Simvastatin (SIM) particles are liposoluble drugs with large particle sizes, resulting in poor compatibility with electrospun polycaprolactone (PCL)/polyethylene glycol (PEG) nanofibers, so that part of them will be exposed to the electrospun nanofiber surface, which is easy to cause the burst release of drugs. Therefore, in this paper, stearic acid (SA) with good biocompatibility was innovatively added to increase the dispersion uniformity of SIM in the spinning solution, thus improving the performances of SIM-loaded PCL/PEG nanofiber membranes (NFMs). Accordingly, the effects of SA addition on the morphologies, mechanical properties, wettability, and drug release properties of the SIM-loaded NFMs were studied. The results showed that after SIM was dissolved in SA solution, the particle size of SIM was significantly reduced and could be evenly dispersed in the polymer spinning solution, thus obtaining the SIM-loaded composite NFMs with the best morphology and performance. The controlled/sustained release of drugs can alleviate or avoid the side effects caused by the initial burst release of drugs, stabilize the blood drug concentration, reduce the toxic and side effects of drugs, and improve the stability and safety of drug treatments. Nanotechnology is one of the important means to achieve this goal. Common drug carriers based on nanotechnology include liposomes, micelles, dendrimers, nanotubes, nanoparticles, nanofibers, and nanogels . Among tElectrospinning is a simple and effective technology for preparing nanofibers, which can control the morphology and structure of nanofibers by adjusting spinning solution components, spinning process parameters, ambient conditions, and post-processing parameters. An electrospinning device is mainly composed of a spinning solution supply device, a spinneret, a high-voltage power supply, and a collector . On applIn the drug delivery system based on electrospinning, the compatibility of drugs and fiber materials will affect the coating and release of drugs. Accordingly, selecting fiber materials compatible with drugs can effectively improve the initial burst release of drugs , and theSimvastatin (SIM) is a liposoluble hydrophobic drug, which can promote the expression of vascular endothelial factor (VEGF) and bone morphogenetic protein (BMP-2) in vivo and in vitro, thus promoting the differentiation of osteoblasts and angiogenesis. Meanwhile, it can also inhibit the role of osteoclasts and be used to repair bone defects . HoweverStearic acid (SA) is a kind of fatty acid widely existing in nature, which has good biocompatibility and is often used to prepare liposome biomaterials . In thisMw = 80000 Da) and penicillin-streptomycin (P&S) were purchased from Shanghai Yuanye Biochemical. Co., Ltd., Shanghai, China, Polyethylene glycol  was obtained from Suzhou Sinopharm Co., Ltd., Suzhou, China, Stearic acid  and sodium dodecyl sulfonate (SDS) (purity ≥ 95%) were purchased from Aladdin Co., Ltd., Hongkong, China, Simvastatin  was provided by Merrill Co., Ltd., New York, NY, USA, Tetrahydrofuran (THF) and anhydrous ethanol (AE)  were purchased from Jiangsu Qiangsheng Functional Chemistry Co., Ltd., Suzhou, China, N-butanol  was purchased from Wuxi Zhanwang Chemical Reagent Co., Ltd., Wuxi, China, Phosphate buffer saline  was purchased from Corning Co., Ltd., Glendale, CA, USA, Minimum essential medium-Alp (α-MEM) was purchased from Shanghai BasalMedia Technologies Co., Ltd., Shanghai, China, Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific, Waltham, MA, USA. Almar blue was purchased from Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China, Calcein acetoxy methyl ester  was purchased from Shanghai Beyotime Institute of Biotechnology, Shanghai, China. Mouse pre-osteoblasts (MC3T3-E1) were provided by Soochow University.Polycaprolactone  was connected to the positive pole of the DC high-voltage power supply, while the stationary copper collector with silicon paper on the surface was connected to the negative pole of the DC high-voltage power supply. The electrospinning parameters were: the flow rate was 1 mL/h, the applied voltage was 15 kV, and the collecting distance was 15 cm. All electrospinning experiments were carried out at 25 °C and relative humidity of 55% for a certain period of time until the spinning solution was completely spun to prepare PCL/PEG NMFs, SIM/PCL/PEG NFMs and SA/SIM/PCL/PEG NMFs with a drug loading capacity of 0.16 g, respectively. The codes of NFMs obtained by electrospinning and their corresponding spinning solution components were shown in The dynamic viscosity and electrical conductivity of spinning solutions were characterized by a viscometer  at a constant speed of 60 r/min and a conductivity meter , respectively. The measurement was repeated five times at room temperature.The morphology of NFMs was investigated by scanning electron microscopy . 10 SEM images and 50 nanofibers at random in each SEM image of every specimen were used for fiber diameter distribution analysis of NFMs by Image J software .−1.The mixture of a crushed specimen and KBr powder was pressed into sheet-like specimens, which were characterized by a Fourier transform infrared (FTIR) spectroscopy  with 64 scans and a wavenumber range of 500–4000 cmThe specimens with a length of 4 cm and a width of 1 cm were prepared from different NFMs, and 10 specimens with the thickness measured and recorded were prepared for each NFM. And then the tensile test of each specimen was carried out, using a universal material testing instrument  at the speed rate of 20 mm/min and with a clamping distance of 20 mm. The Young’s modulus of the specimen was expressed by the slope of its stress-strain curve, and the tensile strength of the specimen was calculated according to the following equation :specimenThe water contact angle (CA) of different specimens was measured by a Krüss DSA100 apparatus . The volume of droplet used for static CA was 6 μL. The average CAs were calculated by measuring 10 specimens for each NFM.The PBS solution containing SDS (0.5 wt%) was used as the in vitro release medium. The addition of SDS was to increase the solubility of hydrophobic SIM, which was conducive to its release in the medium . Two kinCell cultures were carried out using MC3T3-E1, which were cultured in complete medium . NFMs were cut into circular specimens with a diameter of 15 mm. The specimens were fumigated with methanol at 37 °C for 2 h, then transferred to an ultraviolet lamp for sterilization for 30 min, and then put in the sterile operation platform for air drying before being put into the bottom of a 24-well plate. Afterward, 1 mL of cell suspension with a certain concentration was vertically dropped into each well, and 5 specimens were set for each NFM. The 24-well plate with the cells planted was put into the incubator for culture, and the culture medium was updated every two days. Cells cultured on tissue culture polystyrene (TCPs) were used as controls.4 cells/well, placed in a cell incubator , and incubated for 1, 3, 5, and 7 days. At the corresponding time, the 24-well plate was taken out, the medium in the well plate was aspirated, and 200 μL of Alamar blue solution (containing 90 wt% α-MEM medium and 10 wt% Alamar blue indicator) was added to each well, which was put into the cell incubator for 2 h. Afterward, 100 μL of the medium was shifted from each well to a black 96-well plate. The fluorescence value of the solution in the well plate was measured by a microplate reader , the wavelength was set to 590 nm, and the proliferation of living cells was investigated by a confocal laser scanning microscope .Alamar blue, as a cell-proliferation assay, was used to detect the proliferation activity of MC3T3-E1 cells. The cells were seeded on the NFMs at a cell density of 1×10When different polymers are mixed in a solvent, the polymer solution can behave as a mixed clear solution or an unmixed emulsion ,36,37,38The SEM images of SIM and SA/SIM particles were shown in The morphologies of NFMs were investigated by SEM. −1, symmetric C-H and asymmetric stretching vibrations at 2872 cm−1 and 2966 cm−1, and O-H stretching vibration at 3550 cm−1. The FTIR spectra of SA  characterization of NFMs and schematic diagram of material migration during electrospinning were shown in The SIM release curves of NO.1 and NO.2 NFMs were shown in The biocompatibility characterization of NO.2 NFM was shown in In this study, the combination of PCL and PEG became a hybrid biomaterial composite with acceptable mechanical properties and good biocompatibility. SA with good biocompatibility was innovatively added to increase the dispersion uniformity of SIM in the spinning solution, thus improving the compatibility between SIM and polymers, and enhancing the morphology and performance of SIM-loaded PCL/PEG NFMs. Accordingly, the effects of the addition of SIM and SA on the spinning solution properties were investigated, and the effects of SA addition on the morphologies, mechanical properties, wettability, and drug release properties of SIM-loaded composite NFMs were further studied. The results showed that after SIM was dissolved in SA solution, the particle size of SIM was significantly reduced and could be evenly dispersed in the polymer spinning solution, which made NO.2 spinning solution have the smallest viscosity and the highest conductivity. Therefore, NO.2 NFM had the best fiber morphology, the most uniform fiber diameter distribution, the optimum mechanical properties and the relative best hydrophilicity. The initial drug burst release from NO.1 NFM was alleviated during the drug release from NO.2 NFM, and both of the two drug-loaded NFMs had reached sustained release for more than 20 days, suggesting that NO.2 NFM might be a potential candidate in the application of drug delivery system. In addition, the proliferation of MC3T3-E1 cells on NO.2 NFM exhibited good biocompatibility. However, compared with TCPs, NO.2 NFM did not significantly promote cell growth, which would limit its application in tissue engineering. In our future work, the methods and processes of preparing SA/SIM loaded PCL/PEG NFMs with other fiber structures (such as core-shell structures) will be explored to make them more suitable for tissue engineering.


As a vital metabolic and immune organ in animals, the liver plays an important role in protein synthesis, detoxification, metabolism, and immune defense. The primary research purpose of this study was to reveal the effect of breast-feeding, weaning transition, and weaning on the gene expression profile in the goat kid liver and to elucidate the transcriptome-level signatures associated with liver metabolic adaptation. Therefore, transcriptome sequencing was performed on liver tissues, which was collected at 1 day (D1), 2 weeks (W2), 4 weeks (W4), 8 weeks (W8), and 12 weeks (W12) after birth in Laiwu black goats at five different time-points, with five goats at each time point. From 25 libraries, a total of 37497 mRNAs were found to be expressed in goat kid livers, and 1271 genes were differentially expressed between at least two of the five time points. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that these genes were annotated as an extracellular region fraction, exhibiting monooxygenase activity, positive regulation of T cell activation, mitotic spindle mid-region assembly, cytokinesis, cytoskeleton-dependent cytokinesis, regulation of cytokinesis, regulation of lymphocyte proliferation, and so on. In addition, it mainly deals with metabolism, endocrine, cell proliferation and apoptosis, and immune processes. Finally, a gene regulatory network was constructed, and a total of 14 key genes were screened, which were mainly enriched for cell growth and development, endocrine, immune, and signal transduction-related pathways. Our results provide new information on the molecular mechanisms and pathways involved in liver development, metabolism, and immunity of goats. The liver is a central organ that plays a key role in nutrient metabolism, including the metabolism of proteins, fats, and carbohydrates ,2,3. FurDuring embryonic and neonatal periods, the liver functions primarily as a key hematopoietic organ . During During newborn developmental stages, such as breast feeding and weaning transition, the liver undergoes rapid physiological and genetic transformation in response to changes in nutrient intake ,11,12. IThe objective of this study was to reveal the characteristics of the liver transcriptome of postnatal goat kids and to identify genes whose expression is affected by starter feeding and weaning. Therefore, in this study, goat liver samples were subjected to transcriptional analysis at 1 day and at 2, 4, 8, and 12 weeks to examine the differentially expressed genes (DEGs).The experimental animals used in this study were Laiwu black goat, which is a local goat breed with both cashmere and meat produced in the central mountainous area of Shandong Province. It has germplasm characteristics, such as high reproduction rate, excellent meat quality, and strong disease resistance . All goaThe total RNA was extracted from the liver tissues with Trizol reagent, and the genomic DNA was removed by RNase-free DNase I. The Bioanalyzer 2100  and the ND-2000  were used to examine the quality of RNA. The sequencing libraries were constructed using only high-quality RNA samples .RNA-seq transcriptome strand libraries were set up from 5 ug of total RNA using the Illumina TruSeq TM Total Stranded RNA Library Preparation Kit . The paired-end transcriptome libraries were sequenced by Shanghai Majorbio Bio-Pharm Biotechnology Co., Ltd.  using the Illumina NovaSeq6000 system.https://github.com/jstjohn/SeqPrep, accessed on 18 January 2021) and Sickle  were used as default parameters for cropping and the quality control of raw-paired-end reads. The clean reads were then aligned with the goat reference genome (Assembly: GCA_001704415.1) in the direction mode using the HISAT2 software [SeqPrep  clustering of the relationship between samples and transcripts was performed based on the expression levels of the transcripts, and the clustering results were presented in heatmaps. The expression scales of the 25 groups of samples were normalized by the Z-Score, and the corresponding heatmaps were constructed using MEV 4.9.0 .p-value ≤ 0.05. Goatools [In addition, we performed a functional enrichment analysis, including GO and KEGG, to determine which DEGs were significantly enriched in the GO terms and the pathways when compared to the transcriptome-wide background at a Bonferroni-corrected Goatools  and KOBAGoatools  were useAcross the 25 libraries, in accordance with the KEGG results, four clusters of DEGs were significantly enriched in 34 pathways containing a total of 148 genes. All 148 genes were used for the PPI network construction. Their interactions were predicted after in the database of STRING .® 96 System . There were reaction volumes of 20 µL, 0.2 µL of forwarding primer, 0.2 µL of reverse primer, 10 µL of 2 × SYBR Green Pro Taq HS Premix, 2 µL of cDNA, and 7.6 µL of the RNase free water. The reaction conditions of qPCR are divided into two steps, the first step is 95 °C for 30 s, and the second step is 95 °C for 5 s, then 60 °C for 30 s, and this cycle was repeated 40 times. All reactions were performed in triplicate. β-actin (ACTB) was used as an internal reference gene. After amplification, the gene expression was calculated using the 2−ΔΔCT ± SEM [A total of 15 DEGs were selected randomly for RT-qPCR analysis. The primers for controls were designed based on the sequences of the selected DEGs. The primers used in this study are shown in CT ± SEM . CorrectTwenty-five liver cDNA libraries encompassing five stages of the Laiwu Black goats were constructed. A total of 3,106,609,276 raw reads were obtained. After screening, 3,023,865,598 clean reads were obtained . Twenty-As listed in GO and KEGG enrichment analyses were performed on DEGs to further confirm their involvement in the regulation of biological processes. The results of the GO analysis are depicted in The results of KEGG analysis are depicted in Notably, in the first cluster of DEGs in the KEGG pathways, endocrine correlation was recorded. In the second cluster of DEGs in the KEGG pathways, three pathways were associated with amino acid metabolism and another one with endocrine relevance. In the third cluster of DEGs in the KEGG pathways, three pathways were associated with cell growth and death, one with the endocrine system, and one with cancer. Among the fourth cluster of DEGs in the KEGG pathways, 14 were associated with immunity and disease, four with amino acid and lipid metabolism, five with signal transduction, and one with the digestive system. These results indicated differences in the transcript levels in the liver of goat kids. DEGs of the first cluster, as well as the second cluster, were enriched to the same endocrine-related pathway. However, the Q values were different, and the upregulated genes were not identical in both comparisons.To further identify the key regulatory genes that contributed to differential enrichment in the early developmental, metabolic, and immune-related pathways in the goat kid liver, PPI network analysis was performed . Based oCD4, MYC, CCND1, ESR1, ZAP70, CD5, CDK1, IL7R, PLK1, AURKA, CCNA2, CD274, PPARG, and SELL. According to the KEGG enrichment analysis, 38 pathways were significantly enriched, of which three pathways were associated with cell growth and death, four with the endocrine system, 24 with immunity and cancer, and seven with signal transduction. In addition, 15 of these 14 key genes were significantly enriched pathways overlapped with DEG significantly enriched pathways, namely cell cycle (FDR = 0.0000184219185935), progesterone-mediated oocyte maturation (FDR = 0.0000184219185935), primary immunodeficiency (FDR = 0.00023630691446), cellular senescence (FDR = 0.000672336234843), hematopoietic cell lineage (FDR = 0.00226613151582), breast cancer (FDR = 0.00372061978602), cell adhesion molecules (CAMs) (FDR = 0.00478423028459), viral carcinogenesis (FDR = 0.00883270624401), the pathways in cancer (FDR = 0.00950852829045), Epstein–Barr virus infection (FDR = 0.0103220971205), the p53 signaling pathway (FDR = 0.0108061020999), Th1 and Th2 cell differentiations (FDR = 0.0143991514955), T-cell receptor signaling pathway (FDR = 0.017736815761), Th17 cell differentiation (FDR = 0.0178677481378), and the PI3K-Akt signaling pathway (FDR = 0.0185674198687).All 148 genes were used for the PPI network construction, and 690 interactions between 148 genes were obtained. We sorted all genes in the network according to the degree of interaction and filtered out the 14 most important genes as potential key regulatory genes: To validate the accuracy of the sequencing and analysis outcomes, we selected differently expressed mRNAs to validate the qRT-PCR results . qRT-PCRLiver development involves significant changes in gene expression, which is mediated by transcriptional and post-transcriptional control . This orHNF3, MAZ, and Sp1 are required for elevated levels of S-protein gene expression in hepatocytes [During early postnatal development in male rats, decreased β2-adrenergic receptor-mediated glucose mobilization may lead to lowered transcription of the receptor-encoding genes in the liver . It is ratocytes . Researcatocytes . As is ratocytes .Presently, various studies have focused on the metabolism and cellular differentiation of the liver. Studies on the transcriptome of the animal liver based on different developmental periods, as well as dietary structure, have been reported ,44,45,46PPAR-α, PPAR-β/δ, and PPAR-γ, which play important roles in glucose and lipid metabolism [PPAR-γ, there is an interaction between glucose and lipid metabolism in adipose tissue, muscle, and liver, which indicates that PPAR-γ plays an important role in maintaining systemic glucose and lipid homeostasis [FATP) and fatty acid-binding protein (FABP) [FATP, FABP, PPARG, and fatty acid transport-related genes upregulated, and PPARG is the key gene. We accordingly hypothesized that FATP and FABP act on fatty acid transport during lipid metabolism by activating PPARG through transport-related transcription factors acting on adipocytes, contribute to the prevention of various metabolic disorders, and provide energy. We recorded high expression of DE mRNA in the W2 and W8 groups for DEGs and low expression in the other groups (Cluster 2). Notably, the second group of DEGs was also significantly enriched in the PPAR signaling pathway. In our study, genes enriched downstream of the PPAR signaling pathway were also involved in cholesterol metabolism. In addition to lipid homeostasis, the liver is critical for the homeostasis of cholesterol in the body. Excess cholesterol from the diet and newly synthesized cholesterol is known to lead to inappropriate cell membrane dynamics [CCNB1, CDK1, and RRM2 have been reported to be enriched in the p53 signaling pathway and are believed to act as potential biomarkers and therapeutic targets for hepatitis B virus-associated hepatocellular carcinoma [CDK1 was among the significantly enriched genes in the p53 signaling pathway in our study, and CDK1 is also a key regulatory gene. At the heart of the cell cycle, CDKs propel cells through the various phases of the cell cycle. In the G1 phase, cyclin D-CDK4/6 (early) and cyclin E-CDK2 (late) prepare to enter the S-phase, at which point cyclin A-cdk2 takes over and coordinates replication, followed by activation of cyclin A/B-CDK1 to facilitate the transition from the G2 to the M phase [CDK1 can replace other CDKs, and it has been demonstrated to be sufficient to power the mammalian cell cycle [CDK1 functions outside mitosis as a general translational activator. It allows protein synthesis to directly adapt to the rate of cell proliferation [CD4 gene was identified as the top one potential key regulatory gene of PPI network. CD4+ T cells are essential for achieving an effective immune response to pathogens, and they are activated after interacting with antigen MHC complex and differentiate into Th1 and Th2 subtypes, mainly according to the cytokine environment in the microenvironment [CD4 genes were up-regulated with the increase of goat kid ages. We found that CD4 genes were significantly enriched in the Th1 and Th2 cell differentiation signaling pathways and that the PPI regulatory network was a key regulatory gene. As an important immune organ, the liver is involved in regulating T cell differentiation and proliferation, and the proportion of CD4+ T cells in the liver of adult animals is significantly higher than that in the peripheral blood [CD4 gene found in this study indicates that the amount of CD4+ T cells in goat liver and their differentiation are gradually activated and enhanced over time during the neonatal stage of goat kids. We accordingly hypothesized that CD4 genes play an important role in the normal immune responses by acting in the Th1 and Th2 cell differentiation signaling pathways.To investigate the function of DE mRNAs with different expression profiles, we analyzed the DE mRNA expression patterns and identified four clusters. The DE mRNA expression profiles were related to the developmental stages in goats. We found that four clusters of target genes were enriched in the KEGG signaling pathway. DE mRNA was expressed at low levels at birth and at high levels in the liver of weaned goats (Cluster 1). In addition, they were significantly enriched in the PPAR signaling pathway, which is a widely studied nuclear receptor that regulates and controls metabolic changes in humans and animals. The PPAR is a nuclear receptor that mediates nutrient-dependent transcriptional activation and regulates the metabolic networks through energy homeostasis. Through different mechanisms, nutrients can regulate PPAR, thereby ultimately contributing to the prevention of various metabolic disorders . Peroxistabolism . Studieseostasis . Mammalieostasis ,18. The n (FABP) . The livn (FABP) . In our dynamics . We specarcinoma . CDK1 wall cycle . CDK1 fuferation . We hypoironment . The balironment . In our al blood ,58. The CD4, MYC, CCND1, ESR1, ZAP70, CD5, CDK1, IL7R, PLK1, AURKA, CCNA2, CD274, PPARG, and SELL. MYC has been found to be unregulated in almost half of the human solid tumors and leukemias, and increased levels of MYC has been attributed to retroviral transfection or transduction of circulating mammalian cells that reduces the need for growth factors, prevents cell cycle secretion, accelerates cell distribution, and increases the number of cells [MYC is an important transcription factor that regulates the growth, proliferation, and expression of genes involved in different cellular signaling pathways [MYC dysregulation has been causally linked to the development, persistence, and progression of cancer, although possible mechanisms by which oncoproteins play a role in these processes include increased cell proliferation, inhibition of cell death, metabolic regulation, promotion of angiogenesis, and regulation of stem cell formation [MYC genes play an important role in early development-related mechanisms in the liver of goat kids. Thus, we believe that the functions of these key genes deserve further investigation.A total of 690 interactions among 148 genes were obtained in the PPI regulatory network constructed from the KEGG pathway enrichment results. A total of 14 key regulatory genes were screened in order of the degree of interaction: of cells . The oncpathways . MYC dysormation ,62,63. WIn this study, the transcriptome of the goat livers at five developmental stages during the juvenile age were sequenced, and DEGs functional enrichment analysis showed a major concentration in the pathways related to early development, substance metabolism, and immunity. This study provides important information for facilitating the study of the role of the liver in the early development, metabolism, and immunity at a young age. Our results extend the current goat mRNA database and contribute to the better understanding of the mechanisms of early development from the perspective of ruminant liver development.


Ixodes ricinus in particular, has been observed, and TBEV is spreading in bordering countries, e.g., Switzerland. Therefore, we collected sera from wild ungulates during the hunting season (October–December) from 2017 to 2019 in the Susa Valley, Italian western Alps, and screened them for TBEV antibodies by a commercial competitive ELISA test. We collected 267 serum samples by endocranial venous sinuses puncture from red deer, roe deer and northern chamois carcasses. The animals were hunted in 13 different municipalities, at altitudes ranging between 750 and 2800 m a.s.l. The serological survey for TBEV yielded negative results. Borderline results for five serum samples were further confirmed as negative for TBEV by a plaque reduction neutralisation test. To date, our results indicate that TBEV is not circulating in western Piedmont. However, monitoring of TBEV should continue since TBEV and its vector are spreading in Europe. The wide-range distribution of wild ungulates and their role as feeding hosts, make them useful indicators of the health threats posed by Ixodid ticks.Wild and domestic animals can be usefully employed as sentinels for the surveillance of diseases with an impact on public health. In the case of tick-borne encephalitis virus (TBEV), the detection of antibodies in animals can be more effective than screening ticks for detecting TBEV foci, due to the patchy distribution of the virus. In the Piedmont region, northwestern Italy, TBEV is considered absent, but an increase in tick densities, of  Increasing interactions at the human–animal–environment interface are associated with the occurrence of emerging infectious diseases . WildlifIxodes ticks, TBEV is a tick-borne flavivirus of major concern for public health in Europe, and its incidence is increasing [Ixodes ricinus, together with small rodents and insectivores, plays a major role in the maintenance of TBE natural foci in western Europe [Among the broad range of pathogens transmitted by creasing . Ixodes n Europe ,11. TBEVn Europe ,13. Thisn Europe .Ixodes ricinus for TBEV-Eu and I. persulcatus for TBEV-Sib and TBEV-FE [I. ricinus, leading to an increase in associated tick-borne diseases. Accordingly, several endemic European countries have experienced a general upward trend in human TBE cases [Since its first discovery in the late 1930s, five different TBEV subtypes have been identified, differing in their geographic distribution in Europe and Asia ,16,17. T TBEV-FE . These tBE cases . The comBE cases ,21,22, bBE cases ,24. For BE cases . At its BE cases ,26,27, aBE cases ,29. In mBE cases ,32,33. IBE cases ,35,36,37BE cases ,39. IndeBE cases . SeropreBE cases . Recent I. ricinus in the northwestern Alps alongside the circulation of TBEV in neighbouring countries, such as France and Switzerland [In Italy, TBEV is historically present in northeastern regions ,43. The tzerland ,37,38, hTherefore, we investigated the possible occurrence of TBEV in an Alpine valley of the Piedmont region, northwestern Italy, by testing hunted wild ungulates for the presence of TBEV antibodies. Wild ungulates are important hosts for ticks and the fact that they develop a long-lasting immune response can enable the tracing of TBEV exposure over time , helpingSerum samples were collected from red deer, roe deer and northern chamois during three consecutive hunting seasons, from 2017 to 2019 (September to December). As per regulations in force, hunters are bound to present this game on the culling day at the check station of the local Hunting Unit (CA TO2) in Susa Valley, Piedmont. The Piedmont region is an Alpine region bordering France in the east and Switzerland in the north.®, Vacutest KIMA S.r.l., Arzergrande, Italy) at 5 °C for a maximum of 24 h before their processing. Once the blood separated into its components through coagulation process, the samples were centrifuged at 2000× g for 15 min, and the resulting blood sera were collected and stored at −20°C until analysis. In order to evaluate the samples’ quality, we measured the protein content of each sample with the use of an optical refractometer. The carcasses examined in 2018–2019 were also inspected to determine the tick infestation [Blood samples (5 mL maximum when possible) were taken through endocranial venous sinuses puncture, as previously described , using aestation , and datThe obtained sera were heat-inactivated at 56 °C for 30 min before testing. Serological analyses were performed with a commercial competitive immune enzymatic assay , according to the manufacturer’s instructions. This test is designed to measure the total antibody serum titer of all vertebrate species, except mice, against TBEV in serum; its diagnostic sensitivity and specificity were both declared as 95.7%. To confirm or exclude animal exposure to TBEV, borderline results were further subjected to a plaque reduction neutralisation test (PRNT) for TBEV, simultaneously testing for West Nile virus (WNV) and Usutu virus (USUV) to exclude the occurrence of cross-reactive antibodies. Roe deer sera originating from a recent survey on TBEV in the Netherlands , which w2, the virus–serum mixtures were added in duplicate to the 24-well plates. Serum, virus and cell controls were also added to each 24-well cell culture plate. After another hour of incubation at 37 °C and 5% CO2, an agar overlay  and MEM (Temin’s modification) (2X), no phenol red with 4% FBS, 2% penicillin/streptomycin and 5% HEPES, pH 7.4) was added. After four (TBE) or five (Usutu and West Nile) days the plates were fixed with 10% formaldehyde. The plates were then coloured with crystal violet (1% CV/20% ethanol) and washed two times with distilled water. When the plates were dry, plaques were counted. The PRNT50 value was defined as the highest serum dilution that reduced the number of plaques by 50% or more compared with the virus control.A day before performing the PRNT, 24-well cell culture plates were seeded with 2·105 A549 (human lung carcinoma) cells to obtain 80% confluence the next day. A twofold series dilution from 1:8 to 1:2048 was prepared with already inactivated borderline sera from wild ungulates. A virus solution containing 70 plaque-forming units was added in the same volume as the serum dilutions: TBEV ‘Salland’ , WNV  were calculated for ungulate species and different altitudinal ranges. Mean, minimum and maximum protein concentrations were calculated for ungulates species and expressed in g/L.Cervus elaphus) and 52 northern chamois (Rupicapra rupicapra), and the serum samples collected in 2018–2019 were from red deer (n = 142), northern chamois (n = 64) and roe deer . Among the red deer, 33.3% (95% CI = 25.6–41.8) of animals were infested by I. ricinus ticks and were shot at altitudes ranging between 750 and 2200 m; 20.3% (95% CI = 11.3–32.2) of chamois were parasitized and were culled between 1200 and 2200 m, and the two infested roe deer were shot at 1250 and 1600 m a.s.l. . Data on wild ungulates subjected to serological testing is available in the Through serological analyses by the EIA TBEV Ig test, 263 sera samples tested negative for TBEV, and the remaining five samples had borderline results. These latter samples were further confirmed to be negative for the presence of specific antibodies against TBEV, and also for WNV and USUV, by PRNT.I. ricinus ticks being widespread and parasitising ungulates across a wide altitudinal range. While the absence of evidence is not evidence for absence, we hypothesize that, given the number of animals tested in relation to the size of the tick-suitable habitat in this area, the sampling effort was very thorough and higher than, for example in the Netherlands [Our survey indicates that TBEV was not circulating in the study area, at least in the investigated time interval, despite herlands ,50 and tTo assess the occurrence of TBEV, different surveillance approaches have been undertaken across Europe, each of them with advantages and shortcomings . MoleculMonitoring antibodies against the virus in domesticated and wild animals appears to be a more reliable strategy for detecting new TBEV foci, especially in areas where human cases have not yet been reported ,54,59. RHunted wildlife can be a cost and time-efficient source of samples for research and surveillance of pathogens . HoweverR. pyrenaica), although the latter were collected in vivo during the spring–summer period [Regarding the quality of our sera, their mean protein content values were similar to those previously reported in different wild ruminant species ,69,70,71Ovis aries musimon). Notwithstanding, endocranial venous sinuses puncture allowed blood collection in most of the animals investigated, despite the challenging field conditions.The needle length used for blood extraction may also affect the quality of the blood serum. In fact, we observed some anomalies in around one-third of the sera collected, whose protein content was below 20 g/L. These samples yielded uncoloured sera after blood clotting, which may be attributable to some degree of unintentional blood dilution with cerebrospinal fluid taken from the cranial cavity. Similar shortcomings were also observed by Jiménez-Ruiz et al. , who repOur serosurvey indicates the absence of TBEV in the Susa valley, northwestern Italian Alps. However, our study was limited to a small area of the western Alps, and to the period 2017–2019. The need for large-scale routine surveillance in Piedmont region and other naïve areas of Italy is supported by the increase in TBE cases in Europe and by the identification of new endemic areas. In this respect, serosurveillance in wild and domestic ruminants exposed to ticks could be a useful tool for the early detection of TBEV foray. In our study area, where large mammals are routinely subjected to population control plans (hunted game) or sampled for other purposes (sanitary controls in livestock in Alpine pastures), such sampling would be particularly convenient in terms of time and cost savings.


The obtained results show that viticultural practices (defoliation and crop thinning) affected the amino acid and sugar content of Solaris must and thereby the quality of the final wine—mainly in terms of compounds normally related to fruity aroma , non-sugar sweetness , and alcohol content. The content of tyrosol, a natural phenolic antioxidant with a high bioavailability, was increased in the final wine by a combination of defoliation and crop thinning. The results of the metabolomics analysis performed on the must and wine samples from the water stress experiment showed that short-term water deficit significantly affected the concentration of several flavor-related compounds, including glutamate, butyrate and propanol, of the organic acids lactate and fumarate, and of the phenolic compounds caffeic acid and p-coumaric acid. ANOVA simultaneous component analysis showed that the effect of water deficit accounted for 11% (p < 0.001) and 8% (p < 0.001) of the variability in the metabolite concentrations in must and wines, respectively, while viticultural practices accounted for 38% (p < 0.001) and 30% (p < 0.001) of the metabolite variability in must and wines, respectively.Proton nuclear magnetic resonance ( Vitis vinifera (L.), and for this reason, the number of investigations targeting viticultural and oenological aspects related to this white grape variety is quite limited  promoted a similar range of volatile compounds when compared to the wine produced with a single inoculation of S. cerevisiae, and especially M. chrysoperlae was closely associated with fruity, tropical fruit, and elderflower attributes [In 2015, Liu et al. investigated the volatile and non-volatile composition of Solaris wines, as well as the sensory attributes . Recent is wines . Inoculatributes . In 2020Independently of the grape variety, the aroma and flavor profile of wines primarily stems from the chemical composition of the grapes at harvest, the vinification process, aging, and storage conditions ,19,20,211H) NMR spectroscopy must be emphasized for its inherent advantages: it is quantitative, it is unbiased and relatively fast, which makes it a powerful tool for the high-throughput and unbiased analysis of food and beverages, including wine [Over the last decades, chemical analysis by untargeted spectroscopy has become an essential tool for assessing wine quality, and together with multivariate data analysis methods, as done in metabolomics, it has become a powerful tool for elucidating several aspects related to viticulture practices and wine production, origin, and fraud . Metaboling wine . NMR alling wine , to studing wine , terroiring wine , berry sing wine , geograping wine , adultering wine , the imping wine , and theing wine ,40.1H NMR metabolomics. The aims are to describe (1) the baseline metabolome of must and wines as a function of different growing conditions, (2) the changes in the bulk metabolome of must and wine samples obtained from different viticultural treatments, and (3) the changes in the bulk metabolome of must and wine samples from vines that underwent water deficit. A graphical overview of the experimental design is given in In this study, the impact of water deficit (WD), defoliation (DEF), and crop thinning (CT) on the metabolic composition of must and wine samples from Solaris grapes is investigated by 1H NMR spectrum of grape must from the vines grown in the open screenhouse is shown overlaid with a representative 1H NMR spectrum of grape must from vines grown in the field (vineyard). In the high-field region  and is given in the caption to In 1H NMR spectra of wine samples from the screenhouse and field experiments. As expected, the ethanol signals dominate the spectral landscape followed by the intense resonances from glycerol. In the high-field region, where the methyl (−CH3) signal from ethanol resonates, the resonances from several organic acids can be found, including butyrate, lactate, succinate, citrate, malate, and acetate. Furthermore, the signals from shielded methyl protons from the higher alcohols isobutanol  and isopentanol  can be observed. At mid-field, the resonance from tartaric acid is found along with the complex resonances from glycerol, the methylene (-CH2-) ethanol signal, and residual sugars. Trigonelline, 2-phenylethanol, and tyrosol are found at low-field together with the signals from the phenolic compounds caffeic acid and p-coumaric acid, and the nitrogenous bases adenine and cytosine. Metabolite assignment (level 2) is based on literature data  [http://www.ymdb.ca/; accessed on 15 April 2022).l shift) ,39,41,42Must and wine samples were also analyzed by FT-IR spectroscopy. The reader is referred to Aru et al. (2022) for more details on the FT-IR measurements of must samples . WineScaPrincipal component analysis (PCA)  was empl1H NMR datasets from the field experiment . In PCA was also employed to evaluate the impact of water deficit and different viticultural practices on the metabolome of must and wine samples from the field and screenhouse experiments separately. In As for the wine samples D, a weakIn order to scrutinize for possible additional sources of variability that potentially can be related to the observed high inter-sample diversity, PCA scores in p < 0.001) of the metabolite variability in must and 44% (p < 0.001) in wines (p < 0.01) of the total metabolite variability in must samples and 30% (p < 0.001) in wine samples. In the screenhouse experiment, the experimental effect of WD was quantified as approximatively 11% (p < 0.001) and 8% (p < 0.001) of the metabolite variations in must and wines, respectively. Pruning type  and 4% (p < 0.01) in must and wine samples, respectively. In the screenhouse experiment, pruning type accounted for approximatively 23% (p < 0.001) of the metabolite variability in must and 6% (p < 0.001) in wines. Interestingly, the treatment- and pruning type-related variability (and effect size) is attenuated by the fermentation process   was empl process A,B.The chemical changes  occurring during the winemaking process, from must to wine, were analyzed and are described in the following sections. Metabolites identified and quantified in must are amino acids, sugars, and organic acids. Only metabolites whose concentrations significantly changed as result of different treatments are described for wines.The baseline-resolved signals from 11 amino acids , six sugars , and three important organic aids  were quantified in all must samples. Metabolite fluctuations in must samples from the field experiment are shown in p-coumaric acid, the organic acids fumarate and lactate, glutamate, butyrate, and propanol were significantly affected by different water stress treatments. Amongst them, caffeic acid was highest in control and early- stressed samples, while it was decreased by mid- and late-stress. P-coumaric acid exhibited the opposite trend. Fumarate and lactate had opposite trends, with fumarate being lowest in wines from early- and mid-stressed plants, while it was the highest in wines from the same treatment lactate. Glutamate was highest in early- and mid-stressed plants.In 1H NMR metabolomics was employed to investigate the impact of different viticultural practices, including defoliation (DEF), crop thinning (CT), and irrigation (WD) on the chemical composition of must and wine samples from the cool-climate grape variety Solaris. The metabolomics investigation presented here complements our previously published study [In this study, ed study  by descr1H NMR showed inconsistent results with FT-IR measurements and thus has been excluded from all PCA models. The reason for the observed inconsistency can be ascribed to the fact that the buffer solution used for NMR sample preparation contained potassium phosphate salts, which may have altered the natural equilibrium between the three main potassium tartrate-related species present in wines, namely tartaric acid, hydrogen tartrate, and tartrate. Of these, hydrogen tartrate precipitates with potassium.An overview of the chemical changes occurring in must and wines from different growing conditions (field vs. screenhouse) were obtained using PCA, which revealed substantial metabolic differences between field- and screenhouse-grown samples. Concerning the must samples from field and screenhouse, the main metabolic difference was that the latter had higher concentrations of amino acids A. This iAs for the wines, valine, glutamate, and proline were identified in all samples and at higher concentrations in the screenhouse wines B. They ap-coumaric acid were tentatively assigned in the 1H NMR spectra of wines from both the field and screenhouse experiment. It is important to stress that phenols in wine can exist both in the glycosylated and free form—the latter being formed during the winemaking process [1H NMR. Overall, phenolic compounds were found to be slightly higher in the wines from the field experiment. This may be related to a general higher physiological maturity in the slightly warmer screenhouse compared to the field, as the phenolic concentration tend to decrease with increasing maturity [Caffeic acid and  process . The twomaturity . control samples, DEF decreased the amino acid concentration, while CT treatments significantly increased the concentration of phenylalanine, tyrosine, BCAA, and proline . Overall, our results show that, from an agronomical point of view, Solaris has a remarkable ability to tolerate and recover from water stress [p-coumaric acid. Growing conditions (field vs. screenhouse) as well as viticultural practices (defoliation and crop thinning) were found to be the main factors affecting the quality/chemical composition of Solaris must and wines. While the main chemical differences between the must and wine control samples from the field and screenhouse are mainly related to the nutrient-enriched irrigation system in the controlled screenhouse set up, defoliation and crop thinning showed a major impact on the chemical composition of Solaris wines—mainly in terms of compounds normally related to fruity aroma , non-sugar sweetness , and alcohol content. Tyrosol, which might be attractive as a natural phenolic antioxidant with high bioavailability, was increased in wines by a combination of defoliation and crop thinning. The present multidisciplinary study aimed at investigating the impact of water deficit, defoliation, and crop thinning on Solaris’ plant and fruit development (Part 1) as well as on the bulk metabolic composition of Solaris must and wines as measured by FT-IR and r stress . NeverthIn conclusion, our preliminary results show that, although wine preference is subjective, wine quality—in terms of flavor chemistry—can be optimized by fine-tuning viticultural practices and the winemaking process. It must be stressed that our results, although promising, are based on a one-year trial (vintage) and further experiments are required to confirm these findings.1H NMR spectroscopy. A more detailed description of the experimental design is given in Experiments were carried out in 2018 at the Pometum , the experimental orchard of the University of Copenhagen, and consisted of two independent trials. Both studies aimed at investigating the chemical changes occurring in must and wine samples as a result of (1) water deficit (screenhouse experiment) and (2) different viticultural practices, namely defoliation (DEF) and crop thinning (CT) (field experiment). The water deficit experiment was carried out in a screenhouse  where 36Saccharomyces cerevisiae bayanus , and moved to the fermentation room set to 17 °C. Fermentation was monitored via density and temperature control. After fermentation, 5% sulphite solution was added to the wines , which was then transferred to a room set to 3 °C for 1 week of cold settling. Subsequently, samples were analyzed by FT-IR, and a second aliquot frozen at −80 °C for future NMR analysis.A detailed description of must sample preparation and collection is given in Part I . As for Samples were analyzed by FT-IR spectroscopy using the WineScan instrument . More instrument specifications can be found in Part I . Oenolog2O, 99.9%), potassium phosphate monobasic (KH2PO4), and sodium 3-trimethylsilyl-propionate-2,2,3,3-d4 (TSP) were purchased from Sigma-Aldrich .Deuterium oxide  and TSP (5 mM). To ensure homogeneity while avoiding foaming, each sample was gently vortexed for 1 min. For each sample, 600 µL were transferred into a 5 mm O.D. NMR SampleJet tube .For each sample, a total of 3 mL was centrifuged  at 2000 rpm at 4 °C for 30 min to precipitate residual solids. Sample preparation for must and wine analysis by u et al. . Briefly1H-NMR spectra were measured at 298 K using the standard pulse sequence for presaturation of the water signal , a sweep width of 12,626 Hz (21 ppm), a 90◦ pulse, and an acquisition time of 3 s. The relaxation delay was set to 4 s. Spectral data were collected into 64K data points, after 256 scans. The receiver gain was fixed for all the experiments to an adequate value estimated through several tests for the juice and wine samples. NMR spectra were acquired in automation using IconNMR and the SampleJet system . Phase and baseline correction were performed in TOPSPIN. NMR spectra were recorded on a Bruker Avance III 600 operating at a proton Larmor’s frequency of 600.13 MHz and equipped with a 5 mm broadband inverse (BBI) probe . Data acquisition and processing were carried out using TOPSPIN3.6. After temperature equilibration (5 min), 1H NMR spectra were referenced to the TSP singlet at 0.00 ppm and signals aligned using the icoshift algorithm [http://www.ymdb.ca/; accessed on 15 April 2022). Baseline-resolved signals were quantified (relative concentration) by raw sum of spectral intensities. The datasets of metabolite concentrations were imported into the PLS_Toolbox  where the metabolites concentrations were autoscaled and analyzed by principal component analysis (PCA) [NMR data for must and wines were imported separately into MATLAB 2020b  where the lgorithm . A totallgorithm ,39,41,42is (PCA)  and ANOVis (PCA)  (1000 pe


Dalbavancin is a second-generation lipoglycopeptide antibiotic with activity against Gram-positive organisms. Dalbavancin is Food and Drug Administration (FDA)-approved for acute bacterial skin and soft tissue infections (ABSSTIs). There is a lack of substantial data on dalbavancin in more invasive infections, particularly in high-risk populations (patients with intravenous drug use and unstable living conditions). In this retrospective observational study, we reviewed all patients that received at least one dose of dalbavancin in an inpatient or outpatient setting at Parkland Hospital from February of 2019 to August of 2021. The demographics, type of infection, and rationale for dalbavancin were collected at the baseline. Clinical failure was measured by an avoidance of emergency department (ED) visits or hospital readmission at 30, 60, and 90 days. A separate analysis was conducted to estimate hospital, rehabilitation, or nursing facility days saved based on the projected length of treatment. 40 patients were included, and the majority were uninsured (85%), experiencing homelessness (60%), or had intravenous drug use (IDU) (57.5%). Indications for use included ABSSTIs (45%), bloodstream infection (67.5%), osteomyelitis (40%), infective endocarditis (10%), and septic arthritis (10%). Clinical failure was observed in 5 of the 40 patients (12.5%). Nonadherence to medical recommendations, a lack of source control, and ongoing IDU increased the risk of failure. Dalbavancin saved a total of 566 days of inpatient, rehabilitation, and nursing facility stays. Dalbavancin is a reasonable alternative to the standard of care in an at-risk population, offering decreased lengths of stays and cost savings. The uses of second-generation lipoglycopeptides are desirable alternatives to traditional outpatient parenteral antibiotic therapies for patients who otherwise would not qualify or for patients who desire less hospital contact in light of the COVID-19 pandemic.IMPORTANCE This study contributes additional experience to the literature of dalbavancin use in off-label indications, particularly for patients who do not qualify for outpatient parenteral antimicrobial therapy. The majority of the patient population were people who inject drugs and the uninsured. There is difficulty in tracking outcomes in this patient population, given their outpatient follow-up rates; however, we were able to track emergency room visits and readmissions throughout the majority of the local metroplex. The clinical use of dalbavancin at our institution also increased in the midst of the COVID-19 pandemic in an effort to preserve hospital resources and limit health care exposure. In addition, we are able to provide institution-specific cost-saving data with the use of dalbavancin. Dalbavancin is a long-acting, second-generation, lipoglycopeptide antibiotic with potent activity against Gram-positive organisms. Dalbavancin is currently Food and Drug Administration (FDA)-approved for acute bacterial skin and soft tissue infections (ABSSTIs) –2. Growi3–Staphylococcus aureus isolates , and septic arthritis due to suspected or confirmed Gram-positive infections.A total of 40 patients were included , with a median age of 49 (interquartile range [IQR] of 16.25). All patients were greater than 18 years of age. The majority of subjects were PWID, uninsured, and had unstable living conditions. Only 47.5% of the patients underwent procedural/surgical source control. Other baseline characteristics, including comorbidities, are shown in Staphylococcus aureus (MRSA) , methicillin-sensitive S. aureus (MSSA) , Streptococcus species , coagulase-negative Staphylococcus (CoNS) , and Enterococcus species . 11 (27.5%) patients had polymicrobial Gram-positive infections. The clinical indications and causative pathogens are shown in Clinical indications for dalbavancin use included ABSSTIs, BSIOM, IE, and septic arthritis. 23 (57.5%) patients had more than one clinical indication. All included patients had a microbiologically confirmed Gram-positive infection, with the most frequently isolated pathogens being methicillin-resistant A total of 3 dosing strategies were noted in this review. 27 patients received a 1,500 mg intravenous infusion as a single dose, 1 patient received a renally adjusted dose of 1,125 mg as a single infusion, and 12 patients received 1,500 mg intravenous infusions on day 1 and day 8. A total of 52 total dalbavancin doses were administered, 13 (25%) of which were given in an outpatient setting. 10 patients received at least 1 dose in an outpatient setting, 4 of which were treated entirely as outpatients in the infectious diseases (ID) clinic .Clostridioides difficile colitis, which was treated with oral vancomycin, during admission, and the other had sudden onset substernal chest pain during the infusion, which improved with a reduction of the infusion rate. Delayed adverse event monitoring was limited due to the low follow-up rates in the clinic. Only 15 (37.5%) of the included patients were seen in follow-up clinic visits.Only 2 of the 40 patients had an identified adverse drug event. Events were recorded either during their active admission or during their infusion visits in the ID clinic. One patient developed n = 3, 7.5%), ciprofloxacin , cefuroxime , amoxicillin-clavulanic acid , and intravenous piperacillin-tazobactam . One patient received suppressive doxycycline after the dalbavancin therapy, given the paraspinal and epidural abscess that was still present on imaging at the follow-up.The concomitant use of other antimicrobials was seen in 6 (15%) patients. The agents used included metronidazole  were deemed to have a clinical failure. Clinical failures were attributed to either self-discharging prior to source control, inadequate outpatient wound care, or ongoing intravenous/intramuscular drug use. The specific circumstances for the clinical failures can be seen in At the end of the review period, the electronic medical record was reviewed. A total of 3 patients had died, 2 of whom died over 1 year after the administration of dalbavancin and the last of whom died within 3 months of administration. The two patients who were over a year out from treatment died from cardiogenic shock and suicide, respectively. The death of the patient who died within 3 months of treatment was attributed to complications from dementia and/or a chronic sacral decubitus ulcer.Given that all of the patients did not qualify for traditional OPAT, the summation of projected length of treatment was determined to be the total inpatient, rehabilitation, and nursing home days required if the patient were to receive the standard of care (SOC), a total of 998 days. The actual total length of stay for all patients was then subtracted from that amount, resulting in 566 saved days. The mean length of stay was 12 days (±8.13 [standard deviation]), with an average of 14.51 projected days saved (±11.26 [standard deviation]). This resulted in a total of $1,208,410 saved, based on Parkland’s estimated charge data for a single medical-surgical bed (multiplied by the 566 days saved). This excludes the cost of medications for the SOC. This estimate is based on the cost of a hospital bed and nursing for 1 day only. Thus, it is likely grossly underestimated.S. aureus infections   over time  Medical System. The study received approval from the UTSW Institutional Review Board with a waiver of consent.A protocol by which to assess patients’ eligibility for dalbavancin was created to streamline decisions between dalbavancin versus an alternative method to complete OPAT. Per the protocol, infections eligible for dalbavancin included ABSSTIs, OM, septic arthritis, BSI, and native-valve IE due to suspected or confirmed Gram-positive infections. Conditions outside the scope of the protocol included central nervous system infections, including epidural abscesses, uncontrolled abscesses/collections, patients with prosthetic material, including but not limited to prosthetic heart valves, orthopedic hardware, pacemakers, pumps, and brain stimulators, and patients receiving hemodialysis. Patients were excluded from consideration for dalbavancin if they had a history of allergic reaction to dalbavancin or oritavancin, a history of IgE-mediated reaction to vancomycin, were pregnant or nursing, or had Child-Pugh Class B or C liver disease. The OPAT pharmacist or ID consult team made recommendations for dalbavancin, as guided by the protocol, and assessed patients on a case by case basis. All of the patients who received dalbavancin from July of 2019 to August of 2021 were included in the study, regardless of indication. The primary outcome assessed was clinical failure, a composite outcome that was defined by ED visits or hospital readmissions at 30, 60, or 90 days, relating to the original infection. Given the difficulty with follow-ups in this population, this was used as a surrogate marker. Secondary outcomes included adverse drug events (ADE), all-cause ED visits or hospital readmissions, and the total number of inpatient, rehabilitation, and nursing facility days saved. The number of days saved was calculated using the minimal duration of treatment with the standard of care that the patient would have required, as they were not traditional OPAT candidates.Data were collected from the electronic medical record and were stored in a secure internal server. Records were retrospectively reviewed, and the following data were collected: age, race, past medical history, housing status, IDU history, insurance status, inpatient/outpatient setting, length of stay, projected duration of therapy, time to follow-up, and return to ED or readmission within 30, 60, and 90 days. To capture ED visits or readmissions outside our health system, the Care-Everywhere function in Epic was used to screen encounters in all of the major hospital systems in the Dallas metroplex, with the exception of one hospital system. Parkland is a safety net hospital. Thus, most patients return to Parkland if there are ongoing medical needs. Regarding infection and treatment characteristics, the following were collected: the type of infection, pathogen isolated, treatment regimen , surgical management, adverse drug events, number of dalbavancin doses, and location of the dalbavancin infusion. The projected length of treatment was determined by an infectious diseases specialist at the time of consultation.Descriptive analyses were performed for all of the data points using the Microsoft Excel software package. The absolute and relative frequencies (%) were calculated for qualitative variables, and means and standard deviations were calculated for quantitative variables. A separate analysis was conducted to estimate the hospital, nursing home, or rehabilitation days saved as well as the resulting cost-savings, using Parkland’s estimated charge data for a single medical-surgical bed and the cost to Parkland for outpatient skilled nursing facilities/rehabilitation centers.


COL1A1, ALP, BGLAP, SPP1, SP7, and AML3 in differentiated cells compared to the cells cultured in the culture plate. However, butterfly wings were shown to provide a biomimetic microstructure and proper bone regenerative capacity through a unique combination of various structural and material properties. Therefore, this novel platform can be confidently recommended for bone tissue engineering applications.Despite the advent of promising technologies in tissue engineering, finding a biomimetic 3D bio-construct capable of enhancing cell attachment, maintenance, and function is still a challenge in producing tailorable scaffolds for bone regeneration. Here, osteostimulatory effects of the butterfly wings as a naturally porous and non-toxic chitinous scaffold on mesenchymal stromal cells are assessed. The topographical characterization of the butterfly wings implied their ability to mimic bone tissue microenvironment, whereas their regenerative potential was validated after a 14-day cell culture. In vivo analysis showed that the scaffold induced no major inflammatory response in Wistar rats. Topographical features of the bioconstruct upregulated the osteogenic genes, including  Bones are dynamic structures in the human body with limited self-repair capability against physical injuries or traumas . As a reSome common causes of generation of bone defects include motorcar accidents, sport-related injuries, osteoporosis, osteonecrosis, and primary or metastatic malignancies of bones, which impose a significant burden on healthcare systems across the world .The currently applied method, autograft, deals with the repair of the lesion site via harvesting intact bone from the iliac crest and grafting it to the damaged area. Despite several advantages of this method, complications such as morbidity and possible infection of the donor site increase the risk of bleeding and complexity of the operation. Additionally, the limited amount of harvesting material challenges the management of the injury. Therefore, the available approaches cannot solve the problem entirely and impose some adverse complications on the patients ,6.Bone tissue engineering combines material and life science principles to fabricate implantable bioconstructs to replace the damaged bone . This teTissue engineering (TE) scaffolds are meant to create an appropriate niche for cell migration and proliferation, tissue reconstruction, nutrition and growth factors’ delivery, and waste removal from cells . To achiResearchers in the field of tissue engineering have studied many natural and synthetic scaffold materials to find the most appropriate ones for tissue reconstruction. The electrospun scaffold fabricated via PHB with incorporated rGO or PANi is one example of synthetic bioconstructs . In thisIn recent years, production of natural scaffolds derived from plant and animal sources has attracted the attention of TE researchers ,19,20,21Morpho menelaus and Papilio ulysses telegonus as TE scaffolds with improved cells survival and grow [Nowadays, the chitin-based scaffolds are widely used due to their low toxicity, biodegradability, biocompatibility, and renewability . The cheand grow . Moreoveand grow ,38,39,40Papilio demoleus among several species of butterflies due to its chitin abundancy as well as its appropriate wing surface morphology that enables the cells to adhere without the need to decellularization. On the other hand, these butterflies are regarded as a citrus pest and are abundant during spring and summer. As a result, the applied strategy is not only cost-effective but also eco-friendly. In this article, we hypothesized that P. demoleus wings could provide an appropriate scaffold for osteogenic differentiation, according to their biocompatible, porous, non-cytotoxic chitin-based structure. The main objectives of this study are to (1) examine surface properties, polymer structure, and biocompatibility of the wings of P. demoleus butterfly, (2) apply new and more achievable methods to improve the wing’s surface properties, (3) demonstrate its efficacy in directing osteogenic differentiation of Ad-MSCs, (4) introduce the wings of P. demoleus as a novel, eco-friendly, and cost-efficient source of chitin-based scaffold for cell replication and differentiation.We intentionally chose P. demoleus species were collected from citrus gardens located in southwestern Iran in spring. The samples were stored in a closed container in a dry atmosphere while being transferred to the laboratory. The butterfly species had been already approved by the Iranian Research Institute of Plant Protection (IRIPP).Butterflies of 3), and ethanol (96%) were purchased from Merck . Phosphate-buffered saline (PBS) and Collagenase type I were obtained from FUJIFILM Wako Pure Chemical Corporation . Tetrazolium dye MTT 3--2,5-diphenyltetrazolium bromide and Dulbecco’s modified eagle Medium-F12 were purchased from Sigma-Aldrich . Pen-Strep solution and fetal bovine serum (FBS) were obtained from Gibco .Hydrochloric acid (HCl) (37%), sodium hydroxide (NaOH), chloroform   was performed to characterize the morphology of the scaffold surface as well as the cells adhered on the scaffold. Previously, the samples were sputter-coated with gold. The obtained images were analyzed using MIP (Microstructure Image Processing) software to evaluate the surface physical structure, porosity, and distance between pores.To image the topographical features of the scaffold, atomic force microscopy (Veeco Auto Probe CP research) was performed. Using AFM, root mean square (Rq) and average roughness (Ra) were measured on 5 µm × 5 µm slices of the scaffold. Additionally, the obtained results were processed to create three-dimensional images.−1 at 22 °C and under an air humidity of 20%.ATR-FTIR (spectrum 400 Perkin Elmer) analysis was used to determine the surface chemistry of the butterfly wing scaffolds. To do so, the samples were IR irradiated in the spectral range of 450–4000 cm2) at room temperature and measuring the water contact angle after 10 s.A water contact angle test was performed on the surface-treated and untreated wings to validate the efficacy of the applied treatments on their hydrophilicity. To do so, the static contact angle was measured using a JIKAN CAG-20 device by depositing a water droplet on the sample surface (1 cmBrunauer–Emmett–Teller (BET) (BELSORP MINI II) analysis was used to determine the specific surface area, pore volume, and pore size of the scaffold ,43. The To validate the biocompatibility of the wing scaffolds, they were implanted on the skin of rats. Twelve Wistar rats, weighing 263 g on average, were purchased from the Animal Breeding Laboratory and Care Center of Shahid Beheshti University, Iran. The rats were kept in separate cages and were submitted to the same dietary treatment with free access to drinkable water. The rats were eventually anesthetized using chloral hydrate , and circular excision wounds (0.5 cm in diameter) were made on shaved areas on their back to allow subcutaneous implantation of the wing scaffolds.IL-6) and tumor necrosis factor-alpha  were measured in the blood samples using ELISA. For histopathological analysis, four tissue sections were obtained on days 1, 7, 14, and 21 under general anesthesia. The as-prepared sections were fixed in 10% paraformaldehyde  and stained with hematoxylin-eosin. Moreover, some tissue samples were treated to extract RNA using a DENAzist Column RNA isolation kit . Real-Time RT-PCR was performed to detect any changes in the expression levels of IL-6. The cDNA was synthesized by the cDNA synthesis kit . Furthermore, the primer sequences were designed by CLCwork and Oligo7 software  harvested from adipose tissue stem cells  were monitored by a CD14, CD45, CD34, CD73, CD90, and CD105 marker, as similarly shown by Azadian et al. [4 cells per well was used for this purpose and allowed to adhere to the scaffold surface. Subsequently, 500 μL of DMEM F12 medium supplemented with 10% FBS, 2 mM glutamate, 1% penicillin-streptomycin, and 15 mM sodium bicarbonate were added to the wells. The scaffold-cell co-cultures were incubated at 37 °C and 5% CO2. Later, differentiation of MSCs to osteoblasts was investigated at 7, 14 and 21-day intervals. Moreover, control cells were cultured with an osteogenic medium supplemented with dexamethasone (12 μM) (Sigma Aldrich), ascorbic acid (50 μg/mL) , and β-glycerophosphate (10 μM) (Sigma-Aldrich) on tissue culture plates (TCPS).Following the chemical treatment and hydrophilization of the scaffolds, they were sterilized using UV irradiation for 40 min and incubation in 70% ethanol for 2 min. Then, they were washed three times with PBS 1× and 2.5% penicillin/streptomycin for 15 min. The samples were later transferred to a 24-well microplate containing cell culture medium and incubated for 12 h in 5% COn et al. . The celCytotoxicity and cell viability of the scaffolds were quantified using the MTT assay. Human foreskin fibroblasts (HFFs)  were seeded on the scaffold sterilized by UV radiation and 70% ethanol. After several incubation periods of 1, 3, 5, and 7 days, supernatants were aspirated out, and 10 µL MTT solution (50 mg/10 mL PBS) was added to each well containing the scaffolds. Then, 90 µL of culture medium was added to each well, and incubation continued for 4 h. After that, MTT and culture medium were removed. Later, formazan was dissolved in 100 µL DMSO , and well plates were incubated at 37 °C in a shaker incubator for 20–30 min. Finally, optical density was recorded at λ ~ 590 nm using an ELISA reader . The cell adhesion capability and cell proliferation evaluated by MTT assay and Dapi staining , Saso2 cTo investigate the cell viability and cell adhesion on the scaffolds, the cell nuclei were stained with DAPI  . To do so, on the 14th day after the cell culture, the cells present on the scaffold were fixed using 4% paraformaldehyde, were washed three times with water, and then exposed to the DAPI reagent (5 μg/mL) for 10 min. Eventually, the samples were washed twice with PBS and imaged at different magnifications by a fluorescence microscope , Italy) with a UV filter.After 2 weeks of scaffold-cell co-culture, FE- SEM imaging was perfromed to monitor the cells growth on the scaffold. For this reason, the samples were fixed with 4% paraformaldehyde  for 30 min and washed with PBS three times. After that, the scaffolds were dehydrated using gradients of ethanol (20% to 96%). Finally, the scaffolds were gold-coated at 15 mA for 3 min using a Hitachi E-1010 ion-sputtering device.w/v) Alizarin Red S  for 5 min. The dye was dissolved in distilled water (pH 7.2), and the samples were later washed twice with PBS. Finally, the results were inspected by a phase-contrast light microscope.Alizarin Red (AR) staining was used to demonstrate the extent of calcium deposits formed by Ad-MSCs cultured on the scaffold. The cultured cells were fixed with 4% paraformaldehyde and were subsequently stained with 2%  after 7- and 14-days cell culture. First, the total protein content of the samples was extracted manually using the RIPA solution. Afterward, ALP activity was determined according to the manufacturer’s kit. The unit value of the enzyme was normalized to the total protein content. Eventually, the optical absorbance of the samples was measured at λ~405 nm by a microplate reader .COL1A1), alkaline phosphatase (ALP), bone gamma-carboxyglutamate , secreted phosphoprotein 1 , SP7 transcription factor 7 (SP7), and runt-related transcription factor 2  as well as the GAPDH reference gene, was quantified by Real-Time-PCR . Additionally, the primer sequences were designed by the CLCwork and Oligo7 software  and GraphPad Prism 8 software.Total RNA content was determined after 2 weeks of culturing the cells on the scaffold using the DENAzist Column RNA Isolation Kit . RNA quantity and quality were characterized using NanoDrop  and electrophoresis (1.8% agarose gel), respectively. The single-strand cDNA synthesis was performed using the cDNA synthesis kit (Parstous Company), according to the manufacturer’s instructions. Fold changes in gene expression were determined using the comparative Ct method (ΔΔCt) with normalization to the housekeeping gene, The immunocytochemistry (ICC) test was performed using Osteocalcin antibody to characterize osteogenic differentiation. For this purpose, at first, the cells were incubated in PBST (PBS + Triton x100 (0.2%) (Merck)) for 5 min at room temperature to improve the cell migration. After washing with PBS, the well plates were incubated with PBSTw for one hour at 37 °C. After additional washing with PBS (three times), the cells were kept in PBSTW  1% + Glycine) at 37 °C for 30 min. The Human anti-rabbit primary osteocalcin antibody  was added in PBSTW and incubated at 4 °C for 24 h . After washing with PBS (three times), BSA (1%) was added in dark, incubated for 1 h, and washed three times (each time for 5 min). After washing with PBS, supplemented with 0.1% Tween-20, the cells were incubated by secondary antibody (for 2 h) and then washed by PBS. The cells were observed by fluorescent microscope , Italy) using the 430–560 nm filter.p-value of less than 0.05 was considered significant.All experiments were repeated three times, and the obtained data were expressed as mean ± SD. Data analysis was performed by GraphPad Prism 8 and compared using two-way analysis of variance (ANOVA) with a repetition t-test. A After physical removal of the scales, the wings became transparent and less hydrophobic, and thus more prone to chemical treatment. Moreover, the chemical treatment removed the residual scales and made the wings completely transparent. SEM images c,d demonStatistical analysis of the surface is crucial for quantifying the surface features. Therefore, the scaffold roughness was assessed using atomic force microscopy. The results of the surface height distribution analysis showed that the average roughness (Ra) is 27.87 nm, which falls in the nanoscale roughness. The surface skewness, which is an indication for deviation in the surface height distribution from normal distribution, was +0.209. This positive deviation from the normal or Gaussian surface height distribution indicates that the peaks are predominant on the surface, which increases the surface available for the cells to adhere to the surface. The results also indicate an opportune microscale roughness that implies the promising potential of the scaffold to encourage cell adhesion and activation a.−1; presence of a broad and asymmetric double band in the wavenumber range of 3170–3580 cm−1 is an indication of overlapped structural -OH and -NH bands. Symmetric CH3 and asymmetric CH2 stretching bands at 2963 cm−1, CH stretching band at 2883 cm−1, and C=O of amide I band at 1659 cm−1 in the ATR-FTIR spectrum results. Moreover, the bands at 1539 cm−1, 1449 cm−1, and 1379 cm−1 are related to the N-H of amide II, CH2 bending and CH3 deformation, and CH bending and symmetric CH3 deformation bands, respectively. Furthermore, bands at 1308 cm−1, 1156 cm−1, 1037 cm−1, and 743 cm−1 correspond to the amide III and CH2 wagging, asymmetric bridge O2 stretching, CO stretching, and NH out-of-plane bending bands, respectively. The obtained bonds match the standard chemical bonds of chitin-based polymers; accordingly, our scaffold predominantly consists of chitin [One of the main factors governing the cell attachment and proliferation onto a substrate is the chemical cue comprising of surface functionalities, which further controls some physical cues such as wetting and surface hydrophilicity. The chemical analysis of the substrate provides insights into its structure and the functionalities available. As shown in f chitin ,46.The effect of the physico-chemical treatment on hydrophilicity of the butterfly wings was monitored using water contact angle. As shown in P. demoleus wing was characterized by BET. As tabulated in s,BET), total pore volume, and average pore diameter are 20.073 m2 g−1, 0.1461 cm3 g−1, and 29.119 nm, respectively.The porous structure of the Skin erythema and swelling, fever, weight loss, and diet habit changes were monitored for 21 days. Skin reactions were expected to emerge, considering the invasive nature of the applied procedure. However, no exudative discharge and no dramatic swelling were observed. The body temperature of the test group showed a slight increase on days 1 to 3 after implantation; however, a high-grade fever related to infection was absent. A weight loss of 5–10 g was detected, and the food consumption of the rats remained unchanged. In the presence of the scaffold, inflammatory factors such as Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were at a higher level compared to the control on the 1st day, but thereafter they declined. This behavior implies the mildness of the systemic response, originated from the invasive procedure . In concAs verified by a MTT assay, the cells maintain their optimum viability in the presence of the wing scaffold and properly proliferate a. The peA spreading, viable cell population is evident from the analysis of the fluorescent microscopy micrographs. Blue spots present the active areas of cell nucleus viability, confirming the non-cytotoxicity of the scaffold e,f.Micrographs of FE-SEM revealed adhesion of cultured cells with their cytoplasmic process adhered to the surface. Micrographs on days 14 and 21 a–c are eCalcium content as a sign of osteogenic differentiation of mesenchymal stem cells was investigated by Alizarin red staining. The results of this staining showed significant calcium deposition in Ad-MSCs cultured on the scaffold compared to Ad-MSCs cultured on the TCPS d–g.ALP activity was measured to investigate the osteogenic differentiation of MSCs on the scaffolds. Results show that ALP activity of MSCs cultured on the scaffold was significantly greater than MSCs and MSCs with standard osteogenic differentiation treatment cultured on TCPS and gradually increased until the 21st day h.COL1A1, ALP, and SP7 genes were upregulated on day 21 compared to day 14. While the expression of AML3, BGLAP, and SPP1 genes increased until the 14th day, it decreased on day 21, indicating the differentiation of MSCs into mature osteoblasts. Moreover, the expression of ALP gene in the cells cultured on the wing scaffold significantly raised compared to those cultured on TCPS (control+ sample), As verified via RT-qPCR, the expression level of The results of the immunocytochemistry assay showed that osteocalcin protein expirations on the surface of differentiation cells on the scaffold confirm the efficiency of the scaffold in differentiated MSCs to osteoblasts .P.domeleus species because of its abundance as an agricultural pest, a green source, and, the most important, anisotropic wing structure [The Scaly cover of butterfly wings has an anisotropic arrangement in micro-, submicro-, and nano-scales . It is atructure . DescaliThe FTIR survey has confirmed that chitin is the basic and dominant background material of the butterfly wing. Chitin scaffolds have been found to influence osteogenesis-related signaling pathways and stimulate mineralization to facilitate bone regeneration. Chitin scaffolds can provide a proper template for the deposition of calcium phosphate crystals to the mineralization process of the organic matrix in the human body. Moreover, they possess biological properties required for a functional scaffold, such as biocompatibility, biodegradability, low immunogenicity, and chemical stability .Butterfly wing-based scaffolds do not demand high-tech equipment or complex protocols for decellularization, so they are easily treatable, and we treated wings in the briefest way possible. However, complex decellularization approaches are required in the case of cellulose-based scaffolds derived from plants. For example, in our previous studies, we established multi-step approaches to decellularize cabbage leaves and onion to obtain a cellulose-based scaffold for osteogenic differentiation ,49.Moreover, the preparation of synthetic scaffolds is time-consuming, as, for example, in the construction of two kinds of porous ceramics, a novel sintered porous hydroxyapatite, and a porous β-tricalcium phosphate (β-TCP), as well as a collagen-phosphosphoryn sponge as scaffolds for bone cell culture, which took a relatively time-consuming process to prepare . On the 2+ deposition [P. domeleus butterfly disrupts the ability of cells to attach and interact with the scaffold [P. demoleus wings was significantly overcome by the two-step chemical and subsequent chloroform and plasma treatment, causing a decrease from 135° to complete absorption in water contact angle. Moreover, it is evident that physical removal of scales as the natural water barrier reduces the angle (135° to 97°).It is observed that hydrophilicity increase cell-material interaction, cell adhesion, proliferation, infiltration, and osteogenic differentiation. It also contributes to better mineralization and Caposition . Extremescaffold . HydrophSurface roughness affects cell response biology and, in particular ranges of average roughness (Ra), can enhance cell adhesion and proliferation. Surface roughness at the nanoscale mimics the surface of natural tissue and at the microscale increases the bioactivity through the absorption of proteins. Our scaffold exhibits enough roughness at both scales based on the AFM results, making it an appropriate surface for cell activation. In one study, it has been shown that the nanoscale Ra of the silicon surface, between 0 and 64 nm (not above), can positively promote neural cell adherence. Considering suitable cell attachment based on FE-SEM imaging and DAPI staining in our study, the same range can be generalized to Ad-MSCs on the chitin surface. Fluorescent microscopy of DAPI-stained cell-laden wings also reveals an almost uniform distribution of cells on the surface, which validates successful cell attachment ,54,55. DIn vivo experiments on Wistar rats showed that the scaffold is tolerated well by the rats. Mild fever and local allergic reactions were induced, but systemic inflammatory response and agitated behavior were not observed during the follow-up period. Therefore, the scaffold did not induce infection or exudative response, which might indicate anti-microbial and anti-inflammatory traits of it and also confirmed biocompatibility and non-toxicity of the scaffold.Porosity is the key feature to successful bone scaffolding. Material without pores does not serve osteogenic differentiation . This fe2+ ions are necessary elements in the formation of hydroxyapatite in bones, so they are essential components of osseous ECM. We assessed the scaffold’s capacity of mineralization by Alizarin Red staining. Alkaline phosphatase activity is also another determinant in bone ossification. The results of both analyses show the differentiation of MSCs into osteoblasts. Osteocalcin is a calcium-binding peptide that is secreted by osteoblasts. As a non-collagen protein, it is the most abundant protein of organic bone matrix after collagen [Osteogenesis-related activities were surveyed using Alizarin Red staining, immunocytochemistry, ALP enzyme assay, and RT-PCR. Cacollagen . Its precollagen . In lineAML3 gene is thought to play a role in the differentiation process of MSCs into mature osteoblasts since it regulates major signaling pathways related to osteogenesis such as the hedgehog, Fgf, and Wnt signaling. Activation of a major signaling pathway such as Wnt/β-catenin signal cascade leads to upregulation of other essential transcription factors to osteogenic differentiation such as Runx2 [COL1A1 and ALP are early markers of osteogenic differentiation. COL1A1 is crucial to bone phenotyping. Based on RT-PCR results, both show a sustained increasing level of expression from day 7 to 21. BGLAP is the most abundant structural molecule in bone after COL1A1 and is one of the few molecules specific to this tissue [BGLAP is decreasing from day 14 to 21, similar to the positive control, but it shows slightly higher levels of expression than the positive control. Therefore, the scaffold can provide cells with enough facilities to direct their differentiation through osteogenic-related gene expression.The expression of osteogenesis-related genes is substantial to confirm that bone formation is happening in the proposed scaffold. For instance, the as Runx2 ,62. COL1s tissue . The expMorpho menelaus, Papilio ulysses telegonus, and Ornithoptera croesus lydius [In similar studies, Elbaz et al. reported that NIH-3T3 fibroblast cells were attached and survived well on the scaffolds obtained from the wings of three different butterfly species, including s lydius . They has lydius . HoweverM. menelaus and Papilio ulysses telegonus were used as cultures for neural cells, which have been shown to provide enough facilities for cell growth and cell orientation. They have inspected mature Schwan cells and neural ganglia and did not check the effect of physicochemical cues of butterfly wings in guiding stem cell fate [Furthermore, in a recent study, the wings of ell fate . FindingDespite many advances in scaffold fabrication methods, various clinical trials should examine their efficacy and safety as implantable grafts. The ability to establish homeostasis, vasculature link with adjacent tissue, and regeneration are vital needs of a tissue, which future studies should evaluate proposed scaffolds ,64.P. demoleus as the renewable source for developing an appropriate scaffold. In vitro, biological studies confirmed that simply treated P. domeleus butterfly wings can be used as biocompatible, biodegradable, and nontoxic scaffolds for mesenchymal stem cell culture and proliferation. Due to the proper surface morphology revealed by the SEM results and cell adhesion, viability, and differentiation proved by the assays mentioned earlier, the inherent ability of the scaffold to promote bone formation has been established in our study. In conclusion, the scaffold could be used in bone tissue engineering as the optimal candidate for the treatment of bone damages, along with its easy availability and cost-effectiveness.This study introduces the extraction methodology and the biological applicability of the wings of


Cervical cancer is the fourth most common cancer in women worldwide. More than 90% of cases are caused by the human papillomavirus (HPV). Vaccines developed only guard against a few HPV types and do not protect people who have already been infected. HPV is a small DNA virus that infects the basal layer of the stratified epithelium of the skin and mucosa through small breaks and replicates as the cells differentiate. The mucosal types of HPV can be classified into low-risk and high-risk groups, based on their association with cancer. Among HPV types in high-risk group, HPV type 16 (HPV-16) is the most common, causing 50% of all cancer cases. HPV infection can occur as transient or persistent infections, based on the ability of immune system to clear the virus. Persistent infection is characterized by the integration of HPV genome. HPV-16 exhibits a different integration pattern, with only 50% reported to be integrated at the carcinoma stage. Replication of the HPV genome depends on protein E1, an ATP-dependent helicase. E1 is essential for the amplification of the viral episome in infected cells. Previous studies have shown that E1 does not only act as a helicase protein but is also involved in recruiting and interacting with other host proteins. E1 has also been deemed to drive host cell proliferation. Recent studies have emphasized the emerging role of HPV E1 in cervical carcinogenesis. In this review, a possible mechanism by which E1 drives cell proliferation and oncogenesis will be discussed. Persistent infection with some types of human papillomaviruses (HPVs) is the major factor contributing to the development of cervical carcinogenesis . To obsein situ . MoreovePapillomaviridae and currently divided into 5 genera, namely Alpha-, Beta-, Gamma-, Mupa- and Nupapapillomavirus containing members, all of which can infect humans  and low-risk HPVs (Lr-HPVs) - based on the cancer risk associated with their infection. Hr-HPVs include types 16, 18, 31 and 33 , of which are divided into three functional regions - early (E) region, late (L) region, and noncoding part or long control region (LCR) . The earll death . In addill death . HPV E7 ll death . It is tThe E1 protein serves as the primary replication protein of HPV and is an ATP-dependent helicase that binds to the viral origin of replication and unwinds the viral DNA to initiate replication . Across To initiate HPV DNA replication, E1 interacts with E2 , which aThe most well-characterized function of HPV in cervical carcinogenesis is the overexpression of E6 and E7 oncoproteins, which mainly target p53 and pRb tumor suppressor proteins . HoweverThe physical state of the HPV genome, namely integrated, episomal, or mixed, is correlated with cervical carcinogenesis . HPV intvia regulation of FOXO3 in cervical carcinogenesis is still unclear.Failure to induce apoptosis could drive tumorigenesis through multiple mechanisms . In HPV1A variety of mutations in E1/E2 genes have been identified in cervical cancer samples. The clinical stage of patients with the discovered HPV E1 mutations have also been noted in various studies. Many of these variants were linked with lower grade lesions such as noted in previous studies where a 63-bp duplication variant was associated with lower disease progression . Other mvia the host cells’ DNA damage repair pathways and contributes to carcinogenesis , such as, IFN-α and IFN-β, form integral components of the innate immune response, playing important roles in antiviral, anti-proliferative and immunomodulatory functions . HPV18 ESeveral studies have explored possible roles of HPV16 E1 in cervical carcinogenesis and have attributed numerous cellular mechanisms, including increased expression of genes involved in cell survival and apoptosis, inhibition of the antiviral immune response, viral genome maintenance, helicase activity and inactivating mutations . To bettConceptualization, FB, TS, and PB; Writing – Original Draft Preparation, FB, and TS; Writing – Review & Editing, PB, SV, and AC; Supervision, PB; Project Administration, PB and SV; Funding Acquisition, FB, TS, PB, SV and AC; Figure Illustration, TS. All authors contributed to the article and approved the submitted version.The 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, The Overseas Research Experience Scholarship for Graduate Student, Rachadaphiseksomphot Endowment Fund, and Thailand Science Research and Innovation Fund (TSRI), Chulalongkorn University, Bangkok 10330, Thailand and University of Liverpool, UK.We would like to acknowledge that all figures were created with BioRender.com platform.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A 50-year-old female patient presented with post-exercise dyspnea in September 2016, and was subsequently diagnosed with SCLC with multiple brain and spinal metastases. The first-line treatment was etoposide combined with cisplatin and synchronously performed radiotherapy for the brain and spinal cord metastases. She was treated with anlotinib after disease progression in December 2018 and continued to have clinical benefit for nearly 25 months. Unexpectedly, the patient can still benefit from further combination treatment with durvalumab after another disease progression in February 2021. Thus, it may be a potential option to use anlotinib along with immunotherapy after the anlotinib resistance in SCLC, but more clinical data are still needed to confirm it. Moreover, ctDNA dynamic monitoring was performed and reflected the outcome of the process of treatment. Small cell lung cancer (SCLC) is a malignant tumor with aggressive, rapid progression, and metastatic potential, accounting for about 10%–15% of lung cancer cases. At present, chemotherapy is still the main treatment for SCLC, and only a small number of patients can receive second-line treatment with limited benefit. Immune checkpoint inhibitors have shown good clinical effects in the first-line and backward treatment of SCLC, but their absolute benefit for SCLC is still limited . Besides2 (d1–d5) + cisplatin 120 mg/m2 (d1)” for two cycles on 23 September 2016 and 13 October 2016. Evaluation by CT scan showed a partial response (PR) based on the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Concurrent chemoradiotherapy (cCRT) was sequentially delivered, followed by two more cycles of adjuvant chemotherapy. The radiotherapy regimen including GTV/CTV 60/50 Gy/20 times for right lung and right hilar lymph node lesions, CTV 40 Gy/20 times for spinal cord metastasis at T2–3, CTV 30 Gy for intracranial metastasis of the whole brain , and 50 Gy/15 times for GTV intracranial metastasis. During cCRT, the patient developed grade 1–2 gastrointestinal adverse reactions and grade 4 granulocytopenia and thrombocytopenia, so the adjuvant chemotherapy was suspended. The response was categorized as PR  of her brain and spinal cord revealed multiple brain and spinal metastases Figure 2Unfortunately, the patient suffered multifocal metastases on October 9, 2018. PET/CT found a new lesion in the right lower lobe with multiple new hypermetabolic mediastinal lymph nodes. Meanwhile, a plasma ctDNA test was performed and six gene missense mutations were found, including CREBBP, KIT, MUTYH, MYC, PREX2, and SMO. Blood tumor mutational burden (bTMB), defined as the number of somatic, coding, base substitution, and indel mutations per megabase (Mb) of genome examined, was calculated from the GENETRON OncoPanscan 825 Panel NGS platforms. “TMB high” was defined as cases with a TMB of ≥10 muts/Mb, and the bTMB of this patient was 23.33 muts/Mb. Since there were no approved immunotherapy drugs for SCLC, the patient just received another two cycles of “etoposide + lobaplatin” chemotherapy on 18 October 2018 and 12 November 2018. But the response was categorized as progressive disease (PD) (The patient then started taking anlotinib (12 mg d1–d14/q3w) from December 2018 until the scale of the pulmonary lesion shrank and cavitated   .However, the clinical effectiveness of the anti-PD-1 inhibitors nivolumab and pembrolizumab in the third-line treatment of SCLC is still controversial. In the Checkmate032 study, the ORR was 10% in patients with relapsed SCLC treated with nivolumab 3 mg/kg, 23% in nivolumab (1 mg/kg) plus ipilimumab (3 mg/kg), and 19% in nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg) . SimilarIn recent years, the synergistic anti-tumor effect of anti-angiogenic drugs combined with immune checkpoint inhibitors has been supported by several studies. In preclinical  studies,However, although anti-angiogenic or anti-angiogenic drugs combined with immune checkpoint inhibitors for treating SCLC have shown preliminary efficacy, there are still many issues to be discussed, such as the suitable treatment population and the dose of treatment. In the combination therapy, the appropriate dose of anti-angiogenic drugs and the medication regimen are still worth exploring. Lin et al.  found thMoreover, liquid biopsy refers to the analysis of tumor-derived components in body fluids, among which circulating tumor DNA (ctDNA) has been used for dynamic monitoring of tumor changes, therapeutic effects, and patient prognosis in many cancers, including NSCLC , melanomHere, we found that patients with extensive SCLC can benefit from anti-angiogenic therapy plus immunotherapy depending on the situation. The efficacy may be assessed by ctDNA or bTMB, but further research is also needed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Authors HYW, XNW, SXJ, and JNZ collected the clinical information, diagnostic information, therapeutic information, and images of the patients. HYW wrote the manuscript. YH identified the case and submitted the manuscript. JNZ and CHZ revised the manuscript. YJZ and JL proofread the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the Wu Jieping Medical Foundation (No. 320.6750.2020-19-15).We are thankful to HY, KW, and YL for their kindness help in this case. All the authors are thankful to the patients who participated in this case.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.