatic treatment using ICS rather than pleurodesis.On the one hand, the risk of cellulitis was higher in the ICS group, but on the other hand, the implementation of a catheter shows a reduced inpatient LOS.Our analysis identified puncture as both the least and most expensive therapy option depending on outpatient or inpatient treatment. However, determined by the individual patient's situations, it is likely that patients receive the treatment in both settings over the course of their treatment. These circumstances indicate that the modeled cost approach presented in this study is a simplification of the complex reality of possible treatment alternatives. In the context of diverging treatment options such as ICS or puncture for ascites and pleural effusion, potential complications should also be discussed.36 In pleural effusion, the most common complications associated with catheter placement involve subcutaneous and pleural infections, potential catheter rupture, and hemorrhage.37The most common complications of abdominal paracentesis include ascitic fluid leakage, hemorrhage, infection, and drainage of protein and electrolytes, as well as bowel perforation.38 In consequence, these patients are readmitted, resulting in increased treatment costs. Consideration of the individual circumstances of the patient  is also necessary for treatment selection to minimize the risk of aforesaid complications.Among patients with malignant pleural effusion, pleurodesis failure rates are known to be ∼20% to 25% over the subsequent three months.Home-centered treatment options such as drainage can reduce readmission rates and improve the quality of life of patients. In this way, patients spend less time in hospitals or outpatient clinics, hence lowering the risk of hospital-associated infections and increasing patient safety. Studies have shown that treatment at home is usually preferred by patients at end of life, thus also leading to a gain in quality of life. In addition to avoiding hospitalizations and readmissions, a catheter can also lead to significant improvement in overall health and relief of symptoms.40 The ASEPT pleural catheter appears to be comparable and, according to Dhaliwal et al., may ultimately provide cost savings through advantages in design.39It also offers ease of use according to home care nurses. Several authors have demonstrated successful treatment of malignant pleural effusion with the PleurX catheter—a system that can, however, become costly over time.Although we applied the best health economic standards, our study has some limitations. First, the RWD were collected from a single academic institution, thus reducing the results' generalizability and external validity. However, a large sample of over 4000 cases has been analyzed. Second, both in- and outpatient therapy options were included; however, distribution to the groups was not randomized. This leads to various biases. Third, outpatient treatment was simulated. Fourth, certain assumptions had to be made, for example, transaction costs for patients traveling to hospitals and that home care was provided in all ICS cases.Fifth, we were not able to analyze individual patient journeys, for example, from development of symptoms to death, and were not able to identify the reasons for the LOS. Nevertheless, with the help of simulations, an economic evaluation was performed from a metaperspective, allowing a general comparison of underlying treatment options. Finally, another limitation is that we were not able to include data concerning  complications and overall medical institution visits of individual patients.41In conclusion, the present study is one of the first German cost studies on this topic, paving the way for future research. We suggest that future research should collect and analyze RWD on clinical and patient-reported outcomes—in particular, EQ-5D-5L data—to provide a more holistic health economic perspective. However, there are challenges involved in conducting clinical trials in a palliative setting, including, but not limited to, the definition of the palliative phase, recruitment, increased attrition, and uncertainty.Considering demographic changes, disruptions in financing of the health system, and a shift toward the outpatient setting, it is important to continuously assess whether ICSs can contain costs with better outcomes for patients. Our study paves the way for this.


The COVID-19 pandemic has not only strained healthcare systems in Africa but has also intensified the impact of emerging and re-emerging diseases. Specifically in Equatorial Guinea, mirroring the situation in other African countries, unique zoonotic outbreaks have occurred during this challenging period. One notable resurgence is Marburg virus disease (MVD), which has further burdened the already fragile healthcare system. The re-emergence of the Marburg virus amid the COVID-19 pandemic is believed to stem from a probable zoonotic spill-over, although the precise transmission routes remain uncertain. Given the gravity of the situation, addressing the existing challenges is paramount. Though the genome sequences from the current outbreak were not available for this study, we analyzed all the available whole genome sequences of this re-emerging pathogen to advocate for a shift towards active surveillance. This is essential to ensure the successful containment of any potential Marburg virus outbreak in Equatorial Guinea and the wider African context. This study, which presents an update on the phylodynamics and the genetic variability of MARV, further confirmed the existence of at least two distinct patterns of viral spread. One pattern demonstrates a slower but continuous and recurring virus circulation, while the other exhibits a faster yet limited and episodic spread. These results highlight the critical need to strengthen genomic surveillance in the region to effectively curb the pathogen’s dissemination. Moreover, the study emphasizes the importance of prompt alert management, comprehensive case investigation and analysis, contact tracing, and active case searching. These steps are vital to support the healthcare system’s response to this emerging health crisis. By implementing these strategies, we can better arm ourselves against the challenges posed by the resurgence of the Marburg virus and other infectious diseases. Filovirus) and holds the distinction of being the first Filovirus ever discovered [Rousettus aegyptiacus of the Pteropodidae family, serves as the natural host of MARV and RAVV. Human infection with MVD is often associated with prolonged contact with mines or caves inhabited by bat colonies [Throughout the COVID-19 pandemic, numerous African countries, including Equatorial Guinea, experienced distinct zoonotic outbreaks . The Marscovered . It is ascovered , is the scovered . While tscovered . The Egycolonies . The Worcolonies . Since icolonies . On 13 Fcolonies . As of 1colonies . Confirmcolonies . Bata, lcolonies . The inccolonies . Diagnoscolonies . Moreovecolonies . Indeed,Moreover, a continuous and uninterrupted surveillance of viral genetic variations enables scientists and public health officials to trace the emergence and spread of new viral strains. Additionally, monitoring genetic diversity facilitates the identification of clusters or subtypes of the virus circulating in different regions. This knowledge helps in understanding transmission patterns and guiding targeted public health interventions. Our results strongly advocate for active surveillance to ensure the effective containment of any potential outbreak of this re-emerging viral pathogen, not only in Equatorial Guinea but also throughout the African continent. Enhancing genomic surveillance in the country, coupled with measures like alert management, case investigation, contact tracing, and active case search, plays a pivotal role in bolstering the health care system’s response to this emerging health crisis. https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/, accessed on 15 June 2023) [To investigate the evolutionary relationship of Marburg virus over time, a dataset containing all genomes belonging to MARV has been built, downloading all genomes currently available in NCBI Virus portal (ne 2023) . The datPhylogenetic reconstruction was performed by following Scarpa et al. . The genTo obtain the time-calibrated tree, to determine the evolutionary relationship among variants and estimate the time of divergence, we employed Bayesian Inference (BI) with the software BEAST 1.10.4 . This kiTo identify possible subgroups within genetic clusters and assess the genetic variability among genomes, a principal coordinate analysis (PCoA) was conducted using GenAlEx 6.5 . The PCoThe Bayes Factor test indicated that the coalescent constant size, under the lognormal uncorrelated relaxed clock model, provided a significantly better fit to the data compared to other tested models (2lnBF = 11.2). The phylogenetic tree obtained with MrBayes and the time-scaled phylogenetic tree obtained by using the software Beast provide the same topology; therefore, only the latter is showed . The BayThe Bayesian skyline plot (BSP) graph  depicts Analyses of principal coordinates (PCoA) , based oPCoA  revealedHuman-to-human transmission of Marburg virus (MARV) occurs from direct contact (through skin lesions or mucous membranes) with blood, secretions, organs, or other body fluids of infected persons and with surfaces and materials  contaminated with these fluids; health care workers can also be infected while treating patients with suspected or confirmed MVD. Indeed, the first case of the MVD was reported in 1967 when lab personnel working with African green monkeys became infected in Germany and Serbia simultaneously. Burial ceremonies involving direct contact with the body of the deceased may also contribute to transmission of Marburg virus. The incubation period ranges from 2 to 21 days, and MVD starts abruptly, with high fever, headache, and severe malaise. Severe hemorrhagic manifestations appear between five and seven days after the onset of symptoms, and fatal cases usually have some form of bleeding, often from multiple areas. Supportive care  and the treatment of specific symptoms improve survival . DespiteThis pattern has also been substantiated by the PCoA graph, which fully supports the geographical structuring, identifying three distinct groups that align with the main clades observed in the tree . Interestingly, genomes from both Uganda and the Democratic Republic of the Congo are present in each cluster, although the Democratic Republic of the Congo genomes are represented by only a single genome in two of the clusters. As for the genomes from Uganda, they are well represented in all of the main clusters, including ones from relatively recent sampling dates . The largest distance depicted in the graph is between the cluster D (RAVV) and the clusters A + B + C. Indeed, they share a common ancestor dated back about 230 years ago with broad range of credibility interval, as found in previous studies ,30. SuchIn general, the BSP displays a flattened genetic variability. The genetic distances matrix further supports this observation, as the largest value of genetic distance within the internal clusters was found to be 0.003 (±0.0001). Specifically, the BSP graph displayed an initial prolonged phase of flattened genetic variability, with no variations accompanied by a decrease in the viral population size during the 2000s. Afterwards, there was an increase in genetic variability and, consequently, the viral population size reached a plateau between 2005 and 2015. However, a subsequent decrease in genetic variability took place, continuing up to the present day. The fluctuations in genetic variability depicted in the graph reflect the diversification observed in the tree, particularly in relation to the more recent clades. Such fluctuations are the result of localized outbreaks occurring over time. Typically, during an outbreak, the genetic variability and viral population size rise until reaching a plateau and then decrease again. Ever since the first documented case in 1967, numerous outbreaks of MVD have occurred, following initial contact with wild animals and contraction of the disease. Despite trials with a wide range of medications to date, no concrete solution has been achieved. As noted, the most reliable method of treatment remains supportive care with careful monitoring and isolation of the patient. Disease-modifying drugs and inhibitors of viral protein have shown some promising results in many patients and can be administered to those affected; however, they might not be the final answer to this deadly virus. Vaccine research likely remains a more reliable option for creating a vaccine to protect populations especially vulnerable to the disease. With the discovery of the filoviruses now over four decades old, one could question why there is still no approved vaccine available for human use. While several experimental vaccine approaches have been tested for Ebola virus and, to a lesser extent, for MARV, no progress seems to have been made towards clinical trials to date. Given that filoviruses can only be handled in high-containment facilities available in a few countries world-wide  and that, until 2014, they had not caused more than a few thousand human fatalities, there was likely never sufficient commercial interest or funding available for the development of licensed countermeasures. The situation changed following the EBOV epidemic that devastated West Africa from 2013 to 2016; clinical trials for the most promising countermeasure approaches were accelerated and funding was made available for the licensure process. The problem now is not waiting until a devastating Marburg epidemic occurs in some African regions or worldwide. In light of the recent major and lethal outbreaks in Africa regions, it is crucial for health authorities to formulate a clear and applicable pandemic strategy for the current times, where an uncontrolled endemic and pandemic can rapidly escalate an epidemic, causing chaos for health facilities already strained by numerous challenges. In this context, it should be highlighted that understanding the genetic variability and evolutionary patterns of viruses has become increasingly vital in the context of constant monitoring and effective public health surveillance, for instance see Nelson et al.  for InflIn conclusion, it is crucial to continuously monitor the genetic variability of viruses to inform proactive and evidence-based public health interventions. By being alert and flexible in response to viral mutations, we can successfully navigate and respond to novel waves of infections, minimizing their impact and preserving population health. In this context, it is essential to promptly gather current data related to the virus resurgence in Equatorial Guinea and to determine the current cluster. Once the samples of 2023 are accessible, it becomes crucial to sequence them, and analyze and incorporate them into existing studies. This enables us to determine the group of belonging and their potential for spread, thus enhancing our understanding of the virus’s behavior and transmission patterns. Analyzing all available genomes over times, we found at least two different specific patterns of viral spread. One pattern demonstrates a slower, yet persistent and recurring, virus circulation, while the other showcases a faster but episode-confined spread. Understanding these patterns helps to provide a comprehensive view of the present status of Marburg virus (MARV) diffusion. Regular genomic surveillance coupled with real-time data analysis offers invaluable insights into the virus’s evolutionary dynamics, aiding informed decision making for public health responses. This allows authorities to enact suitable measures, such as adjusting testing strategies and bolstering contact tracing efforts, to effectively mitigate new waves of infection and prevent rapid spread within communities.