Emerging evidence suggests that soluble epoxide hydrolase (sEH) inhibition is a valuable therapeutic strategy for the treatment of numerous diseases, including those of the liver. sEH rapidly degrades cytochrome P450-produced epoxygenated lipids (epoxy-fatty acids), which are synthesized from omega-3 and omega-6 polyunsaturated fatty acids, that generally exert beneficial effects on several cellular processes. sEH hydrolysis of epoxy-fatty acids produces dihydroxy-fatty acids which are typically less biologically active than their parent epoxide. Efforts to develop sEH inhibitors have made available numerous compounds that show therapeutic efficacy and a wide margin of safety in a variety of different diseases, including non-alcoholic fatty liver disease, liver fibrosis, portal hypertension, and others. This review summarizes research efforts which characterize the applications, underlying effects, and molecular mechanisms of sEH inhibitors in these liver diseases and identifies gaps in knowledge for future research. Ephx1–4, where Ephx1 and Ephx2 encode microsomal and soluble EHs , and Ephx3 and Ephx4 encode EH3 and EH4, which are not well characterized. EHs are expressed across all domains of life, including both mammalian and non-mammalian animals [Since their discovery in the 1970s, mammalian epoxide hydrolases (EHs) have become an increasingly popular focus of research due to their role in disease pathology . This fa animals , fungi,  animals ). EHs ca animals ,9,10 and animals , and can animals  and tiss animals . However animals .ClinicalTrials.gov NCT03486223) and smoking-related endothelial dysfunction [ClinicalTrials.gov NCT00847899). Another sEHI, EC5026, is under development to reduce neuropathic pain in humans (NIH NIDA 1UG3DA048767-01).The potent biological effects of epFAs are abrogated due to their rapid hydrolysis by EHs—particularly sEH, the main EH responsible for endogenous lipid epoxide degradation . sEH is function . AnotherThere is accumulating evidence that sEH expression is induced in many liver pathologies, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, and portal hypertension (PTH) ,43,44,45https://www.ncbi.nlm.nih.gov/pmc/) was searched for key literature sources  mice were also fed a control or HFD. Compared to controls, HFD + t-AUCB mice had decreased steatosis as shown by liver hematoxylin and eosin (H&E) staining and reduced liver triglyceride content. Unlike Iyer et al., Liu et al. demonstrated a marked improvement in liver inflammation in HFD mice given t-AUCB or in Ephx2−/− mice. Specifically, decreased macrophage accumulation as determined by F4/80 immunohistochemistry and decreased mRNA expression of numerous pro-inflammatory cytokines  were observed. In this study, t-AUCB was also administered in a prevention paradigm, where the inhibitor was added to the drinking water prior to the animals being placed on a longer term 16-week HFD feeding protocol. In this paradigm, the NAFLD phenotype was similarly abrogated—decreased liver organ hypertrophy, decreased steatosis and liver/plasma triglycerides, and decreased inflammatory cytokine expression were reported in this model. Further, markers of inflammatory pathway activation (JNK and p38) were reduced. Conversely, when mice were injected with adenoviruses encoding human sEH, metabolic syndrome was exacerbated—liver/plasma lipids were increased, pro-inflammatory cytokine production was increased, and JNK and p38 proteins were increased. sEH inhibition or deletion also caused a reduction in HFD-induced adipose tissue inflammation, suggesting a role for the adipose-liver axis in sEH-mediated liver pathology. A study by Yao et al. also supports a pathogenic role for sEH in NAFLD [Cpt1α, Acox1, and Mcad).There are several pre-clinical studies demonstrating the beneficial effects of sEH inhibition in NAFLD and associated metabolic abnormalities. A report by Iyer et al. investigated the efficacy of sEH inhibition in metabolic syndrome using male Wistar rats fed a high-carbohydrate, high-fat (HCHF) diet ad libitum with or without the sEHI t-AUCB administered in the drinking water . Comparein NAFLD . Here, ain NAFLD ,60. In mS-pyrazol-1-yl) benzenesulfonamide (PTUPB) reduced inflammasome activation, as evidenced by a decrease in Nlrp3/NLRP3 and Asc expression, compared to HFD-fed mice without PTUPB. HFD-induced expression of downstream inflammasome targets pro-caspase 1, pro-IL1β, pro-IL18, and caspase 1 p10 were also decreased by PTUPB [Mechanistically, evidence generated by Sun et al. suggests that sEH inhibition may reduce hepatic inflammation by blocking inflammasome activation . While tby PTUPB . AdditioEphx2−/− mice and wild-type (WT) mice administered TUPS [Hepatic endoplasmic reticulum (ER) stress is a known cellular consequence of NAFLD and metabolic syndrome. During ER stress, accumulation of unfolded proteins in the ER lumen activates the unfolded protein response (UPR), a signaling pathway which acts to either mitigate the burden of unfolded proteins or trigger apoptosis . ER strered TUPS . SpecifiLopez-Vicario et al. further corroborated the ability of sEH inhibition to attenuate hepatic steatosis and inflammation  . The ER Liver fibrosis is a pathological feature of multiple liver diseases, including NASH, severe ALD, viral hepatitis, and cholestatic liver disease. Progressive fibrosis ultimately leads to liver cirrhosis and subsequent liver failure . The sca4)-induced liver fibrosis in mice given TPPU in the drinking water [4 and reversal back to control levels by TPPU. More mechanistically, TPPU decreased expression of genes associated with HSC activation such as Col1a2, Col3a1, Itag2 (integrin α2), and Tsp2 (thrombospondin 2). TPPU also decreased both the mRNA expression and activity of matrix metalloproteases (MMPs), enzymes that positively correlate with fibrosis. CCl4 is also a potent inducer of liver inflammation and ER stress, both of which contribute to HSC activation. Harris et al. also showed that TPPU attenuated CCl4-induced markers of inflammation (Cxcr4 and Ccr2), ER stress , and fibrosis (Tgfβ1), indicating that sEH inhibition may inhibit liver fibrosis both directly by acting on pro-fibrotic mediators and indirectly by acting on contributing factors. The use of an additional sEHI (t-TUCB) as well as Ephx2−/− mice validated these results, suggesting a pathological role for sEH in liver fibrosis. Similar protection against CCl4-induced liver fibrosis and portal pressure were obtained by Zhang et al. with the sEHI t-TUCB [4-induced HSC activation markers  and markers of inflammation . In addition, readouts of oxidative stress, another cellular consequence of CCl4 administration associated with increased liver fibrosis, showed that t-TUCB decreased CCl4-induced malondialdehyde formation and rescued the CCl4-mediated loss of superoxide dismutase and glutathione.One such report by Harris et al. demonstrated the ability of sEH inhibition to improve carbon tetrachloride . In addition to improving hemodynamics and endothelial function, t-TUCB also ameliorated the underlying fibrosis/HSC activation and inflammation, as indicated by decreased αSMA and NFκB protein levels, respectively. Therefore, this study suggests the ability of sEH inhibition to protect against PTH both by directly improving endothelial function, and thereby hemodynamics, but also indirectly by improving the underlying fibrosis and inflammation.A study by Deng et al. aimed to determine whether sEH inhibition could also ameliorate PTH in rats . PTH is ith IHVR . t-TUCB Ephx2−/− mice [One immunological manifestation of severe liver disease is sepsis—a condition characterized by dysregulated response to inflammation following invasion of gut bacteria and associated bacterial products . Like PT−/− mice . Despite−/− mice ,42,43, A−/− mice .A study by Chen et al. provides further evidence that sEHI-mediated protection against sepsis is model dependent by showing the beneficial effects of TPPU in a surgical cecal ligation and puncture (CLP) model . This poCyp2j2 improves NAFLD endpoints like steatosis, inflammation, and oxidative stress [sEH may be responsible for the metabolism of some toxic xenobiotics, but the evidence reviewed here suggests a pathogenic role for sEH in liver diseases . Indeed,e stress .Il6 and Mcp1) in RAW264.7 cells in a dose-responsive manner [Il6 and Mcp1 expression. These results suggest an immunomodulatory role for sEH in either preventing or permitting macrophage activation, depending on the epFA or dihydroxyFA considered.In vitro studies suggest a link between individual epFAs and mechanisms underlying liver pathology such as lipid/cholesterol accumulation, insulin signaling, ER stress, autophagy, and inflammation. With respect to lipids and cholesterol, Mangels et al. showed a beneficial role for LA-derived 12,13-EpOME in cholesterol homeostasis in HepG2 cells . In thise manner . InteresMechanistically, epFAs may act via several receptors, including peroxisome proliferator-activated receptors alpha and gamma  ,81. ThesAnother important point to consider is that there may be differences in the biological activities of epFAs based on the parent PUFAs. Data reviewed here suggest that omega-3-derived epFAs may have greater efficacy than omega-6-derived epFAs in blocking cellular characteristics of liver pathology. This idea is analogous to the more established concept that omega-3 PUFAs themselves are beneficial in liver disease, whereas omega-6 PUFAs are more pathogenic . SpecifiEphx2−/− animals.While the initial discovery of epoxide hydrolases occurred approximately fifty years ago, the pathogenic role of sEH in liver diseases has been defined only recently. Clearly, sEH inhibition is an attractive therapeutic option in a spectrum of liver diseases, but future research in some key areas is needed to further elucidate its applications, limitations, and molecular mechanisms. One area that should be addressed is sex-specific responses to sEH inhibition in liver diseases. None of the studies reviewed here included female animals, despite the well-known sexual dimorphism of some liver diseases such as NAFLD in both humans and rodents . ImportaEPHX2 gene associated with increases or decreases in sEH activity [The ultimate goal of sEHI research in preclinical animal models is translation to human diseases. It is important to mention that humans have various polymorphisms in the activity ,97. Specactivity ,99,100, sEH inhibition is a promising therapeutic strategy for the treatment of numerous liver diseases, including NAFLD. sEHIs show an efficacy in treating liver diseases that is typically not specific to one inhibitor nor one disease model. In vitro studies similarly show impressive efficacy of individual epFAs whose degradation is prevented by sEHIs in vivo, drawing a clear connection between sEH inhibition, epFAs, and attenuation of liver pathology . Continu


Staphylococcus aureus (MRSA) across the human, animal and environmental compartments in East Africa.The emergence and spread of antimicrobial resistance (AMR) present a challenge to disease control in East Africa. Resistance to beta-lactams, which are by far the most used antibiotics worldwide and include the penicillins, cephalosporins, monobactams and carbapenems, is reducing options for effective control of both Gram-positive and Gram-negative bacteria. The World Health Organization, Food and Agricultural Organization and the World Organization for Animal Health have all advocated surveillance of AMR using an integrated One Health approach. Regional consortia also have strengthened collaboration to address the AMR problem through surveillance, training and research in a holistic and multisectoral approach. This review paper contains collective information on risk factors for transmission, clinical relevance and diversity of resistance genes relating to extended-spectrum beta-lactamase-producing (ESBL) and carbapenemase-producing Enterobacteriaceae, and Methicillin-resistant blaCTX-Ms (45.7%), SCCmec type III (27.3%) and IMP types (23.8%), respectively.The review of the AMR literature (years 2001 to 2019) was performed using search engines such as PubMed, Scopus, Science Direct, Google and Web of Science. The search terms included ‘antimicrobial resistance and human-animal-environment’, ‘antimicrobial resistance, risk factors, genetic diversity, and human-animal-environment’ combined with respective countries of East Africa. In general, the risk factors identified were associated with the transmission of AMR. The marked genetic diversity due to multiple sequence types among drug-resistant bacteria and their replicon plasmid types sourced from the animal, human and environment were reported. The main ESBL, MRSA and carbapenem related genes/plasmids were the The high diversity of the AMR genes suggests there may be multiple sources of resistance bacteria, or the possible exchange of strains or a flow of genes amongst different strains due to transfer by mobile genetic elements. Therefore, there should be harmonized One Health guidelines for the use of antibiotics, as well as regulations governing their importation and sale. Moreover, the trend of ESBLs, MRSA and carbapenem resistant (CAR) carriage rates is dynamic and are on rise over time period, posing a public health concern in East Africa. Collaborative surveillance of AMR in partnership with regional and external institutions using an integrated One Health approach is required for expert knowledge and technology transfer to facilitate information sharing for informed decision-making. Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL)-producers. For instance, MDR invasive non-typhoidal Salmonella (iNTS) disease poses a major challenge to the clinical management of infections in resource-limited settings especially as alternative more effective antibiotics remain either unaffordable or simply unavailable for majority of patients , ESBL and MRSA , ESBL (13.1%), and MRSA (24.25%) were reported in studies conducted from 2013 to 2016, 2009–2012, and 2013–2016 respectively. The rise of ESBL, CAR, and MRSA carriage rates in East Africa pose a threat to public health particularly in the geographical areas where the rates are very high. A limitation of this analysis is the fact that these data were collected from unlinked cross-sectional studies conducted in different geographical areas in East Africa; consequently, some important information may have been missed.Figure The genetic diversity at antibiotic resistance loci could determine a population’s capacity to cope with future challenges of the antimicrobial drugs used for infection control . The genThe high MRSA carriage rates among patients and health workers in the East Africa is a concern given poor infection prevention and control measures in our settings . In KenyE. coli in humans, animals and their environments are conducted by postgraduate students of the SACIDS Foundation for One Health at the Sokoine University of Agriculture and Muhimbili University of Health and Allied Sciences in Tanzania. This work is attempting to understand the transmission dynamics of AMR genes and their virulence [The World Health Organization, the Food and Agriculture Organization of the United Nations and the World Organization for Animal Health has advocated for a holistic and multisectoral approach to address the AMR problem. This has been strengthened by the collaboration of regional consortia involving local and regional academic and research institutions in Africa, and a joint dedication of efforts to fight against infectious diseases in animals, humans and their environments. Regional consortia such as the SACIDS Foundation for One Health in collaboration with the London School of Hygiene and Tropical Medicine, London International Development Centre, Royal Veterinary College, Chatham House of the Royal Institute of International Affairs in the United Kingdom and the American Society of Microbiology of the United States, have partnered to address AMR problem through research and training. Strategically, surveillance is being supported by phenotypic and genomic data, to understand the flow of resistomes across human, animal and environmental compartments. For instance, research programs focused on genomic epidemiology of ESBLs producing irulence , and wilThe implementation of effective and sustainable AMR surveillance programmes in Africa is hampered by a lack of infrastructure and other resources required to perform optimal surveillance . Most laThe high genotypic diversity of the AMR genes among the bacterial isolates might suggest possible exchange of strains or a flow of genes among different strains due to transfer by mobile genetic elements or multiple sources of resistance bacteria. Such levels of diversity prompt the calling for immediate interventions, including guidelines concerning antibiotics use and regulations governing their importation and sale. However, antibiotic use and regulation is likely to be a very complex system because other factors such as cultural and ecological conditions favour transmission of bacteria. There is a high likelihood that people will have AMR bacteria irrespective of how antibiotics are used. Therefore, control strategies against AMR need to be tailored, beyond antibiotic use and availability, to local practices that affect bacterial transmission. Moreover, the trend of ESBL, MRSA and CAR carriage rates is dynamic and are on rise over time period, posing a public health concern in East Africa. In addition to that, phenotypic and genotypic drivers of AMR should be investigated within an integrated One Health framework. Such investigations will inform transmission dynamics of resistomes across compartments, and facilitate information sharing for informed decision making; ultimately, reducing the spread of AMR genes in bacteria. Further, the application of advanced techniques such as whole genome sequencing for detection of AMR could cut a bridge between clinical research and care, so that case management and treatment decisions can be informed and personalised.


Mycobacterium bovis. During early-stage infection, M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected via nested-polymerase chain reaction (PCR) experiments. Little research has focused on immune responses in nested PCR-positive (bTB PCR-P) or nested PCR-negative (bTB PCR-N) M. bovis-infected cattle. Here, we investigated the transcriptomes of peripheral blood mononuclear cells (PBMCs), with or without stimulation by purified protein derivative of bovine tuberculin (PPD-B), among bTB PCR-P, bTB PCR-N, and healthy cattle using RNA-Seq. We also explored the potential value of PBMC transcripts as novel biomarkers for diagnosing bTB. Numerous differentially expressed genes were identified following pair-wise comparison of different groups, with or without PPD-B stimulation (adjusted p < 0.05). Compared with healthy cattle, bTB PCR-P, and bTB PCR-N cattle shared 5 significantly dysregulated biological pathways, including Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, Hematopoietic cell lineage, Osteoclast differentiation and HTLV-I infection. Notably, dysregulated biological pathways of bTB PCR-P and bTB PCR-N cattle were associated with cell death and phagocytosis, respectively. Lymphotoxin alpha and interleukin-8 could potentially differentiate M. bovis-infected and healthy cattle upon stimulation with PPD-B, with area-under-the-curve (AUC) values of 0.9991 and 0.9343, respectively. B cell lymphoma 2 and chitinase 3-like 1 might enable differentiation between bTB PCR-P and bTB PCR-N upon stimulation with PPD-B, with AUC values of 0.9100 and 0.8893, respectively. Thus, the PBMC transcriptome revealed the immune responses in M. bovis-infected cattle (bTB PCR-P and bTB PCR-N) and may provide a novel sight in bTB diagnosis.Bovine tuberculosis (bTB) is a chronic disease of cattle caused by  Mycobacterium bovis infection  is a serious disease in cattle populations that is primarily caused by nfection , 2. M. bd humans , 4 and id humans . Despited humans .M. bovis has escaped eradication   alone or in combination with interferon gamma (IFN-γ)-release assay (IGRA). Cattle with positive TST and/or IGRA test results are slaughtered. However, due to the limitations of these tests and the significant economic burden produced by this eradication strategy, dication . In humadication , tubercuacterium , mainly acterium . Previouand milk . Prior r (PCR-P) , 12. HowBlood transcriptomic profiling has been widely used to examine host-immune responses to infection based on an unbiased analysis and comprehensive overview . RecentlM. bovis-infected cattle and evaluated the diagnostic value of PBMC transcripts. PBMCs isolated from cattle with different infection statuses  were treated with bovine purified protein derivative of bovine tuberculin (PPD-B) or phosphate-buffered saline (PBS) for 6 h. By RNA-Seq, we compared the transcriptomes of PPD-B-stimulated and -unstimulated (PBS-treated) PBMCs between the three groups. Finally, several potential biomarkers were validated for differentiating bTB PCR-P and bTB PCR-N or infected cattle and healthy cattle, as outlined in Here, we investigated peripheral blood mononuclear cell (PBMC) transcriptomes in naturally M. bovis infection (n = 243) and that were negative for M. bovis (n = 125). bTB was detected by TST and IGRA testing, as we described previously  of the mpb70 gene with the primers M70F, 5′-GAACAATCCGGAGTTGACAA-3′, and M70R, 5′-AGCACGCTGTCAATCATGTA-3′). Then, a nested PCR was run using 1 μl of the previous reaction mixture to amplify a 208-bp fragment within the 372-bp region of the mpb70 gene with the primers M22F, 5′-GCTGACGGCTGCACTGTCGGGC-3′, and M22R, 5′-CGTTGGCCGGGCTGGTTTGGCC-3′  were chosen from herds with a recent history of eviously . Nasal sovis n = 5. bTB wa2, harvested for treatment 6 h post-stimulation. Harvested cells were washed by ice-cold PBS for three times and then stored at −80°C in TRIzol reagent  until further use.About 50 ml of whole blood with anticoagulant (heparin) was collected aseptically from each animal. PBMCs were isolated using bovine lymphocyte-separation medium  by density-gradient centrifugation, per the manufacturer's instructions. Red blood cells (RBC) were removed by RBC lysis, the cells were washed by PBS for three times. And then cells from each animal were counted and seeded into 12-well tissue culture plates at 5 × 106 cells/ml OpTmizer™ T-Cell Expansion SFM (Gibico) containing 1% L-Glutamine (Gibco) and 1% penicillin-streptomycin . Subsequently, the PBMCs were stimulated with 300 IU PPD-B ; unstimulated PBMCs (PBS-treated) from the same cattle were run as a negative control. Subsequently, PBMCs were incubated at 37°C with 5% COTotal RNA was extracted using the TRIzol reagent (Invitrogen), following the manufacturer's instructions. The time frame from blood collection to RNA extraction was within 24 h. The quantity and quality of RNA samples were checked using a NanoDrop 2000 spectrophotometer  and an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip Kit . All total RNA samples had an RNA integrity number >7.0, an A260/280 ratio >1.8, and an RNA quantity > 3.0 μg.M. bovis-infected cattle that were nested PCR positive, 3 M. bovis-infected cattle that were nested PCR negative and 3 healthy cattle). According to the manufacturer's protocol, the libraries for Illumina sequencing were made with the TruSeq Stranded mRNA sample prep kit  and sequenced using an Illumina HiSeq 2000 system.A total 18 RNA-Seq labraries were prepared which comprised PPD-B stimulated and unstimulated across 9 cattle in different infection statuses of bTB  using TopHat2 software (http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes database (KEGG) (http://www.genome.jp/Kegg/).For the sake of obtaining high-quality clean reads, the adapter sequence and low-quality sequences were trimmed. All clean reads were mapped to the software , 18. Thesoftware , and thesoftware . For theCt−ΔΔ method  primers . Those total RNA samples were the same samples used in RNA-Seq library preparation in section RNA-Seq Library Preparation and Sequencing. For qPCR, triplicate 20 μL reactions containing 10 μL 2 × QuantiNova SYBR Green PCR Master Mix (Qiagen), different pairs of 0.7 μM primers , and 1 μt method . All priM. bovis-infection cattle (including 17 bTB PCR-P and 17 bTB PCR-N cattle) and 17 healthy cattle. The RNAs were extracted to measure gene-expression levels using the 2CT−ΔΔ method by qRT-PCR, with unstimulated PBMCs serving as a calibrator, and β-actin serving as the reference gene . M. bovis-infected bTB PCR-P and 20 bTB PCR-N cattle , as well as 20 age- and sex-matched healthy cattle (HC) for this study. Three cattle in each group were used for RNA-Seq, and the remaining 17 cattle were studied in biomarker-verification experiments.Screening results indicated that, of the 368 cattle, 138 cattle were positive for TST, 106 cattle were positive for IGRA, and 32 cattle were positive for nested PCR. Based on the TST, IGRA, nested-PCR and the serological diagnosis results , we selected 20 naturally q < 0.05)  were identified based on our screening criteria  as a stimulus to analyze the adaptive immune responses of M. bovis-infected cattle with different infection statuses. Interestingly, in the PPD-B-stimulated groups, a relatively large number of DEGs was observed. Compared to the HC cattle, we identified 1585 DEGs (776 upregulated and 809 downregulated) and 217 DEGs (205 upregulated and 12 downregulated) in bTB PCR-P and bTB PCR-N cattle, respectively. We also identified 488 DEGs (199 upregulated and 289 downregulated) between bTB PCR-P and bTB PCR-N cattle  and 318 DEGs (249 upregulated and 69 downregulated) in bTB PCR-P and bTB PCR-N cattle, respectively. A total of 188 DEGs (67 upregulated and 121 downregulated) were identified between bTB PCR-P and bTB PCR-N cattle . Even thN cattle .Overall, our current results revealed numerous DEGs in the pair-wise comparisons of different groups with and without stimulated. Volcano plots were also drawn . With thIFIT1) gene was not detected in unstimulated PBMCs from bTB PCR-N cattle by qRT-PCR because of the low expression level. Our results showed that the expression of most (13/14) of the selected genes was consistent with the log2 (fold-change) values determined by RNA-Seq and qRT-PCR -stimulated PBMCs.To gain specific biological insights into different bTB infection statuses, we analyzed the DEGs using the GO and KEGG databases. To increase the rigor of our analysis, we focused solely on The significantly enriched GO categories  are summarized in Given the broad and variable differences in PBMC mRNA-expression levels between healthy and bTB cattle for the PCR-positive and -negative pairs, KEGG analysis was performed to identify dysregulated biological pathways by pooling DEGs into host-gene pathways. The 20 most significantly affected pathways of the three pair-wise comparisons revealed changes in the different biological states (PCR-positive and -negative) of bTB . For theFurthermore, Venn diagram analysis revealed 16 DEGs shared between the bTB PCR-P, bTB PCR-N, and HC groups . HeatmapM. bovis infection and distinguishing the stage of M. bovis infection. Based on the fold-changes, the bioinformatics and Venn diagram analyses of the DEGs, and combined with our previous proteomic results  obtained by RNA-Seq were plotted in The PBMC transcriptomic profiles observed in this study provided an unbiased, comprehensive overview of host factors that are dysregulated in the progression of bTB infection. Given this phenomenon, we explored the diagnostic value of host PBMC genes in detecting  results , 9 PBMC  results . The expLTA, IL-8, BCL2, and CHI3L1 genes serve as valuable, novel biomarkers for bTB diagnosis. The mRNA-expression levels of LTA and IL-8 induced by PPD-B were significantly higher in bTB cattle (including bTB PCR-P and bTB PCR-N) than in HC cattle  induced by PPD-B was significantly higher in bTB cattle than in HC cattle (IFNG was 1.0000 (BCL2L1 (AUC = 0.7024) and TNF-α (AUC = 0.6851) showed poor diagnostic performance , indicating that it may not be bTB-specific. CRP mRNA expression was not high enough to be detected which means it is unable to meet the essential requirements as biomarker.Protein IFN-γ has been used commercially as a valuable biomarker for diagnosing bovine tuberculosis. In this study, the mRNA-expression level of C cattle , and thes 1.0000 . This reformance , althougformance . The TLRIFNG) and inappropriate transcripts , we validated 4 novel potentially reliable biomarkers for bTB diagnosis. PPD-B-stimulated LTA and IL-8 could potentially differentiate M. bovis-infected cattle from HC, and PPD-B-stimulated BCL2 and CHI3L1 have the potential power in differentiating bTB PCR-P cattle from bTB PCR-N cattle.Collectively, after excluding a known biomarker  determined to be nested-PCR-positive or -negative. We identified 188 and 488 DEGs in unstimulated and PPD-B-stimulated PBMCs between bTB PCR-P and bTB PCR-N cattle, respectively (TP53INP1 (tumor protein p53 inducible nuclear protein 1) and TPRKB (TP53RK binding protein) were significantly downregulated which downregulated p53-mediated apoptosis that suppressed cell death . KEGG analysis identified dysregulated biological pathways in bTB PCR-P and bTB PCR-N cattle associated with cell death and phagocytosis, respectively. Previous findings indicated that M. tuberculosis inhibits host cell apoptosis as one of its pathogenic mechanisms (BCL2 and BCL2L1) increased significantly and the level of expression of apoptotic factors  decreased significantly, we hypothesized that, like M. tuberculosis, M. bovis inhibits apoptosis. Phagocytosis plays an important role in the immune response against tuberculosis. Interestingly, we found the DEGs associated with phagocytosis were significantly enriched in bTB PCR-N cattle and were associated with biological processes (phagocytosis and engulfment) and pathways (phagosomes and lysosomes). Although phagocytosis efficiently kills mycobacteria, mycobacteria also inhibit phagosome–lysosomes fusion for intracellular survival .TX, LJ, and JD designed the experiments. LF, WL, HJ, XGa, XS, YJ, SH, LZ, and XGu performed the experiments. TX, LJ, LF, HZ, and WL analyzed the data. LF, TX, and LJ wrote and revised the paper.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.


Mycobacterium tuberculosis (Mtb) infection causes tuberculosis (TB), a disease characterized by development of granulomas. Granulomas consist of activated immune cells that cluster together to limit bacterial growth and restrict dissemination. Control of the TB epidemic has been limited by lengthy drug regimens, antibiotic resistance, and lack of a robustly efficacious vaccine. Fibrosis commonly occurs during treatment and is associated with both positive and negative disease outcomes in TB but little is known about the processes that initiate fibrosis in granulomas. Human and nonhuman primate granulomas undergoing fibrosis can have spindle-shaped macrophages with fibroblast-like morphologies suggesting a relationship between macrophages, fibroblasts, and granuloma fibrosis. This relationship has been difficult to investigate because of the limited availability of human pathology samples, the time scale involved in human TB, and overlap between fibroblast and myeloid cell markers in tissues. To better understand the origins of fibrosis in TB, we used a computational model of TB granuloma biology to identify factors that drive fibrosis over the course of local disease progression. We validated the model with granulomas from nonhuman primates to delineate myeloid cells and lung-resident fibroblasts. Our results suggest that peripheral granuloma fibrosis, which is commonly observed, can arise through macrophage-to-myofibroblast transformation (MMT). Further, we hypothesize that MMT is induced in M1 macrophages through a sequential combination of inflammatory and anti-inflammatory signaling in granuloma macrophages. We predict that MMT may be a mechanism underlying granuloma-associated fibrosis and warrants further investigation into myeloid cells as drivers of fibrotic disease. Mycobacterium tuberculosis (Mtb), a bacterium that infects over a third of the world’s population. The only available vaccine for TB has limited efficacy and drug treatment involves several antibiotics that are taken for several months. These drugs can have significant side-effects and a lack of compliance can lead to drug resistance in Mtb. A hallmark of Mtb infection is the development of clusters of cells that form around infected macrophages to contain the infection called granulomas. Older granulomas, or granulomas in patients treated with antibiotic, often become fibrotic and this can cause chronic lung problems long after the Mtb infection has cleared. The process that drives a fibrotic outcome has been difficult to assess in vivo. Herein we combined wet-lab and computational experimentation to identify a novel mechanism leading to peripheral fibrosis in granulomas. We find that macrophages transforming into myofibroblast-like cells, may be a key pathway to granuloma-associated fibrosis, a phenomena that has not been well characterized in vivo. Further we identified factors that can inhibit fibrosis development during TB that could be therapeutic targets during TB treatment to limit the risk of long-term tissue damage in TB.Tuberculosis is a disease caused by  Mycobacterium tuberculosis (Mtb). Control of the global TB epidemic has been limited by many factors including a complicated drug regimen, development of antibiotic resistance, and the absence of a fully protective vaccine against infection and disease [Mtb infection and these lung lesions are composed of immune cells that exist in organized microenvironments around mycobacteria-infected cells. Granulomas serve as staging areas for immune responses that kill bacteria and limit their dissemination, but because they are also rich in potential Mtb host cells, they are niches for bacterial persistence and replication [Mtb infection [Tuberculosis (TB) is a deadly infectious disease caused by inhalation of the bacterium  disease ,2. Granulication –5. Fibronfection ,6,7. Fibnfection . Fibrosinfection . Moreovenfection ; thus, iMtb infection and granuloma development have been difficult to identify in the context of TB. This problem is compounded by the biology of TB in humans where granulomas form in lungs, which cannot be easily sampled, and on a more basic level, there are challenges associated with a lack of consensus regarding how to identify and characterize human fibroblasts in tissues [The mechanisms that promote fibrosis during  tissues ,11. In s tissues ,13, the  tissues ,14,15. I tissues ; however tissues ,18. Circ tissues ,20. FurtMtb infection can be tightly associated with granulomas, and it is also common for fibrosis to be present only on the periphery of granulomas [Previous work from our group explored the role of fibroblast to myofibroblast differentiation in granuloma fibrosis . These ranulomas . There ianulomas . HoweverGranSim, our next-generation computational model, to explore a possible role for MMT in fibrosis at the lesion level, and then classified cells in the fibrotic cuff of fibrotic granulomas from non-human primates (NHPs) to identify their origin. The computational model [Mtb burden. Our results suggest that MMT could occur in granulomas and raise the possibility that this process could be targeted therapeutically to improve resolution of TB and the efficacy of drug treatments.To generate our hypotheses we used al model , which wal model  and macral model  allow usNon-human primate studies: Formalin-fixed paraffin-embedded (FFPE) tissue sections were cut from samples obtained from macaques as previously described [α-smooth muscle actin (αSMA) , CD11c , and CD31 . Coverslips were applied using Prolong Gold mounting medium containing DAPI (ThermoFisher Scientific) and slides were imaged using a Nikon e1000 immunofluorescent microscope  with a scanning stage. Images were saved as TIFF-format images, and processed with the Fiji build of ImageJ and the Template Matching plugin to align samples [The Institutional Animal Care and Use Committee of University of Pittsburgh approved all experiments. The samples came from animals enrolled in completed studies. While in-study, animals were housed and maintained in accordance with standards established in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. The University of Pittsburgh is an AAALAS certified program. escribed ,7. Antigescribed ,22. Tiss samples ,26.αSMA expression and morphology. We then used an iso-clustered, unsupervised classification analysis from toolbox of ArcPro2.4 to the images with classification parameters to maximize the spectral and spatial detail in the images. The classes that resulted were collapsed into two categories. First, we labelled those cells that by linear dimensions  and staining for αSMA represented fibroblastic structures. Second, all other cell classes were grouped into a separate category called ‘other’. This produced a layer in GIS that we collapsed only to the category of interest  that was converted to vector data (polygons). Because structures other than those of interest in the granuloma images also have similar spectral characteristics as fibroblasts (i.e. same color and brightness), we then filtered the images using two additional filters: 1) polygon geometry and 2) length and area. These known fibroblast polygon parameters from control data spatially filtered out non-fibroblast like polygons. This process distinguished fibroblast from non-fibroblast-like structures and their locations within the images. Finally, we used a spatial-selection polygon to further filter-out non-fibroblast polygons, which refined the identification of fibroblasts. From these methods, we identified the size and shape, number and location of fibroblasts in the granuloma images.Images of stained granulomas were imported into ArcPro 2.4 , a Geographical Information Systems (GIS) software as we have done previously . These iThe process of identifying fibroblasts and other cell classes above helped with the identification of granuloma borders. We then used an unsupervised iso-clustering approach from ArcPro 2.4’s toolbox to digitally classify granuloma images into feature classes based on color and brightness of each pixel. This technique resulted in eleven unique classes, representing different cell-types, cellular debris and image background . We further combined these eleven feature classes into three feature classes that revealed differences between tissue preparation background, granuloma, and extra-granuloma locations. This combination of three feature classes revealed a narrow (20-100micron), border between granuloma and extra-granuloma cells and structures. We further enhanced granuloma border location by constructing a minimum-bounding polygon around the fibroblast collar.Noise arises when classifying an image using GIS, particularly as IHC images are imperfect 2D representations of 3D object. The slices capture parts and pieces of cells in the Z-plane, and thus deriving exact dimensions for all cells is not possible. This is referred to as edge effect. As the cell’s length is long, 1–2 um differences are negligible with large samples sizes. However, width on such a narrow object is greatly affected by edge effect. Therefore, the width is derived from a calculation but the length is exact. As Area = Length x Width, so Width = area /length. It is standard to multiply the quotient by 2 to capture the 3D edge effect.GranSim. This model has been curated and used for testing hypothesis in TB since 2004. The model has been developed in conjuction with an experimental collaboration with the laboratory of Dr. JoAnne Flynn at the University of Pittsburgh and has been validated against large and numerous datasets regarding the immune response to M. tuberculosis within lungs of non-human primates (NHP) leading to the formation of granulomas [. Briefly, GranSim tracks the immune response in lungs following infection with Mtb that ultimately results in either emergence of a granuloma . GranSim is an agent-based  model (ABM) that is comprised of 5 features: Agents-are individually tracked immune cells as follows: four macrophage states , three T cell classes , fibroblasts and myofibroblasts (see below for more details), as well as cytokines IFN-γ, TGFβ, IL10, TNFα, chemokines CCL2, CXCL9 and CCL5 and individual bacteria in 3 states  [The Environment: Or environment represents a section of lung tissue that is 4 mm x 44 mm in size, which allows for simulated granulomas to grow as large as typical in vivo granulomas. The grid is 2D  and the lung grid is partitioned into 20 micron x 20 micron microcompartments, as that is the typical size of our immune largest immune cells under study, macrophages and fibroblasts. The lung grid also is populated with blood vessel ports that were placed based on studies of healthy NHP lungs where cells, chemokines, or cytokines can enter into the lung space. Rules: Rules are the probablistic interactions between cells and each other as well as the environment. These are based and validated on hundreds of observed interactions of primate immune cells and molecules in vivo and both calibrated and validated by extensive NHP datasets;. Parameters: The model is parameterized by hundreds of parameters that are estimated from data and further studied using uncertainty and sensitivity analyses; Finally, ABMs are defined by the time step of the fastest process which in our case is diffusion of molecules, that runs on a 6-second time scale. The model when simulated, processes rules and agents at the cell and molecular scale and leads to emergent behavior of a single granuloma at the tissue scale.In this work we build the macrophage to myofibroblast transition model into our existing hybrid multi-scale agent-based model (ABM) of granuloma formation, anulomas ,23,28 (s caseum) ; The EnvGranSim architecture fibroblasts and myofibroblasts as well as TGF-β, a major signaling molecule involved in MMT. We also have studied M1 to M2 macrophages with our model using a sub-model that tracks NFkb and STAT1 signalling within macrophages [Rules) in their environment. Resting macrophages become activated if they are exposed to IFN-γ, which also programs the macrophage agent to register that it has been STAT1 stimulated, or TNF-α. Resting and Activated macrophages become infected if they take up Mtb agents, and Infected macrophages become Chronically Infected if they contain more than a threshold number of bacteria . If a macrophage is exposed to IL-10, it downregulates some phagocytic capabilities and becomes STAT3 stimulated. The level of gene stimulation is a macrophages property that allows the model to track each agent’s history; however, it is the real-time environmental conditions that effect the state of a macrophage at each time step. Warsinske et al. presented the addition of fibroblasts to GranSim by the inclusion of the following rules: Fibroblasts can divide if they bind a sufficient amount of TGF-β, have not bound more than a maximum amount of IL10, and are within close enough proximity to the granuloma. Fibroblasts can differentiate into myofibroblasts if they have not proliferated in the current time step, have bound a sufficient amount of TGF-β1, have not bound more than a maximum amount of IL10, and are present in a sufficient concentration of TNFα to indicate close proximity to the granuloma. Fibroblasts are motile cells but myofibroblasts remain stationary. To facilitate macrophage to myofibroblast transformation (MMT) in GranSim we created a function that forces resting macrophages to irreversibly switch phenotypes and become myofibroblasts immediately if they receive both STAT1 and STAT3 signals through the appropriate pathways while they remain negative for Mtb exposure , and the Qt framework for visualization . The ABM is cross-platform  and runs with or without visualization software. GranSim model simulations were performed on the XSEDE Comet supercomputer (see Acknowledgment).GranSim simulation at five-day intervals and used the radius of the granuloma to determine if a macrophage agent was located in a central or peripheral location within a granuloma. To determine the radius, the granuloma boundary was determined using our previously published algorithm [generalized linear model (GLM) from the stats package in R [We recorded the location and status of every macrophage in the lgorithm . Macrophage in R  with a bSpartan package in R [p-values are less than 0.05, calculated by a Z-test. A positive PRCC indicates that the parameter is associated with an increase in numbers of fibroblasts while negative PRCCs are parameters are associated with a decrease in the number of fibroblasts. The use of LHS and PRCCs in sensitivity analysis is reviewed in [We used global sensitivity analysis to identify the influential parameters driving MMT in the simulation. Latin Hypercube Sampling (LHS)  was usedage in R . We clasiewed in ,36.Previous studies from our group demonstrated that including only fibroblasts as a driver of fibrosis results in collagenization that spreads into the lung environment and cannot adequately reproduce the tightly maintained spatial structure of peripheral granuloma-associated fibrosis . Given tin vivo [To explore the potential drivers of MMT in fibrosis we recorded the position and status of all the macrophages in our computational granuloma, and classified them as being either peripheral, central, or outside of the granuloma according to their coordinates on the grid, thereby aligning them with types of fibrosis previously identified in vivo . Our groin vivo . Using tgeneralized linear model (GLM) to identify the macrophage properties (listed in Mtb (p < 0.05). We used Latin Hypercube Sampling (LHS) to vary model parameters driving STAT1, STAT3, NFκB, as well as other general granuloma parameters, and performed simulations under each parameter set . We used the results from these LHS simulations to identify locations where the MMT-induced fibrosis could occur under criteria of joint STAT1 and STAT3 stimulation as well as non-exposure to Mtb in different settings. We then highlighted STAT1/STAT3 simulated macrophages that were not exposed to Mtb in images of simulated granulomas to determine where MMT could occur in different granulomas. This analysis revealed that STAT1/STAT3+ Mtb- macrophages exhibit spatial distributions analogous with central-, peripheral-, full, and no fibrosis as observed in published imaging studies  signaling must come first and the average time between the initiation of STAT1 signaling and initiation of STAT3 signaling is 7.23 days. We did observe instances where macrophages in the granuloma periphery underwent STAT3 signaling before STAT1 (as indicated by the negative error bars in Previous research has suggested that macrophages that are able to transition to become fibrotic through MMT are M1 polarized macrophages –41 but oMtb macrophages that transition to myofibroblasts) has the potential to alter the observed patterns of fibroblasts arising from MMT from the macrophage locations predicted  in the granuloma reduce the probability of a granuloma becoming fibrotic. Similarly, increasing the amount of TGFβ stimulation required to downregulate a T cell or macrophage also decreases the number of fibroblasts in the simulation. Taken in combination, the sensitivity analysis and PRCCs suggest that fibrosis is a marker of healing in the TB granuloma, and that inflammation inhibits the development of fibrosis. This is in agreement with what is commonly accepted in other disease that fibrosis arises as a dysregulated healing response [A key strength of modeling is the ability to predict what mechanisms are driving model outcomes. Here, we are interested in identifying what model factors lead to increased numbers of myofibroblasts. To this end, we performed a sensitivity analysis using the LHS/PRCC approach as we have done previously . Our resresponse –45.Mtb infection and identified key features in relation to what we had observed computationally. The granulomas we selected were scored by a veterinary pathologist as we have done previously [To validate our predictions that cells undergoing MMT were M1 macrophages we studied properties of cells in the fibrotic cuff of lung granulomas from NHPs that were necropsied 10–12 weeks post eviously  and we sαSMA), a marker for myofibroblasts [αSMA + cells on the periphery of the fibrotic granulomas that is conserved between samples  .ex-vivo when peripheral blood monocyte-derived macrophages are polarized to become M1 macrophages and given fluid from healing but not chronic wounds [Mtb infected lung. Through complementary IHC staining of fibrotic granulomas from NHPs infected with TB 10–12 weeks after infection, we identified a population of CD11c+αSMA+ cells in the fibrotic region of granulomas suggesting the involvement of cells that are derived from sources other than lung resident fibroblasts.A better understanding of the processes that regulate development of fibrosis in TB has important implications for treatment and disease resolution. Fibrosis has been difficult to investigate in TB however because there is not a small animal model that recapitulates end-stage disease and human granulomas are not readily accessible for study. NHP granulomas replicate human TB, including the fibrotic outcomes associated with drug treatment and can serve as an alternative system where the basic immune mechanisms can be investigated. To address this problem, we characterized the phenotype of fibrotic NHP granulomas and used a systems biology approach to identify the basic biology of fibrosis in TB. Our work suggests that MMT may lead to peripheral fibrosis in granulomas, and although not well understood, MMT may underlie aspects of TB that have been difficult to explain up to this point. MMT has been demonstrated to occur c wounds . To dateGranSim can be inhibited by exposure to Mtb and by parameters that drive inflammation e.g. the recruitment rate of IFNγ-producing T cells and the rate at which TGFβ downregulates inflammatory cytokine production. It is important to note that our computational model includes only STAT3 as an abstraction for all anti-inflammatory regulators, thus while GranSim predicts both STAT1 and STAT3 are involved in MMT, these pathways are actually representative of any alternative anti-inflammatory pathway with similar dynamics to the STAT3/IL-10 pathway and a combination of these pathways could be responsible for driving MMT in vivo, and further experimentation is needed to fully explore this hypothesis. This has been previously reported for pSMAD3/TGF-β signaling and MMT in a murine model of renal fibrosis [in vitro studies identifying M1 cells as sources of MMT in biology [et al (sample shown in Our results suggest that MMT occurs in macrophages that receive inflammatory (M1-polarizing) signals before receiving secondary anti-inflammatory signals. We also show that MMT in fibrosis  and cannfibrosis . It rema biology ,49,50 su biology . The disThe transformation of macrophages into myofibroblasts has important implications for granuloma function, interaction between networks of cells within the granuloma, and granuloma-scale immunity. When protective, this process may occur in a way that gradually increases fibrosis and serves to contain bacterial populations. However, this would occur in combination with persistent, but gradually decreasing, macrophage antimicrobial function. This would be accompanied by increasing levels of stiffness and extracellular matrix deposition and restricted diffusion of chemotactic ligands out of Mtb-rich regions coupled with limited migration of new immune cells into these areas. This approach may be preferable to a rapid influx of conventional fibroblasts that rapidly attenuate macrophage antimicrobial activity or produces a difficult-to-resolve scar that leads to permanent loss of lung function. Alternatively, inappropriately timed or positioned macrophage to fibroblast transition could limit antibacterial activity by restricting T cell access to Mtb-infected macrophages and decrease bacterial exposure to macrophage-expressed antimicrobial factors. This could lead to liquefaction of the granuloma and potential dissemination of bacteria.in vivo investigation for the role of myeloid cells in fibrotic lung disease, including idiopathic pulmonary fibrosis. Reducing granuloma associated fibrosis could improve lung function and shorten TB treatment by improving the ability of antimycobacterial drugs to access difficult-to-target microenvironments where mycobacteria exist. Alternatively, there is the possibility that inhibiting fibrosis could increase dissemination of bacteria and hinder their clearance, so more work is needed to understand the trade-off of protective versus pathologic roles that fibrosis plays in TB. Moreover, additional work investigating how collagen is deposited in the granuloma needs to be performed before the impact of reducing fibrosis on disease burden can be predicted. The role of collagen deposition in the granuloma is not well characterized in vivo and further work in this area is required to fully understand the impact of reducing granuloma fibrosis, particularly since the conservation of immune balance is particularly pertinent in TB [We demonstrate a potential role for macrophages in development of granuloma-associated fibrosis and our work provides a basis for further nt in TB .S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.


Furcraea bedinghausii) and Acrylic hydrogels (H) and reinforced acrylic hydrogels with fique nanofibres (HRFN) were synthesized, using the solution polymerization method. The extraction was carried out using a combined extraction method . The raw material (NAT-F), bleached fibers (B-F), hydrolyzed fibers and fibers treated with ultrasound (US-F) were characterized by infrared spectroscopy (FTIR) and thermal stability analysis; also, in order to have a comparison criterion, a commercial microcrystalline cellulose sample (CC) was analyzed, which demonstrated the extraction of fique cellulose. The surface morphology of the NAT-F and the B-F was determined by scanning electron microscopy and the average particle size of the nanofibers was made through transmission electron microscopy. In H y HRFN the strain percent and compression resistance (Rc) were measured. The fique nanofibers showed diameter and length averages of 25.2 ± 6.2 nm and 483.8 ± 283.2 nm respectively. Maximum degradation temperature was 317 °C. HRFN presented higher compression resistance (16.39 ± 4.30 kPa) and this resistance was 2.5 greater than the resistance of H (6.49 ± 2.48 kPa). The results indicate that fique lignocellulosic matrix has potential application for obtaining polymeric type composite materials.Cellulose nanofibers were obtained from the Colombian fique ( Its application is oriented to the manufacture of personal hygiene products, medical3, environmental6 and agricultural applications8. Hydrogels for application in agriculture are usually synthesized based on acrylates or similar monomers , because cross-linked polymers with high absorption capacity and bloating rate are obtained9 and low toxicity11. One of the main limitations of hydrogels for agricultural applications is their low mechanical strength13. The pressure exerted by the plant and the soil layer on the hydrogel influence the loss of swelling capacity, elasticity and stiffness15. To maintain polymer elasticity, long-chain molecules and adequate interbreeding are needed to dissipate external mechanical stresses on the hydrogel16. To improve mechanical properties, manometric-sized reinforcement materials such as: inorganic nanoclafers, carbon nanotubes, nanoparticles of inorganic compounds are usually used17. However, one of the disadvantages of using reinforcement materials is the decrease in water absorption capacity18. As consequence, an alternative is the use of micro-scale cellulose and nano as a reinforcement material in hydrogels19. Several studies show that the addition of cotton nanofibers20, chitosan21, wood22 into acrylic base hydrogels, increases, mechanical strength and swelling capacity due to increased surface area, elastic character of fibers and the presence of hydroxyl groups within the polymer matrix24. The addition of nanofibers in hydrogels increases compression resistance by up to 50%22, and the ability to recover its original shape (elasticity) from the application of external forces, up to 80% compared to its original height14.Hydrogels are polymers with a crisscrossed three-dimensional structure which allows to absorb, store and release water molecules25. Agricultural activity generates a large amount of crop waste, including fique26. The fique agribusiness in Colombia focuses on the production of fibers27, however, the fibers represent only 4% of the leaves, the remaining 96% corresponds to waste generated in the process, such as juice (70%) bagasse or cabuyasa (30%)28. Consequently, fique lignocellulosic residues are an alternative source for nanofiber isolation. Fique fibers are characterized by their high cellulose content (50–74%), specific strength, biodegradability29, low density (1.5 g cm−3 compared to fiberglass 2.5 g cm−3)30, and is considered one of the most tensile resistant (511–635 MPa) compared to other natural fibers31.For the use of cellulose nanofibers as reinforcing agents, cellulose sources that are available, sustainable and inexpensive should be considered, therefore, an alternative is the use of agro-industrial waste25. These pretreatments are necessary because lignin and hemicelulose coat27 cellulose chains and lead to different qualities and morphologies of nanofiber25. To break the amorphous region of the chains and isolate cellulose nanofibers, several methods have been used since 2008 including acid hydrolysis, enzyme processes, oxidation, high-pressure homogenization, high-intensity ultrasound and steam blasting processes32. Recently new approaches to pretreatment have been developed that do not hydrolyze or oxidize the cellulose fibers, which include the use of deep eutectic solvent, NaOH swelling, Imidazole, Carboxymethyl cellulose adsorption, but their detailed mechanisms remain to be investigated33.Chemical pretreatment of cellulosefibers is essential to improving nanofibrillation and decreasing energy consumption34. The use of combined methods decreases cellulose degradation and optimizes morphology and aspect ratio32. To increase the yields in obtaining nanocellulose is required to separate cellulose chains from the crystalline part, one of the methods used for this purpose is the ultrasound technique. The transfer of ultrasonic energy to the cellulose chains generated by the cavitation process, is similar to the energy to break a hydrogen bond (100 kJ/mol), therefore, when applying this technique can disintegrate cellulose fibers from the crystalline part, of micrometric size to nanometric size36.In the isolation of nanofibers the use of concentrated chemicals or mechanical methods for long periods of time degrade cellulose decreasing the degree of polymerization of chainsThe objectives of this paper were to obtain fique nanofibers using chemical methods combined with ultrasound radiation and to evaluate the effect of fique nanofibers as reinforcing material in acrylic hydrogels. Morover, fibers and nanofibers study was made using infrared analysis (FITR), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA); additionally, the strain and resistance to compression of the reinforced hiydrogels are discussed.26 where after the delignification process the lignin content decreased from 23.3 to 2.8% and the cellulose content increased from 52.3 to 83.6%.Cellulose and lignin contents of NAT-F were 45, 42 and 21.34% respectively, and B-F were 91.92% and 4.14% respectively. The fiber bleaching process was efficient as it managed to eliminate more than 80% of the lignin present in NAT-F. These results were similar to the results reported by Ovalle et al.37 lignocellulosic materials without any treatment do not show homogeneity due to the presence of compounds such as lignin, hemicellulose, pectin, among others, which act as binders, preventing defibrillation of cellulose chains. Additionally, roughness in bleached fique fiber is due to nanofibrils which are connected through hydrogen bonds, to form larger units of microfibrils37.SEM micrographs were performed to have the particle size of the different products obtained during the nanofibrilation process from fique. The Fig. Infrared spectrum of the NAT-F is shown in Fig. −1 for the raw material corresponds to hydroxyl groups (–OH) and the band at 2,918 cm−1 corresponds to C–H stretch present in cellulose, hemicellulose and lignin38. The carbonyl group band appears at 1,740 cm−1, and its correspond to bond vibration present in the ester and carboxylic groups (–COOCH3 and –COOH) characteristic of hemicellulose, ferulic and p-coumaric acids, which belongs to lignin39. The band at 1,238 cm−1 corresponds to –C–O–C– bond vibration present in the bonds between aromatic ring and methoxy groups in lignin40. The bands at 1,649, 1,460 and 1,421 cm−1 for raw material, corresponds to the C=C bond stretching present in aromatic rings of lignin41. Finally, at 1,028 cm−1 for raw material refers to C–O–C bond stretching of β-1,4-glycosidic ring bonds among d-glucose units in cellulose37.Infrared spectrum of the NAT-F shows two absorption zones. The absorption band that was obtained from 3346 cm−1) caused by the removal of lignin and hemicellulose due to chemical treatment. It should be mentioned that, in addition, an increasing in peaks intensity around 1,030, 3,332 and 2,900 cm−1, indicate the presence of cellulose and become more pronounced in the H-F and US-F spectra. It is observed, in addition, in B-F spectrum the appearance of peaks in 1,160, 1,315 and 897 cm−1. Additionally, these peaks become more intense due to the effect of acid treatment followed by treatment with ultrasound radiation. The presence of peak at 1,428 cm−1 and an increasing in peaks intensity around 1,367 and 1,315 cm−1 in US-F spectrum is in line with that reported by Avolio et al.42 and Candra et al.37, where is indicated that acute signals around 1,426 and 897 cm−1, 1,370 and 2900 cm−1 reflect the crystalline band of cellulose. In addition, Fan et al. 43 report that sharp peaks around 1,420 and 894 cm−1 indicate the presence of crystalline structures, a widening of these bands reflects a more disordered structure that would correspond to amorphous cellulose.To remove the lignin, hemicelluloses, waxes and other components was applied an alkali treatment (delignification) and to remove completely the lignin the bleaching treatments was performed, obtaining B-F. The FTIR spectra of B-F, H-F, US-F and CC. B-F FTIR spectrum shows the disappearance of several peaks  results Fig.  are summ27 used 5% KOH, 1%NaClO2 and 1%HCl as a bleaching medium. The authors demonstrated by FTIR the absence of peaks of lignin and hemicellulose in bleached fibers, however, thermogravimetric analysis showed the presence of shoulders around 300 °C indicating the presence of traces of hemicellulose. On the other hand, the H-F and US-F samples show a more pronounced point of maximum degradation temperature, 340 °C and 317 °C respectively. The degradation temperature of H-F is approximately to DC, while the degradation temperature of US-F is lower than the degradation temperature of DC (338 °C), typical value of cellulose44. Our results are close to those reported by Ovalle et al.26; who obtained nanofibers from fique residues by the oxidation process with TEMPO combined with ultrasound and whose degradation temperature was 310 °C, temperature lower than the degradation temperature of the cellulose. According to previous reports45, the low degradation temperatures of the cellulose nanofibers is related to number of free terminal chains which decompose at lower temperatures. Therefore, in accordance with what was reported by Barbash et al.34 and Roman and Winter46, the isolation of cellulose nanofibers by hydrolysis with H2SO4 causes a significant decreasing in degradation temperature and an increasing in range of degradation temperature and this is due to the inclusion of sulfate groups in cellulose chains. This behavior was evident in this study. Nevertheless, Rämänen et al.47, indicate that the effect of decreasing in thermostability is decreased when sulphate groups were neutralized previously to drying process. The mentioned authors also report that drying method is another factor that influences the thermal stability of cellulose nanofibers, and reports that differences in thermostability are due to fibers reordering due to water elimination. When samples are dried in a conventional manner with air, nanofibers are free to form a homogeneous phase with a defined distance among particles. While drying process through lyophilization requires initial freezing. In lyophilization, nanofibrils cannot be rearranged during water sublimation process, obtaining clusters of dispersed nanofibers and a higher specific surface area47, which affects thermal stability. In addition, at low temperatures the fibrils regrouping occurs randomly leaving a large part of free cellulose chains45. The above mentioned is important to consider in this study since samples once the hydrolysis process was finished, they were subjected to drying in a forced convection oven and, after ultrasound treatment, they were subjected to drying through lyophilization.A thermal stability analysis was performed to demonstrate the efficiency of bleaching, hydrolysis acid and ultrasound treatment treatments Fig. . Three s2SO4 process combined with ultrasound leads to fibers individualization to diameters smaller than 100 nm, which initially ranged from 14 to 21.4 μm. The results of length and aspect ratio (L/D) of the nanofibers were: 483.8 ± 283.2 and 19.2 ± 12.2 respectively.Particle sizes of 25.2 ± 6.2 nm in diameter were obtained for fique nanofibers, which is evidenced in micrographs obtained from TEM analysis Fig. . It is o2SO4 process combined with ultrasound leads to fibers individualization to diameters smaller than 100 nm, which initially ranged from 14 to 21.4 μm. The results coincide with what was reported by49 where it is argued that to be considered nanofibers they must have a diameter between 10 to 100 nm while length ranges between 100 and 1,000 nm.Treatment with H50, agglomeration generally depends on Van der Waals attractive forces among nanoparticles. These results are comparable in variability to the report by Trifol et al.51 where fique nanofibers production with a diameter of 27.0 nm and length of 658 nm is reported.TEM micrograph shows separated and agglomerated nanoparticles. According to what was reported by Song et. al27 reports nanofiber sizes of fiche residues between 8 and 17 nm using bleaching combined with ultra-stex treatment. Similarly, Ovalle et al.26, who also worked with fique residues by the method of oxidation by TEMPO and ultrasound, found similar results. While the authors do not specify the diameter of the nanofibers exactly, it is reported that elongated and interlaced structures were obtained, with diameters of around 100 nm and lengths of several micrometers. Although the aforementioned authors used methodologies other than hydrolysis acid, the fiber sizes obtained show low efficiency in the separation of nanofibers from both the amorphous part and the crystalline part of the cellulose structure.On the other hand, the study made by Gomez et al.52, Aspect ratio values ranged from 15.1 to 42.7 can be used as reinforcing agents in composite materials. Therefore, it can be inferred that fique nanofibers can be an alternative for material reinforcement. Likewise, the application of Colombian fique nanofibers for the synthesis of acrylic base hydrogels has not been reported. In addition, nanofibers with an L/D ratio below 60 have the capacity to form a percolated network that is maintained by hydrogen bond interactions, this contributes to an increasing in rigidity and, therefore, fibers with these characteristics have potential as reinforcement using low proportions53.To use nanofibers as reinforcement materials length is an important parameter for determining the diameter ratio (L/D) since the value of this parameter depends on the effect on the mechanical properties on the material to be reinforced. L/D of the nanofibers were 483.8 ± 283.2 and 19.2 ± 12.2 respectively. According to Pelissari et al.The Fig. 54. Nanofibers dispersed uniformly in the hydrogel help to support higher loads, this could be due to the bonding between the polymer chain and the hydroxyl groups of the nanofibers11. Our results are consistent with reports that indicate that concentrations of nanofibers less than 6.7% increase the resistance to compression, higher values of nanofibers in the hydrogel reduce the dispersion of the nanofibers and decrease the crosslinking points14. Acrylamide base hydrogels have been synthesized using commercial cellulose nanocrystals of 10 nm in diameter and a length of 120 nm as reinforcement14. Similarly, Mahfoudhi and Boufi10 obtained acrylate base hydrogels—acrylamide and eucalyptus pulp nanofibers with 5 nm of diameter and 1 mm in length. The addition of these nanofibers increased the stiffness, strength and elasticity of the hydrogel. The authors also reported that at a concentration of 10% nanofibers, the elastic module and compression resistance were up to 10 and 13 times higher than hydrogel without fibers. Our study showed that the addition of fique cellulose nanofibers to hydrogels improves mechanical strength. However, the compression resistance found by Mahfoudhi and Boufi10 were considerably higher than our results.The HRFN supported higher stresses than H because of the HRFN showed higher Rc (16.39 ± 4.30 kPa), and this value is 2.5 times better than the resistance of H (6.49 ± 2.48 kPa). This is explained because the nanofibers interact with the polymer matrix, which favors the stability of the network10. This indicates that studies are required to optimize the size and concentration of fique nanofibers added to the hydrogel, and also the use of other methods of pretreating the fibers. Fique nanofibers would be an excellent environmentally friendly option to add value to waste generated in the fique production chain.Fique fibers in their native state are characterized by their high specific strength compared to other fibers so it could be expected that there are concentrations of fique nanofibers (different from those added to hydrogels in our research) that can generate mechanical resistances equal to or greater than that obtained by Mahfoudhi and Boufi20, the presence of cellulose nanofibers in hydrogels increases the number of hydrophilic groups, facilitating the diffusion of liquids into the matrix. Consequently, they induce the formation of pores in the structure of the polymer. In fact, the incorporation of nanofibers reduces the crossing points of the hydrogel and dilates the gaps of the network24 favoring the penetration of water within the polymer network.Figure Finally, in this investigation it was evident that the combined process of acid hydrolysis with ultrasound radiation allowed the obtaining of fique nanofibers with diameters less than 100 nm. And that hydrogels reinforced with fique nanofibers considerably improved the compressive strength compared to commercial hydrogels, therefore the nanocellulose obtained from fique has great potential to be used as a reinforcing material in polymeric materials that allows to improve its mechanical properties. However, other research must be done to optimize the concentration of nanofibers needed to maximize the mechanical properties of hydrogels.The raw material was obtained in fique crops at San Bernardo . The samples were washed with distilled water to remove impurities. The fique was cut into pieces of approximately 1 cm in length. After, fique pieces were dried at 65 °C until constant weight, then were milled in a knife mill and finally sieved to particle size of 0.5 mm (NAT-F).38.A mixture of NAT-F and sodium hydroxide aqueous solution (4% w/v) in a ratio 1:25 respectively, was refluxed during 4 h at 90 °C under mechanical stirring, washed several times with distilled water until the alkali was completely removed. Subsequently, the material obtained was bleached using a solution (1:1) of aqueous sodium chlorite (1.7% w/v) and acetate buffer solution . A mixture of delignified fiber and chlorite/buffer solution in a ratio 1:20 respectively, were heated at 80 °C under mechanical stirring and reflux during 6 h. Both, alkali and bleaching treatments were performed in duplicate. Finally, fibers were vacuum filtered, washed until neutral pH with distilled water and dried in a forced convection oven at 50 °C until constant weight2SO4 6.5 M) was stirred for 24 h at 50 °C under reflux. Subsequently, the mixture was diluted with distilled water and neutralized with NaOH 4% until pH 6.0 to 7.0; the precipitate was filtered and washed with distilled water. For fibrillation process, several suspensions of the obtained cellulose (1 g) were prepared in distilled water (100 mL) and subjected to ultra-turrax treatment at 10,000 rpm during 15 min (H-F). The suspensions (in an ice bath) were homogenized using an ultrasonic homogenizer , equipped with a ½″ diameter cylindrical probe, at a frequency of 20 kHz with a power of 400 W for 30 min. Finally, the obtained nanofiber suspensions were dried using a freeze-dryer  operating with a 0.110 mbar vacuum, 48 h, − 40 °C condenser temperature, and 0.03 °C min−1 (US-F)38. The nanofibers were stored in hermetic containers at 5 °C for further analysis.In a bottom flask, a mixture of 5 g of B-F and 125 mL of sulfuric acid solution (H55.The contents of cellulose and lignin of NAT-F and B-F were determined by the method reported by Va SoesSurface morphology from NAT-F and B-F were observed in a JEOL table microscope JCM 50000 (Japan). Previously to SEM analysis, samples were coated with gold using the high vacuum mode.−1. Commercial microcrystalline cellulose (CC) was used as a control treatment.Infrared spectra were obtained for NAT-F, B-F, H-F and US-F. Fourier transform infrared (FTIR) spectra were recorded on a spectroscope  in a wavelength ranged from 400–4,000 cm−1, with nitrogen supplying (20 ml min−1) and alumina crucibles with approximately 8 mg of sample.TGA were obtained for NAT-F, B-F, H-F and US-F. In addition, TGA analysis of CC was carried out. Thermogram were obtained in a computer DSC/TGA 2STAR system,  with a temperature ranged from 30 to 600 °C at a heating rate of 10 °C/minTo determine the diameter of nanofibers, images were obtained in a transmission electron microscope (TEM) . Dispersions of 0.1% by weight nanofibers in water were prepared and mounted on copper grids. Diameters of nanofibers for each sample were estimated using the image processing software (Image J). One hundred measurements were taken to obtain the diameter distribution. The length of nanofibers was determined using Zetasizer  and to measure length, 0.1% by weight suspensions were prepared in distilled water, which were subjected to an ultrasound bath for 30 min. Size measurements were made in quintuplicate, using 1 ml of suspension in a polystyrene cell with a detection angle of 90° at 25 °C.2S2O8) (0.3% by weight with respect to the amount of monomers) and the crosslinker N,N-methylene bis acrylamide (NMBA) (0.085% by weight with respect to the amount of monomers) were added to the mixture. Finally, the suspension was refluxed in a 500 mL reactor, equipped with reflux condenser, under nitrogen atmosphere and magnetic stirring. The polymerization reaction was carried out at 70 °C for 6 h. The final polymeric product was cut to reduce the size to approximately 1–2 mm, then washed with ethanol and dried at 70 °C until constant weight56.Acrylic hydrogels (H) and reinforced acrylic hydrogels with fique nanofibres (HRFN) were synthesized, using the solution polymerization method. Initially, suspensions of fique nanofibers were prepared in 80 mL of water (0 and 5% by weight with respect to the monomer concentration) and dispersed using Ultra Turrax at 9,000 rpm 10 min. On the other hand, 14.0 g of acrylamide (AM) was mixed with 22.0 g of potassium acrylate (AK), obtained by neutralizing 16.0 g of acrylic acid (AA) with potassium hydroxide (KOH). The nanofiber solutions and the monomer solutions (AM and AK) were mixed and stirred during 10 min and subsequently. The initiator potassium persulfate  and strain percent were performed in a universal testing machine (Goobrand 50 IR), according to the methodology proposed by Araki et al.H and HRFN al 3% was observed with a scanning electron microscope (JEOL JSM6490) with an acceleration voltage of 20 kV and high vacuum conditions. Additionally, samples were embedded in resin, their water content was extracted by lyophilization and were coated with gold to study their morphology.


The SEM images confirmed that the prepared nanofibers exhibited uniform morphology with an average diameter of 144 ± 19 nm. The inclusion of JFP in pullulan was confirmed by TGA and FTIR studies. XRD analysis revealed that the increased crystallinity of JFP/pullulan nanofiber was observed due to the formation of intermolecular hydrogen bonds. The tensile strength and water vapor permeability of the JFP/pullulan nanofiber membrane were also enhanced considerably compared to pullulan nanofiber. The JFP/pullulan nanofibers loaded with hydrophobic model drugs like ampicillin and dexamethasone were rapidly dissolved in water within 60 s and release the encapsulants dispersive into the surrounding. The antibacterial activity, fast disintegration properties of the JFP/pullulan nanofiber were also confirmed by the zone of inhibition and UV spectrum studies. Hence, JFP/pullulan nanofibers could be a promising carrier to encapsulate hydrophobic drugs for fast-dissolving/disintegrating delivery applications.The fast-dissolving drug delivery systems (FDDDSs) are developed as nanofibers using food-grade water-soluble hydrophilic biopolymers that can disintegrate fast in the oral cavity and deliver drugs. Jelly fig polysaccharide (JFP) and pullulan were blended to prepare fast-dissolving nanofiber by electrospinning. The continuous and uniform nanofibers were produced from the solution of 1% ( Fast-dissolving/disintegrating drug delivery systems (FDDDSs) is an emerging field involved in developing fast-dissolving nanofibrous films for oral administration in pediatric and geriatric patients ,2. In reThe main advantage of these nanofibers is that they can dissolve/disintegrate quickly to deliver the desired drug without swallowing and the need for water. The drug releases occur rapidly in the oral cavity, which aids rapid absorption and improves bioavailability . GeneralThe electrospinning process is a versatile and straightforward technique for preparing polymeric nanofibers with the desired size (nanometers to micrometers) to load and release the drug in a sustained manner . ElectroRecent studies demonstrated that the electrospinning technique has been widely employed in health care systems including drug delivery, diagnostics, theranostics, tissue engineering, and wound healing ,22,23,24Aureobasidium pullulans grown in starch and sugar media [(Ficus pumila var. awkeotsang), also known as Aiyu locally, is a native plant species grown in Taiwan’s mountains. The polysaccharide extracted from jelly fig achenes (JFP) is a pectinous polysaccharide mainly composed of galacturonic acid with a low degree of methylation [Over the past decades, there is growing interest in using natural polymers to develop biomaterials, such as edible films and biodegradable polymer scaffolds. Pullulan is a food-grade and water-soluble polysaccharide produced by ar media . Pullulaar media . Besidesar media . Jelly fhylation . The JFPhylation .Blending different natural polysaccharides showed various synergic effects that would be useful for the preparation of nanofibers. For example, the ternary blending hydrogel made by agar/κ-carrageenan/konjac glucomannan exhibited significant improvements in mechanical and water barrier properties . The ble(Ficus awkeotsang Makino) were purchased from the local market and used for the polysaccharide extraction. Pullulan was procured from Tokyo Chemical Industry Co., Ltd. . Nonionic surfactants Triton X-100 and X-305 were bought from Sigma-Aldrich . All other chemicals used in the experiment were analytical grade also purchased from either TCI or Sigma-Aldrich if not specified.The achenes of Jelly fig Ficus awkeotsang Makino achenes with slight modifications [JFP was extracted from the ications . Brieflyw/w) JFP powder and 6–12% (w/w) of the pullulan powder were dissolved in deionized water and stirred continuously for 4 h at 70 °C using a magnetic stirrer hot plate to obtain a homogeneous solution. The JFP/pullulan solution was used for electrospinning at a flow rate of 0.008 mL/min. The 10 mL plastic syringe and stainless-steel needle with an internal diameter of 0.52 mm were used to eject the solution. The voltage was applied to 27 kV. The ground wire was connected to the stainless-steel plate collector to maintain a constant electric field. The distance between the injection needle and the collection plate was 20 cm. The whole electrospinning process was carried out uniformly with a temperature of 24 °C and relative humidity of 50%. The resulting nanofibers were stored in a dry box with moisture control.A total of 1% , and the photomicrographs were recorded. The nanofiber samples were adhered to the sample platform using double-sided inspection carbon tape and coated with a layer of platinum on the surface of the sample for analysis. The voltage used in this process was 15 kV. The magnification of the micrographs was ranging from 3000–10,000×. The average diameter of the nanofibers was estimated from 50 SEM images using Image J software.1. The two characteristic peaks of 1615 and 1744 cm⁻1 were used to analyze the structure of the JFP and the peak areas were used to determine the degree of methylation (DM) of JFP.FT-IR spectrometer  was used to analyze the structure and functional groups of polymers. The analysis was performed by scanning the sample in the range of 400–4000 cm⁻Thermogravimetric analysis  was conducted to assess the thermal stability and composition of materials. 8 to 10 mg sample was kept in a ceramic tray used to measure thermal stability from room temperature to 800 °C.X-ray diffraction study  was performed to analyze the phase recognition and crystallinity of the sample. The maximum employed power of the XRD spectrometer was 18 W (60 kV-300 mA). The scanning range and scanning rate were 5°–90° and 2–10°/min, respectively.2) was strained at a 1 cm/min pulling speed until rupture. The obtained tensile strain and elongation ratio were used to investigate the influence of JFP addition to pullulan on mechanical properties.The texture analyzer  was used to examine the tensile strength of the nanofiber membrane. A test strips of nanofiber membrane (2 × 1 cm2 (0% RH) was placed inside the cup, and a saturated NaCl solution (75% RH) was added to the glass chamber to maintain 75% relative humidity (RH) gradient across the nanofibers. Under steady-state conditions, the permeable membrane was weighed at regular intervals for 24 h. The water vapor permeability of the samples was evaluated in triplicate using the following equation:2), t is the time of permeation (h), and ∆p is the water vapor pressure difference between the two sides of the sample (kPa).The water vapor permeability (WVP) was measured using the method based on the standard ASTM E96-80  . The expThe water solubility of nanofibers was determined at room temperature (25 ± 1 °C) by adding water dropwise on pullulan and JFP/pullulan nanofiber samples (1.5 cm × 1.5 cm) placed in glass Petri dishes. The dissolution property was recorded by continuous shooting mode .E. coli) and Gram-positive bacteria (B. cereus) grown on Luria broth agar plates.To test the applicability of prepared JFP/pullulan nanofiber membrane in the FDDDS, ampicillin and dexamethasone were employed as model encapsulants for in vitro release studies. The in vitro release profile of ampicillin and dexamethasone was assessed at 25 ± 1 °C. A total of 1 mg ampicillin and 40 mg dexamethasone were dissolved into a 10 mL JFP/pullulan solution before electrospinning JFP/pullulan nanofibers. The in vitro release of ampicillin and dexamethasone from the prepared nanofiber membrane was examined by antibacterial test and spectrometry. Briefly, 4 mg of the drug-loaded or blank JFP/pullulan nanofiber was dissolved in 10 mL of distilled water, and then a 1 mL solution sample was taken for the UV/Vis spectrometric analysis with scanning wavelength from 190 to 300 nm. The prepared nanofiber membranes would be a useful candidate for oral drug delivery applications particularly, in pediatric and geriatric patients. Therefore, the water was used as a release medium. Further, the antibacterial activities of the drug-loaded and blank JFP/pullulan nanofiber membranes were assessed by the disc diffusion method using Gram-negative  pullulan solution and from 0.391 to 0.257 Pa·s for the 12% (w/w) pullulan solution as the shear force rate decreased from 1 to 100 L/s. The 10% (w/w) and 12% (w/w) pullulan solutions exhibited a Newtonian behavior within this shear rate range. While 1% (w/w) JFP was added to the 10% (w/w) and 12% (w/w) pullulan solutions, the viscosity increased significantly from 0.626 to 1.162 Pa·s, and the shear thinning behavior was observed. This result indicated that adding 1% JFP to the pullulan solution increased the chain-chain interaction or entanglement between JFP and the pullulan macromolecules. The results also showed that the addition of both surfactants decreased the viscosity of the JFP/pullulan solutions, but the surfactant added solutions still exhibited shear-thinning behavior and considered as pseudoplastic fluids.Besides, nonionic surfactants were commonly used to enhance the electrospinnability of an aqueous mixture of polymers ,43,44. Tw/w) of JFP to 10% and 12% pullulan solutions showed an increased conductivity from 302.8 and 407.3 μS/cm. The data revealed that JFP has polyelectrolyte properties. When the JFP/pullulan solutions were added with Triton X-305 and Triton X-100, the conductivity was increased because of the higher hydrophilic-lipophilic balance (HLB) value of the added surfactants [The 10% pullulan solution showed the lowest conductivity of about 8.8 μS/cm. However, the conductivity increased to 27.5 μS/cm, while the concentration of pullulan increased to 12%. Then, the addition of 1%  produced beaded fibers due to the lack of polymer chain entanglement at those concentrations [w/w) was blended with 10% and 12% (w/w) of pullulan to obtain ultrafine fibers without beads (w/w) to 10% and 12% pullulan solution increased the fiber diameters from 155 ± 22 nm to 189 ± 23 nm  and the peak at 3443 cm−1 to the OH bond. However, the carboxylate (COO-) and methyl esterified carboxyl (COOMe) groups were noticed at 1615 and 1744 cm−1, respectively. The degree of methylation of polysaccharides can be calculated from the characteristic peak areas of carboxylic acids and methyl esterified carboxyl groups [The functional group analyses of the JFP samples are given in l groups . The degl groups .−1 were attributed to α- and α- groups. The peaks found at 851, 1658, and 3330 cm−1 represented the structure of α-d-glucopyranose units, the –OCO– group, and the -OH groups. The data was in agreement with previous studies of pullulan [d-glucopyranose units at 754, 930, and 851 cm−1 respectively. The FTIR spectrum of JFP/pullulan nanofibers was shown in −1 and confirmed the existence of JFP in the prepared nanofibers.FTIR analysis of pullulan c showed pullulan ,36. The The TGA spectra showed the first degradation curve at 100 °C for water loss in JFP and pullulan A. Then, The X-ray diffraction pattern of JFP powder consists of two peaks at 25.1° and 27.9° representing the amorphous and crystalline areas of JFP B. The puThe mechanical properties of the JFP/pullulan nanofiber were studied as depicted in −5, g·mm/Pa·h·cm2) than JFP/pullulan nanofibers , which is probably due to the difference in the molecular structure of JFP and pullulan. JFP showed more affinity towards water molecules and increased the water vapor permeability due to the presence of -COOH and –OH groups [The WVP of pullulan and JFP/pullulan nanofibers were displayed in H groups . TherefoH groups . The resAntibiotic and anti-inflammatory drugs are commonly used in wound dressing, but the dissolution of poorly water-soluble drugs is a persisting challenge . SeveralE. coli) and Gram-positive (B. cereus) bacteria, and the results are displayed in E. coli (left) and B. cereus (right) compared to blank JFP/pullulan nanofibers. This study concludes that the electrospinning process did not degrade the ampicillin molecules and improved their water solubility.The applicability of JFP/pullulan nanofibers in FDDDSs was also assessed by in vitro release of encapsulated model drugs dexamethasone and ampicillin. The antibacterial activity of the drug-loaded nanofibers was tested against both Gram-negative  jelly fig 12% (w/w) pullulan solution along with surfactant Triton-X 305 produced continuous, uniform and bead-free nanofibers with an average fiber diameter of 144 ± 19 nm. FTIR and TGA analysis proved the successful blending of jelly fig and pullulan. The XRD pattern revealed that the formation of intermolecular hydrogen bonds between the jelly fig and pullulan molecule and increased crystallinity. The improved water vapor permeability of jelly fig/pullulan nanofiber was noticed due to the presence of carboxyl and hydroxyl groups in jelly fig moiety. The dissolution test substantiated that jelly fig/pullulan nanofibers can be rapidly soluble in water (60 s). The antibacterial activity and existence of ampicillin/dexamethasone in jelly fig/pullulan nanofibers were also confirmed through the dissolution test. Moreover, the encapsulated hydrophobic drugs can also be quickly dispersed into the surroundings. The results showed that the JFP/pullulan nanofibers can dissolve rapidly in a water medium and could be useful in oral drug delivery. Overall, the present investigation concluded that JFP/pullulan nanofibers could be an alternative material in FDDDSs.Novel fast-dissolving nanofibers were fabricated from the blends of JFP and pullulan by electrospinning. The jelly fig polysaccharides were extracted from achenes of