Enterocytozoon hepatopenaei (EHP) causes slow growth syndrome in shrimp, resulting in huge economic losses for the global shrimp industry. Despite worldwide reports, there are no effective therapeutics for controlling EHP infections. In this study, five potential druggable targets of EHP, namely, aquaporin (AQP), cytidine triphosphate (CTP) synthase, thymidine kinase (TK), methionine aminopeptidase2 (MetAP2), and dihydrofolate reductase (DHFR), were identified via functional classification of the whole EHP proteome. The three-dimensional structures of the proteins were constructed using the artificial-intelligence-based program AlphaFold 2. Following the prediction of druggable sites, the ZINC15 and ChEMBL databases were screened against targets using docking-based virtual screening. Molecules with affinity scores ≥ 7.5 and numbers of interactions ≥ 9 were initially selected and subsequently enriched based on their ADMET properties and electrostatic complementarities. Five compounds were finally selected against each target based on their complex stabilities and binding energies. The compounds CHEMBL3703838, CHEMBL2132563, and CHEMBL133039 were selected against AQP; CHEMBL1091856, CHEMBL1162979, and CHEMBL525202 against CTP synthase; CHEMBL4078273, CHEMBL1683320, and CHEMBL3674540 against TK; CHEMBL340488, CHEMBL1966988, and ZINC000828645375 against DHFR; and CHEMBL3913373, ZINC000016682972, and CHEMBL3142997 against MetAP2.The compounds exhibited high stabilities and low binding free energies, indicating their abilities to suppress EHP infections; however, further validation is necessary for determining their efficacy. Enterocytozoon hepatopenaei (EHP) infections in Pacific white shrimp, Litopenaeusvannamei, have become a pandemic in recent years [Penaeus monodon, in Thailand in 2009 [nt years . The pat in 2009 ,3,4. EHP in 2009 ,5. The p in 2009 ,7. EHP,  in 2009 .Toxoplasma gondii [Trypanosoma brucei [Leishmania sp. [Although EHP infections do not cause significant mortality in shrimp, the pathogen dramatically retards the growth of penaeid shrimp and is therefore emerging as a critical threat to the shrimp farming industry, owing to the loss of more than 7.4 million USD annually . Signifia gondii , Trypanoa brucei , and Leiania sp. . TherefoL. vannamei available in the UniProt Knowledgebase (https://www.uniprot.org/ accessed on 5 January 2022), and the results demonstrated that all the selected proteins have <25% identity with the proteins of the host. Therefore, the compounds identified against the five proteins of EHP are less likely to inhibit the proteins of the host.A total of five potential druggable target proteins of EHP were identified, of which three proteins, namely, cytidine triphosphate (CTP) synthase, thymidine kinase (TK), and dihydrofolate reductase (DHFR), are associated with the biosyntheses of pyrimidine nucleotides and purine nucleotides. Of the other two proteins, methionine aminopeptidase (MetAP) is an exopeptidase, while aquaporin (AQP) is responsible for the maintenance of cellular osmotic pressure. The sequences of these 5proteins were compared with the proteome data of The three-dimensional structures of all the five proteins of EHP were generated using AlphaFold 2 . The strArabidopsis thaliana (PDB ID: 5I32), and 98% query coverage and 26% identity with human AQP (PDB ID: 1FQY). Residues Phe22, Asn69, Pro70, Glu138, Gly197, and Asn201 were found to be conserved in EHP AQP (Drosophila melanogaster (PDB ID: 6L6Z) and human (PDB ID: 5U03), respectively, and 29% and 27% identity, respectively. Residues Lys24, Tyr295, Cys377, Phe351, and Arg453 were conserved in the CTP synthase of EHP (Coxiella burnetii (PDB ID: 3TQ8) and human (PDB ID: 3F8Y), respectively, and 30% and 28% identity, respectively. Furthermore, residues Leu27, Ala29, Met43, Trp45, Asp51, Lys56, Arg71, and Thr138 were found to be conserved in the DHFR protein of EHP  and human (PDB ID: 1R58), respectively, and 51% and 43% sequence identity, respectively. Residues Phe80, Pro81, His91, Ile201, His202, His244, Met246, Pro275, and Tyr304 were conserved in the MetAP2 protein of EHP (The sequence homologs of the query proteins were determined with BLASTp against the Protein Data Bank (PDB). EHP AQP (A0A1W0E445) had 95% query coverage and 29% identity with the AQP protein of  EHP AQP . Similare of EHP . The DHFn of EHP . The Metn of EHP . The binn of EHP . No signThe ZINC Purchase library and the ChEMBL database, comprising 12,941,912 and 1,791,953 drug-like molecules, respectively, were screened against the predicted ligand-binding pockets of the 5 druggable targets of EHP. After the primary screening, approximately 1100 drug molecules were selected against each protein target from the ZINC15 library and ChEMBL database based on Lipinski’s rule of five. The selected molecules were further screened using docking-based VS. Compounds with affinity scores ≥ −7.5 and ≥ 9.0 protein-ligand interactions were selected. Totals of 247, 183, 198, 98, and 128 compounds were finally selected against CTP synthase, TK, MetAP2, DHFR, and AQP, respectively .The ADMET properties of the screened drug-like molecules were subsequently predicted. The results demonstrated that most of the compounds qualified the Golden Triangle rule, Lipinski’s rule of five, and Pfizer’s rule. Molecules with predicted carcinogenicity, rat oral acute toxicity, AMES toxicity, and mutagenicity potentials were removed from the library prior to the final screening. Totals of 156, 120, 115, 73, and 81 compounds were finally selected against CTP synthase, TK, MetAP2, DHFR, and AQP, respectively . The selThe selected molecules were further screened based on their EC using Flare v5.0.0, and compounds with EC scores > 0.25 were selected. For each protein, the top 20 ligands were generally selected from the docked conformations for further analysis . The PatThe intermolecular interactions between the selected compounds and the binding sites of the target proteins are depicted in A total of 5 compounds, CHEMBL3674540, CHEMBL1683320, CHEMBL391279, ZINC000031750813, and CHEMBL4078273, were similarly selected against TK, with binding affinities ranging from −9.5 to −8.1 kcal/mol . All theA total of five compounds, namely, CHEMBL56533, ZINC000016682862, ZINC000828645375, CHEMBL3901573, and CHEMBL108166, were selected against EHP DHFR. All the compounds formed hydrogen bonds with different amino acids and were stabilized in the binding pocket . The calMD is a powerful computational method for predicting and analyzing the stabilities of protein-ligand complexes and for studying atomic movements with respect to a macromolecule. The stabilities and behaviors of the protein-ligand complexes were analyzed in a dynamic environment based onroot-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (Rg), and molecular mechanics/generalized Born surface area (MM/GBSA) energy. The simulation trajectories were subjected to a principal component analysis (PCA), and a cross-correlation matrix of the resultant MD trajectories was also constructed and analyzed.The RMSD values of the protein-ligand complexes were plotted graphically for understanding the structural stability and integrity of the complexes. The results demonstrated that the average RMSD values of the 5 compounds selected against AQP ranged from 1.84 to 3.68 Å. Compounds CHEMBL3703838, ZINC000002243083, and CHEMBL3140193 showed high stabilities during the simulation, with an RMSD fluctuation of 0.5 Å . SimilarThe compactness of the protein-ligand complexes during the simulation was determined by measuring the values of Rg. The average values of Rg for the 5 compounds complexed with AQP ranged from 17.89 to 18.30 Å . SimilarThe average atomic mobility of the protein backbone during the MD simulations was measured using the values of RMSF. The average RMSF values of the 5 compounds complexed with AQP ranged from 0.92 to 1.53 Å . FurtherThe cross-correlation matrix of the resulting trajectories was analyzed for understanding the dynamical correlations of conformational motion of the protein-ligand complexes. The positive regions of the matrix are associated with the strongly correlated motions of residues moving in the same direction, while negative regions are linked with anti-correlated movements. The correlations of motion of the residues of AQP complexed with the five ligands were generated and displayed as a correlation matrix, depicted in The binding free energy represents the sum total of all the interaction energies, including the van der Waals energy, polar solvation energy, electrostatic energy, and solvent-accessible surface area SASA energy. The binding free energies of all the complexes were estimated using the MM/GBSA approach . The binThe slow growth syndrome of shrimp caused by EHP infections is a major threat to sustainable shrimp farming in Asia at present. The disease has caused significant economic losses for shrimp farmers not only in Thailand but also in other parts of Southeast Asia since 2009. Shinn (2019)  calculatT. gondii disrupts the lytic cycle [T. brucei [3, which is subsequently utilized for the ATP-dependent conversion of UTP to CTP. A structural analysis of Drosophila CTP synthase (dmCTP synthase) revealed that the guanine base of GTP interacts with the Leu107 and Leu444 of another monomer. Further studies revealed that GTP forms three hydrogen bonds with Arg481. In addition, the π–π interaction between the guanine ring and Phe373 also contributes to the binding. The ribose ring of GTP interacts with Phe50 and Arg479 via hydrogen bonds. The triphosphate moiety of GTP also forms three hydrogen bonds with Lys306, Tyr307, and Arg376. The binding of glutamine via interactions with Phe37 and Cys399 further stabilizes the binding of GTP [Plasmodium sp., Trypanosoma sp., and Toxoplasma sp., includingα-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, glutamate γ-semialdehyde, azaserine, and 2,4-diaminopentanedioic acid, have been identified and reported in previous studies [CTP synthase catalyzes the synthesis of cytidine 5′-triphosphate (CTP) from uridine 5′-triphosphate (UTP) in the final step of the production of cytidine nucleotides from both the de novo and uridine salvage pathways . CTP synic cycle . CTP syn. brucei , malaria. brucei , and gia. brucei . A struc. brucei . The GATg of GTP . A compag of GTP . Potent  studies ,26,27.T. cruzi [Cryptosporidium hominis [P. falciparum . Although pyrimidine biosynthesis occurs ubiquitously in EHP and shrimp, the DHFR enzyme is divergent enough to allow the design of inhibitors specific to EHP DHFR. In this study, we identified five compounds with high binding affinity to the folate binding pocket of EHP DHFR. Of these compounds, ZINC000016682972 had the lowest free energy of binding and exhibited high stability with EHP DHFR. Further analysis revealed that MTX and TMP bind to the same region of DHFR, which was selected as the druggable site for docking-based VS against EHP DHFR in this study. DHFR inhibitors, including riluzole, 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine, 7-((2-thiazol-2-yl)benzimidazol-1-yl)-2,4 diamino quinazoline, and proguanil, which specifically bind to the folate-binding pocket of DHFR, have been identified for suppressing infections caused by L. major, T. brucei, Staphylococcus aureus, and the malarial parasite, respectively [The DHFR catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). Tetrahydrofolates serve as co-factors for the synthesis of purines, pyrimidine (thymidylate), and for the re-methylation of homocysteine to methionine. Reduction in DHFR enzymatic activity diminishes the THF pool inside the cell, which slows DNA synthesis and cell proliferation, eventually leading to cell death ,29. DHFRT. cruzi  and Cryp hominis . Severalectively ,32,33.E. bieneusi, E. cuniculi, Entamoeba histolytica, and Vittaforma corneae [E. cuniculi (EcMetAP2). A structural analysis of EcMETAP2 revealed that it has a metal-binding domain and a methionine-binding domain. Residues Asp130, Asp141, His210, Glu243, and Glu339, which are responsible for coordinating Fe2+ ions, were found to be completely conserved in all the MetAP protein sequences, including EcMetAP2.MetAP2 catalyzes the removal of N-terminal methionine residues from nascent proteins . Inhibit corneae ,38,39. AThe residues comprising the methionine-binding pocket include Phe97, Pro98, His109, Ile217, and His218 inthe peripheral loops and His261, Val263, Pro292, and Tyr324 in the subdomain. Only two residues, His109 and His218, are completely conserved across the entire MetAP protein family . The metFumagillin/TNP-470 is a well-known inhibitor of MetAP2 that binds to the methionine-binding pocket of EcMETAP2 via hydrophobic interactions with Phe97, Pro98, Ilu217, His261, Val263, Pro292, and Tyr324, which are equivalent to residues Phe80, Pro81, Ile201, His244, Met246, and Tyr304 of EhpMetAP2 . HoweverL. major [T. brucei [P. falciparum, Giardia intestinalis, and T. cruzi [The TK key enzyme catalyzes the transfer of the γ-phosphate of ATP to 2′-deoxythymidine (dThd), forming thymidine monophosphate (dTMP) via the salvage pathway in microsporidia . InhibitL. major  and T. b. brucei . The gen. brucei . Glu98, . brucei ,47. The T. cruzi ,49,50.2 effectively inhibits the germination of Anncaliia algerae spores [A. algerae was, therefore, identified as a potential druggable target in an earlier study. [Xenopus laevis by binding to a region similar to that identified in the present study [AQPs are transmembrane channels that transport water and/or small solutes, such as glycerol, nitrates, and urea, across cellular membranes. A total of 13 AQP isoforms (AQP0 to AQP12) have been identified in humans . Of these spores . The AQPr study. . In thisr study. ,59. A sint study .https://www.uniprot.org/ accessed on 5 January 2022). The three-dimensional structures of all the five proteins were generated using AlphaFold 2 [The potential druggable protein targets of EHP were identified using two different approaches. The complete protein information of EHP was retrieved from the UniProt Knowledgebase and subjected to a functional enrichment analysis for identifying the candidate proteins that are involved in major metabolic pathways, using the online tools in DAVID for a KEGG enrichment analysis. The protein targets were finally selected using an approach similar to that used in earlier studies ,62, whichaFold 2 . The stehaFold 2 .The druggable pockets in the five protein targets were identified using two separate approaches. The amino acid sequences of the five proteins were first used for identifying all the sequence homologs in the PDB with the NCBI BLAST server , using dhttp://biosig.unimelb.edu.au/easyvs/ accessed on 25 January 2022) [The three-dimensional structures of the five proteins of EHP were used as targets for screening drugs from the ZINC15  and ChEMry 2022) . A sequehttps://admetmesh.scbdd.com/ accessed on 10 August 2022) [All the screened molecules were subjected to an ADMET analysis for predicting the pharmacokinetics and toxicity properties using the SwissADME server (st 2022) . ADMET sThe compounds were initially screened based on the following criteria: affinity scores ≥ 7.5 and numbers of interactions ≥ 9. These compounds were re-screened based on their EC . The res+ or Cl− ions at a molar concentration of 0.15 M. The systems were subjected to energy minimization for relaxing the water molecules and intramolecular steric clashes at a temperature of 300 K under 1 bar pressure. The systems were subsequently equilibrated for 20,000 ps while imposing positional restraints of 700 kJ/mol. All the simulations were performed under the NPT ensemble by maintaining the temperature at 300 K using the Langevin thermostat [The stabilities of the protein-ligand complexes were verified with 100 ns MD simulations using the Amber ff19SB  force fiermostat , with a ermostat  at a refermostat  and the ermostat  cloud-babind) were calculated using the following equations [complex, ΔGreceptor, and ΔGligand represent the free energy of the complex, receptor, and ligand, respectively.gas) comprises the internal energy (ΔEint), electrostatic interactions (ΔEele), and van der Waals interactions (ΔEvdw) energy terms. The solvation free energy (ΔGsol) comprises the polar energy (ΔGGB) and non-polar energy (ΔGSurf) terms. TΔSgas represents the contribution of conformational entropy.The binding free energies of the docked complexes were calculated using the mechanics/generalized Born surface area (MM/GBSA) approach . The binquations ,81:ΔGbinL. vannamei and P. monodon. Shrimp production in Asia has declined by 10–20% owing to EHP infections, which has resulted in significant economic losses. In addition, the infection is rapidly spreading to new geographical locations. The shrimp farming industry will suffer substantial economic losses if the scenario of EHP infections remains unaltered, which will have serious effects on the global socio-economic structure. In this study, a total of fifteen compounds  were identified against five potential druggable protein targets of EHP. The compounds had high binding affinities and low free binding energies, as indicated by the results of extensive the insilico analyses. The compounds formed stable complexes with the respective protein targets and were predicted to have insilico inhibitory potentials. The results of the computational analyses obtained in this study will be experimentally validated by in vitro and in vivo studies in future.EHP is an intracellular parasite that is responsible for the slow growth syndrome of the Penaeid shrimps


Substantia nigra pars compacta (SNc) dopamine neurons are required for voluntary movement and reward learning, and advanced age is associated with motor and cognitive decline. In the midbrain, D2-type dopamine receptors located at dendrodendritic synapses between dopamine neurons control cell firing through G protein-activated potassium (GIRK) channels. We previously showed that aging disrupts dopamine neuron pacemaker firing in mice, but only in males. Here we show that the amplitude of D2-receptor inhibitory postsynaptic currents (D2-IPSCs) are moderately reduced in aged male mice. Local application of dopamine revealed a reduction in the amplitude of the D2-receptor currents in old males compared to young, pointing to a postsynaptic mechanism. Further experiments indicated that reduced D2 receptor signaling was not due to a general reduction in GIRK channel currents or degeneration of the dendritic arbor. Kinetic analysis showed no differences in D2-IPSC shape in old versus young mice or between sexes. Potentiation of D2-IPSCs by corticotropin releasing factor (CRF) was also not affected by age, indicating preservation of one mechanism of plasticity. These findings have implications for understanding dopamine transmission in aging, and reduced D2 receptor inhibition could contribute to increased susceptibility of males to SNc dopamine neuron degeneration in Parkinson’s disease. Dopaminergic behaviors decline with advancing age, with movement deficits, apathy, and depression commonly observed in the elderly population5. Moreover, aging is the single largest risk factor for development of Parkinson’s disease (PD)6; a disease that features motor symptomatology as a consequence of degeneration of SNc dopamine neurons7. While a modest level of dopamine cell death is thought to occur with normal age11, it is insufficient to explain the ~ 60% loss in extracellular dopamine that has been reported in the striatum12, suggesting a contribution from declining physiological and/or morphological factors in single neurons. Indeed, we have previously reported that aging affects pacemaker firing frequency and regularity in dopamine neurons from male mice13, while firing in female mice is largely preserved14. Additionally, we have observed a striking decrease in nimodipine-sensitive (presumably L-type) calcium channel currents in dopamine neurons from aged males, while currents through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and small-conductance calcium-activated potassium (SK) channels are unaltered. It is less clear how age affects other channels and receptors, and no study to date has explored individual synaptic inputs to dopamine neurons across the lifespan.Dopamine neurons of the substantia nigra pars compacta (SNc) are integral to motor function and reward learning15, where D2 type autoreceptors are able to sense the local release of dopamine16. Evoking dopamine release through electrical or optogenetic stimulation produces a D2 receptor-mediated inhibitory postsynaptic current (D2-IPSC) that is action potential- and calcium-dependent18. This form of dendritic neurotransmission proceeds through D2 receptor activation of Gi/o proteins and type 2 inhibitory G-protein-activated inwardly rectifying potassium (GIRK) channels20. The resulting inhibitory conductance hyperpolarizes the cell and strongly reduces firing rate21. D2 receptor signaling in the midbrain is susceptible to multiple forms of short- and long-term plasticity and is regulated locally by neuropeptides and neuromodulators27. For example, the stress related-molecule corticotropin releasing factor (CRF) transiently increases the amplitude of both D2- and GABAB receptor-mediated currents in a postsynaptic manner consistent with activity at the level of the GIRK channel23. The effects of advanced age on D2-IPSCs have not been systematically studied, however humans exhibit a decline in D2-like receptor availability at varying rates depending on the brain region30, with perhaps no decline at all in the SNc31.Dopamine neurons in the midbrain communicate with each other via dendrodendritic synapsesThe aim of the present work was to delineate the extent to which pre- and postsynaptic components of dopamine neurotransmission in the SNc are affected by advancing age using whole cell patch clamp recordings in mouse brain slices. We hypothesized that dopamine signaling would be reduced both pre- and postsynaptically resulting in smaller and slower currents, and that this deficit in dopamine transmission could affect dopamine cell function in advanced age. We did observe a small but significant reduction in D2 receptor currents in aged males that was postsynaptic and at the level of the D2 receptor. However, both the kinetics of D2-IPSCs and CRF-induced plasticity were preserved with advanced age, as was dendritic branching of individual dopamine neurons. These results indicate that most aspects of dendritic dopamine neurotransmission are remarkably resilient to normal aging.1,62 = 0.195, p = 0.661; age, F1,62 = 0.375, p = 0.543). We did observe a significant age-sex interaction . We then normalized each cell to its maximum amplitude, as a difference in the shape of stimulus–response curves could indicate alterations in presynaptic sensitivity or dopamine release probability. Data indicated an expected increase in amplitude with greater stimulus , which plateaued at the highest stimulus intensities. However, no difference was observed between experimental groups , nor was there a group-stimulus interaction . This suggests that the modest differences in aging were not accompanied by an observable decrease in the stimulus–response relationship across groups, arguing against presynaptic differences. An analysis of the kinetics of the D2-IPSCs indicated that the average normalized traces from each group were essentially superimposed . These results suggest that the amplitude and time course of D2-IPSCs are largely conserved across age and sex, and the subtle difference observed in peak amplitudes cannot be explained by altered IPSC kinetics or a shift in the stimulus–response relationship.We acutely prepared horizontal midbrain slices containing the SNc from young (2–8 month) and old (20–25 month) C57Bl/6N mice of both sexes. We then performed whole cell voltage clamp electrophysiological recordings of SNc dopamine neurons (holding voltage −55 mV). In the presence of synaptic blockers, we next used trains of five electrical stimuli to elicit D2-IPSCs Fig. a over a 51 = 1.61, p = 0.114). Analysis of the normalized time course  with main effects of time  and age . Posthoc analysis revealed a significant simple effect of age during washout from prolonged drug application, indicating a possible decrease in the ability to clear large amounts of dopamine from the extracellular space.Since D2-receptor currents tended to be smaller in old males compared to young, we next investigated if this could be due to alterations in postsynaptic D2 signaling in aged males. We voltage clamped dopamine neurons at −55 mV and bath-applied a high concentration of dopamine (100 µM) for 7 min. On average, peak amplitudes were larger in cells from young versus old males [Fig. 32. Analysis of the resulting outward current indicated that neurons from old males exhibit smaller currents compared to young male mice . When the currents were normalized to their peak amplitude  that would indicate a difference in kinetics. These results indicate that rapid, local application of dopamine produces smaller amplitude currents in cells from aged males.Since bath application of dopamine is relatively slow and current amplitude peaks at a time that desensitization is starting to occur, we repeated this experiment with rapid, local activation of dopamine receptors. We used iontophoresis to rapidly apply dopamine  near the soma as previously described17. We next sought to determine the effect of age on GIRK channel activation mediated by a different receptor. GABAB receptors also activate GIRK channels in dopamine neurons, producing a potassium conductance that is partially overlapping with the D2 receptor-mediated conductance33. We therefore recorded from SNc dopamine neurons in slices from young and old male mice and sequentially bath applied two concentrations of the GABAB receptor agonist baclofen (3 and 30 µM) while in voltage clamp  and recorded GABAB receptor currents in SNc dopamine neurons from young and old males. Results showed no difference in response between young and old male mice ; unpaired t-test, t78 = 0.569, p = 0.571; Fig. GIRK channels are activated by D2 receptors in dopamine neurons and are responsible for the bulk of the D2-IPSC34. Aging has been associated with decreased responsivity of central and peripheral catecholaminergic systems to acute stress and progressive hypothalamic CRF deficiency in the rat36. In humans, decreasing CRF concentrations have been observed with aging in the cingulate gyrus 37. In the ventral tegmental area, CRF-positive axon terminals establish asymmetric (excitatory) synapses on neurons that stain for tyrosine hydroxylase, a dopamine neuron marker38. CRF can act through CRF-R1 and CRF-R2 receptors in dopamine neurons40, and has been reported to alter their firing rate and pattern43. We have shown that CRF increases D2-IPSCs amplitudes, and this effect is blunted in mice that have been exposed to repeated restraint stress or injections of psychostimulants23. Therefore, we next proceeded to determine if CRF-induced modulation of dendritic dopamine signaling is affected by aging. We recorded from SNc dopamine neurons and applied CRF at a concentration that strongly potentiates D2-IPSCs . This suggests that this form of D2-IPSC plasticity in dopamine neurons is preserved in aging.Corticotropin releasing factor (CRF) is a neuropeptide involved in both peripheral and central stress response. Along with its role in initiating the hypothalamic–pituitary–adrenal (HPA) axis loop, CRF is also released in extrahypothalamic sites within the brain44 and by our group in both sexes of MitoPark mice, a genetic animal model of PD45. Here, we measured dendritic morphology of male SNc dopamine neurons located along the rostrocaudal and mediolateral axis of the SNc  by filling cells with biocytin included in the recording pipette, conjugating with streptavidin, and performing confocal imaging. Using the Sholl analysis method46 we analyzed dendritic arbors between 20 and 400 µm from the soma .We next sought to determine if differences in somatodendritic morphology could contribute to reduced D2 receptor currents in old males. Altered dendritic morphology has been described in SNc dopamine neurons from adult (5.5–17 months of age) versus juvenile (2–4 weeks) female mice33 = 0.610, p = 0.546; number: t33 = 1.10; p = 0.278. Since retraction of dendrites is more likely to happen towards the end of the branches, any subtle shrinkage with age could be reflected in a change of neurite length (or number) at some level of the dendritic arbor , inner branches  or terminal branches . We conclude that SNc dopamine neuron morphology is preserved throughout the adult lifespan in males, and that the reduced D2 receptor currents that we observed in old males are not due to shrinkage of the dendritic arbor.Another way to analyze dendritic morphology is by measuring the length and number of primary, inner and terminal branches Fig. c. If somB receptors with baclofen or iontophoresis of GABA did not decline with age. Most other parameters measured were also unchanged by age, including CRF-induced enhancement of D2-IPSCs and the quantification of dendritic arbor morphology of single dopamine neurons. As we have previously noted13, perhaps the most striking finding is the number of parameters that were not changed by age. However, the fact that many experiments yielded negative results gives us confidence that the observed decrements in D2 currents with age are not due to experimental considerations, such as a phenomenological consequence of the brain slicing procedure.This is the first report of the effects of aging on dopamine D2 receptor transmission in SNc dopamine neurons from mice. D2-IPSC amplitudes were moderately reduced in dopamine neurons from aged males, while the shape of the stimulus–response curve and the IPSC itself were identical across all groups. Iontophoretic application of dopamine proximate to the recorded neuron produced smaller outward currents in aged males, suggesting a postsynaptic decrease in D2 receptor signaling. This reduction did not appear to be caused by impairments at the level of the GIRK channels, as activation of GABA14, and also observed a reduction in the nimodipine-sensitive (presumably L-type) calcium channel conductance with age13. Our current results demonstrate that aging can affect synaptic input to dopamine neurons in addition to intrinsic conductances, as evidenced by a reduction in postsynaptic D2 receptor currents. This effect could potentially impact the precision and/or frequency of dopamine neuronal firing, along with other altered ionic conductances or synaptic inputs. We and others have previously shown that synaptically-evoked dopamine release produces a pause in firing47. We have also long noted a slight but reliable increase in firing rate upon application of the D2 receptor antagonist sulpiride, suggesting modest tonic dopamine inhibition even in brain slices (data not shown). This is consistent with recently published findings from other groups showing an inward current in response to the D2 receptor antagonist sulpiride48 and an apparent rightward shift in firing rate histograms in the presence of sulpiride, although the latter was not directly compared49. SNc dopamine neurons are notoriously sensitive to insults due to their high energy demands, vast neuritic arbors, large calcium oscillations, and handling of dopamine metabolites, and could be particularly susceptible to oxidative stress52. Age and sex are the two leading risk factors for development of Parkinson’s disease, with men exhibiting a 1.5–2.0 times higher incidence than women53. Increased firing in aged males due to reduced D2 receptor signaling could therefore conceivably interact with other factors to initiate or accelerate processes associated with neurodegeneration due to normal aging and/or diseases such as Parkinson’s.We previously found that dopamine neuron firing fidelity is only compromised in aged malesp = 0.863). General G protein/GIRK channel activity was also not obviously affected by age. Responses to bath application of two concentrations of the GABAB agonist baclofen or to GABA iontophoresis did not vary with age, and the kinetics of the D2-IPSC (which could reflect rapid changes in G protein activation or inactivation) were identical across sex and age. We did observe a reduced response to exogenous dopamine in aged males, indicative of reduced D2 receptor responses, although the effect sizes were modest and differences hovered near the edge of statistical significance. Responses to bath applied dopamine seemed to indicate a population of high-responding dopamine neurons in young mice that were not present in old mice, although peak amplitudes exhibited merely a trend toward significance. Interestingly, after several minutes of application, currents observed during washout were significantly increased in aged males. This is not likely due to decreased DAT function, as the kinetics of the D2-IPSC are strongly sensitive to reduced DAT-mediated uptake54 but were unaffected here. In contrast, peak currents elicited by iontophoretic (rapid) application of exogenous dopamine exhibited a significant reduction in aged males. Although the two data sets looked qualitatively similar, the larger effect of age with local application could be due to the different time course of the respective responses. One limitation in our interpretation is that there are currently no reliable selective antibodies for measuring total or surface expression of endogenous D2 receptors55. Regardless, the evidence in hand indicates that the most likely explanation for reduced D2-IPSC amplitudes are due to effects at the level of the postsynaptic D2 receptor. In humans, studies using imaging and/or D2/D3 receptor radioligand tracers indicate variable rates of D2-like receptor decline in normal aging depending on the brain region. Cortical areas seem to show the largest decline in D2-like receptor availability with age (6–16% per decade) while subcortical and striatal regions are relatively spared (1.5–5% per decade)30. Interestingly, a postmortem study (age 19–88 years) found that D1-like and D2-like receptor density in substantia nigra does not change with age31.There are several potential explanations that could explain smaller D2-IPSC amplitudes in old males: (1) decreased presynaptic dopamine release, (2) altered postsynaptic D2 receptor activity, (3) altered GIRK channel activity, or (4) morphological changes, such as reduced dendritic branching or a decrease in synapse number. Although each of these factors does not preclude influences from the others, our findings are most consistent for effects at the level of the postsynaptic D2 receptor. The shapes of the stimulus–response relationship were completely unaffected by age or sex, arguing against presynaptic factors such as reduced release probability. This is also consistent with our results (not shown) using paired trains of stimuli 3 s apart to evoke D2-IPSCs in neurons from male mice which indicated no effect of age  remained stable during aging62. We previously demonstrated that SNc dopamine neurons show no change in cell capacitance with age13, which would argue against gross effects on cell surface area or morphology. In this study we focused on just two age groups in adult mice and cannot make conclusions about dendritic morphology at other times. For instance, Rafols et al.63 used Golgi staining to show that total dendritic length of aspiny neurons in the striatum gained branching complexity from 3 to 20 months old, but decreased thereafter. Another study in C57BL/6N mice found no differences in dendritic parameters in cortical barrels across the entire lifespan64, while a study in the basolateral amygdala of rats showed hypertrophy of the dendritic arbor with aging65. Perhaps unsurprisingly, age effects on dendritic arborization exhibit differential patterns based on cell type and brain region, from dendritic morphology not changing at all to growing or regressing early or late in life66. Of note, here we did not go beyond 25 months as we did not want to bias the data toward a long-lived population of mice.Normal aging is characterized by cognitive impairment that could involve neuronal loss, however many studies have shown minimal cell loss during aging35. Other rodent studies also report reduced negative feedback of the HPA axis with aging in response to elevation of glucocorticoids, resulting in a pattern of prolonged activation in response to stress67. In humans, reports of age-related changes in HPA axis function are conflicting68, and little is known about age-related alterations in CRF function in the SNc in humans or rodents. D2-IPSCs are subject to multiple forms of pre-22 and postsynaptic plasticity including long-term depression induced by the peptide neurotensin25 and a transient enhancement produced by CRF application23. The CRF enhancement occurs postsynaptically through activation of CRF-R1 and apparently occurs at the level of the GIRK channel, as GABAB signaling is also potentiated. Further, either repeated exposure to psychostimulants (cocaine or methamphetamine) or restraint stress decreases the magnitude of the CRF response23. Here we show that aging does not affect enhancement of D2-IPSCs produced by bath-application of CRF. Deficits in synaptic plasticity  have been described with aging that could in part explain cognitive deficits characteristic of advance age70. For example, extracellular recordings demonstrate that LTP from the perforant path to the dentate gyrus declines more rapidly in 28–34 month-old rats and synaptic enhancement correlates with the ability to perform a circular platform task71. Norris and colleagues72 reported increased long-term depression in 20–24 month-old rats following low frequency stimulation of CA3 fibers to CA1. In this same work, low frequency stimulation was able to reverse LTP in aged but not in adult animals. Others have proposed that deficits in synaptic plasticity appear early in brain aging70. LTP is impaired in 7–10 month old rats when stimulating the Schaffer-commissural projections from a region of hippocampal CA3 that projects only to the basal dendrites of CA173. While it is likely that brain areas exhibit differential sensitivity of synaptic plasticity to advancing age, we have provided evidence that a neuropeptide (CRF)-induced enhancement of D2-IPSCs and a presynaptic short-term depression (indicated by paired pulse ratios) in dopamine neurons are not affected by age.We also explored age effects on CRF-induced enhancement of D2 receptor transmission. In male rats, CRF mRNA levels decline with age in the paraventricular nucleus of the hypothalamus, and aging induces greater ACTH and corticosterone responses following a CRF challenge, consistent with hypothalamic CRF deficiencyB receptor signaling, and multiple measures of somatodendritic morphology. The overall conclusion is that dendrodendritic transmission in the SNc is largely preserved with age.In summary, our results indicate a modest reduction of postsynaptic D2 receptor current amplitudes in SNc dopamine neurons from aged males. Most of the other parameters measured were unaffected by age, including IPSC shape, indicators of presynaptic dopamine release, plasticity induced by CRF, GABAMale and female C57BL/6N mice aged 2–8 months (“young”) and 20–25 months (“old”) were used in this study. Mice were provided by the National Institute on Aging (NIA) aged rodent colony, and these mice or their first and second-generation offspring were used for all experiments. All mice were group-housed in ventilated standard cages with ad libitum access to food and water. Animal rooms were maintained on a reversed 12-h light/dark cycle (lights off at 9:00 AM) with the temperature held at 26 °C. Procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Oklahoma Medical Research Foundation (OMRF). Studies were conducted in compliance with ARRIVE guidelines.2O2 and 5% CO2) cutting solution for 45 s. The brains were quickly extracted and placed in a cutting solution containing the following (in mM): 2 KCl, 7 MgCl2, 0.5 CaCl2, 1.2 NaH2PO4, 26 NaHCO3, 11 D-glucose, and 250 sucrose. Horizontal slices (200 µm) containing the ventral midbrain were obtained using a vibrating microtome (Leica VT1200S). Slices were incubated for 30 min at 33–34 °C with carboxygenated artificial cerebrospinal fluid (aCSF) that also contained the NMDA receptor antagonist MK-801 (30 µM). aCSF contained (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 21.4 NaHCO3, and 11.1 D-glucose. Slices were left to stabilize at room temperature for at least 30 min.On the day of the experiment, mice were anesthetized with 2,2,2-tribromoethanol (0.25 g/kg i.p.) and transcardially perfused with ice-cold carboxygenated (95% 74 with wide extracellular waveforms (> 1.1 ms), their characteristic hyperpolarization-activated inward cation current (IH) of > 100 pA75, and ultimately by measuring D2-IPSCs, which have not been reported in other cell types in wild type mice. Recording pipettes (2.0–2.2 MΩ resistance) were constructed from thin wall capillaries (World Precision Instruments) with a PC-10 puller . Whole-cell recordings were obtained using an internal solution containing the following (in mM): 115 K-methylsulfate, 20 NaCl, 1.5 MgCl2, 10 HEPES-K, 2 ATP, 0.4 GTP, and 10 BAPTA, pH 7.35–7.40, 269 –272 mOsm.Slices were then placed in a recording chamber attached to an upright microscope (Nikon Instruments) and maintained at 33–34 °C with aCSF perfused at a rate of approximately 2 ml/min. SNc dopamine neurons were visually identified based on their location in relation to the midline and the medial terminal nucleus of the accessory optic tract. Dopamine neurons from SNc were further identified by their large soma, slow pacemaker firing (< 7 Hz)B receptor currents were also evoked by iontophoresis in the presence of the GABAA receptor blocker picrotoxin (100 µM). GABA  was ejected as a cation with a pulse of 200 nA for 8–12 s. Leakage was prevented by the application of a negative backing current of -5 to -10 nA.Electrical stimulation was performed with a bipolar platinum electrode with tip separation of 305 μm, placed in the slice ~ 100 μm caudal to the cell being recorded. The membrane voltage was held at -55 mV and synaptic currents mediated by dopamine could be detected in most of the cells using 5 pulses (0.5 ms duration) applied at 40 Hz once every 60 s. For stimulus–response curve experiments, intensity of the electrical stimulation was applied in an incremental manner (0.02–0.05–0.1–0.15–0.2–0.25–0.3 mA). For experiments using bath applied dopamine, peak values were determined from continuous recordings, while data from prolonged application , CGP 56999a , DNQX -dione) , and hexamethonium . For bath application experiments, drug concentrations were as follows: dopamine hydrochloride , CRF , and baclofen . GABA, Mg ATP, Na GTP, Na HEPES, K HEPES, and BAPTA were obtained from Sigma-Aldrich. K-methyl sulfate was from MP Biomedicals, LLC.D2 receptor-mediated outward currents were recorded in the presence of the following receptor antagonists : MK-801 , picrotoxin  was added to the internal solution at a final concentration of 0.25% and allowed to diffuse within the cytoplasmic compartment for 10–12 min with the series resistance stable at < 10 MΩ. Once cells were filled, and for a maximal preservation of intact soma and dendrites, the glass pipette was slowly retracted horizontally and then vertically until the pipette was outside the slice. A 5–10 min washout with aCSF was allowed to remove any extracellular presence of biocytin molecules that could interfere with the subsequent staining procedure. The 200 µm brain sections were then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 12 h and washed three times with PBS at room temperature for at least 30 min. All staining was conducted on freely floating sections, and incubations were conducted with gentle agitation at room temperature. Post-fixed tissue sections were blocked with 5% normal donkey serum (NDS) in PBS with 0.2%Tween 20 (PBST) for 1 h. Sections were washed at least three times with PBST and incubated with native Streptavidin protein DyLight 488  in 0.5% fish skin gelatin with PBST for 2 h. The sections were then subjected to two rinses in PBST followed by two washes in PBS before mounted on gelatin-coated slides, and cover slips were applied with ProLong Gold Antifade Mountant . Z-stack images were captured using a Zeiss LSM 710 Confocal microscope  at 20 × magnification with 1 μm step size using Zen software (black edition). Neurites were traced using ImageJ and the Simple Neurite Tracer (SNT) plugin. Neurite tracings were used to calculate dendritic measurements and arborization patterns via Sholl and SNT analysis.Student’s t-tests or one-way ANOVA was performed to analyze differences between two or more groups. Two-way ANOVAs were used to analyze differences in current amplitudes over time between groups, along with Holm-Šídák's multiple comparisons test . Summary data in graphs is presented as mean ± SEM. Significance was set a priori for all experiments at a p value < 0.05.Unpaired


Kallmann syndrome (KS) is a rare genetic disorder manifested by the combination of hypogonadotropic-hypogonadism and olfactory dysfunction. It is usually diagnosed at 14 - 16 years of age due to delayed puberty. However, delays in diagnosis have been reported in a few cases. We presented a 34-year-old man presented with primary infertility as the chief complaint. Physical examination revealed bilateral gynecomastia, Tanner stage 2, and anosmia. Hormonal studies show a hypogonadotropic hypogonadism profile. Genetic testing revealed a normal male karyotype. Abdominal ultrasonography (USG) revealed a small prostate, and testicular USG demonstrated small testicles. Neuroimaging study revealed olfactory bulb agenesis and hypoplasia of the olfactory sulcus. Treatment was done by testosterone replacement therapy, and the patient is now on a regular follow-up. In conclusion, suspected clinical features of KS may guide the diagnosis with comprehensive hormonal and imaging studies. However, the reported patient was diagnosed extremely late. Kallmann syndrome (KS) is a hypogonadotropic-hypogonadism disorder associated with olfactory disorders such as hyposmia or anosmia . It is nPatient information: a 34-year-old Indonesian male presented at Dr. Hasan Sadikin General Hospital Bandung with primary infertility as the chief complaint. He had ever conducted a pregnancy program with his wife. Due to the abnormal hormonal assay results, he planned to do further examinations. He was circumcised. Morning erections were still notable. He did not complain of any disturbances in sexual activity and ejaculation. He did not realize any abnormalities since he was a teenager.He had never smelled anything since he was a child. A family history of anosmia in the family is not found. He admitted that his voice was high-pitched. He had no history of seizures, blurring of vision, color blindness, and hearing loss. He had normal developmental milestones and no intellectual disturbances. He has siblings, and a similar complaint is not reported.Clinical findings: physical examination showed an adult man profile. His body weight, height, and body mass index are 67kg, 160cm, and 26kg/m´, respectively. He had no galactorrhea, but bilateral gynecomastia was noted , LH levels of <0.5 IU/ mL , and FSH levels of 1.83 IU/mL . The prolactin was within the normal limit with a serum level of 4.76 ng/mL . Abdominal ultrasonography revealed a normal bladder with small prostate (prostate . The finprostate . Geneticprostate . UnderdeTherapeutic interventions: the patient was given testosterone replacement therapy to induce virilization. An oral testosterone undecanoate regimen was consumed in a dosage of 40 mg daily for two weeks. Follow-up will be regularly conducted by evaluating the clinical features and hormonal tests.Follow-up and outcome: the patient was evaluated after receiving therapy for two weeks. No significant increment of penile length, testis size, and pubic hair thickness. The size was 2.1 cm, 2.5 ml using an orchidometer, and similar pubic hair thickness, respectively. Treatment was discontinued due to the patient´s economic issue.Patient perspective: the patient was pleased with the care he received throughout therapy. He hoped he would restore fertility.Informed consent: written informed consent was obtained from the patient for participation in our study.We report a case of KS in a man diagnosed late in his age. KS combines an isolated form of hypogonadotropic hypogonadism and a lack of sense of smell. These features are due to defects in olfactory neurons, including olfactory tracts and bulbs, and failed migration of GnRH-secreting neurons to the hypothalamus regions, specifically in the preoptic area. As a result, secondary testicular failure occurs. Genetic mutations are the base of the neurological pathological condition .et al. reported KS patients with other facial anomalies, smooth philtrum, and congenital absence of puncta, which are uncommonly seen in KS [KS can be diagnosed in neonates with micropenis and cryptorchidism. It is mostly diagnosed in adolescents due to the absence of secondary sexual development . The curen in KS . Decreasen in KS . Our casSmall testes and micropenis are commonly found in KS, as in our patient . UltrasoMRI is the first recommended imaging in suspected KS cases. It is superior in visualizing the morphological abnormalities of the olfactory apparatus. Furthermore, it is precise in detecting even very little damage in this area. The accuracy in measuring the volume of the olfactory bulb in various pathological conditions raises its strength . OlfactoTestosterone replacement therapy is the main standard of treatment in KS. The main goal of this patient was to effectively develop fertility. Besides, it also induces the development of secondary sexual characteristics. Immediate treatment is to restore metabolic and bone activity and improve the psychosocial effects in patients with KS . TestostKallmann syndrome is a rare genetic hormonal disorder that is now called olfactogenital dysplasia, characterized by hypogonadotropic-hypogonadism and lack of olfactory function. KS should be suspected clinically in patients with one or both of these features because the late diagnosis of KS will impact the outcome after treatment. Late diagnosis is demonstrated in this case. The prognosis of patients with KS diagnosed early is better than late.


Fibrous materials have unique applications in drug release and biomedical fields. This study reports on the preparation of porous silica nanofibers, using organic nanofibers as templates, and their use for drug release. Different from the commonly used electrospinning method, the organic nanofibers are produced via a self-assembly approach between melamine and benzene-1,3,5-tricarboxylic acid. Silica is then coated on the organic nanofibers via homogenization in a silica sol, a freeze-drying process, and then a sol–gel process. In order to regulate the surface area and mesopore volume of silica nanofibers, cetyltrimethyl ammonium bromide at different concentrations is used as template in the sol–gel process. With the removal of organic nanofibers and the surfactant by calcination, porous silica nanofibers are generated and then assessed as a scaffold for controlled drug release with ketoprofen as a model drug. Poly  is coated on the silica nanofibers to achieve slow burst release and prolonged cumulative release of 25 days. This study demonstrates an effective method of preparing hollow silica nanofibers and the use of such nanofibers for long-term release with high drug loading. Over the years, researchers have been exploring how to achieve a safe and desired drug delivery system. A range of natural and synthetic materials have been widely employed for drug delivery systems, such as biodegradable poly  (PLGA) , biocompAmong the various materials investigated, silica is regarded as “Generally Recognized As Safe (GRAS)” by the Food and Drug Administration (FDA) of the USA. Porous silica materials have therefore been widely used for drug delivery and biomedical applications, due to the further capabilities of adjusting their pore size/shape and pore volume and modifying their surface functionality ,11. VariSelf-assembly has been widely recognized as a versatile approach to fabricate functional and porous materials . The assMotivated by the formation of nanofibers under mild conditions via a self-assembly process ,34,35, oHerein, we report on the preparation of organic nanofibers, which are further used as a template to produce silica nanofibers for tuneable drug release. Melamine and benzene-1,3,5-tricarboxylic acid (BTC) were chosen as the building blocks to construct the melamine-BTC (M-BTC) nanofibers. Silica was then coated on the M-BTC nanofibers via homogenization, freeze-drying, and a sol–gel process. Subsequently, the M-BTC nanofibers were removed via calcination. During the sol–gel process, cetyltrimethylammonium bromide (CTAB) micelles served as a template to enhance the surface area and pore structure of the silica. This facilitated the uploading and release of a model drug, ketoprofen (KPF). To further tune the release behavior, PLGA was coated on the silica nanofibers to act as a release control barrier. This study developed an easy scale-up method for the preparation of organic and silica nanofibers. High drug loading into the silica nanofibers was achieved, which could act as a drug reservoir. A prolonged drug release profile with reduced burst release was then demonstrated.Tert-butanol was provided by Merck. Acetone  and ethanol were supplied by Fisher Scientific. Ketoprofen (KPF) was purchased from Fluorochem Ltd. All chemicals were analytical grade and used without further treatment.Melamine (99%) was purchased from Alfa Aesar. Benzene-1,3,5-tricarboxylic acid , tetraethylorthosilicate , oxalic acid (98%), as well as cetyltrimethylammonium  were bought from Sigma-Aldrich. Poly  (PLGA) RG 503 (M.W. 24000) was purchased from Boehringer Ingelheim. BTC (0.67 g) was dissolved in ethanol (50 mL) at 60 °C. Melamine (0.42 g) was added into deionized water (50 mL) with continuous stirring at 60 °C, until a clear solution was formed. These two solutions were combined together, and then 100 mL deionized water was added. After that, the mixture was stirred overnight at 90 °C and was cooled down to room temperature and then filtered out. Finally, the solid was frozen in liquid nitrogen and then freeze dried for 48 h using a freeze dryer .tert-butanol mixture . Oxalic acid  was then added with continuous stirring for 4 h at room temperature to generate an homogeneous silica sol (4 w/v%) [w/v%) and the mixture was stirred for 3 h at room temperature. The silica sol concentration was also varied (0.8 w/v%) to change the coating condition. M-BTC nanofibers (25 mg) were added into that solution. The mixture was homogenized for 8 min at 12,000 rpm . After that, the mixture was frozen in liquid nitrogen and then freeze dried for 48 h. The freeze-dried materials were further treated in an oven for 24 h at 100 °C to complete the sol–gel process. The calcination process was carried out in a furnace  with the programmed procedure: heating at 2 °C/min to 600 °C, holding at 600 °C for 300 min, then cooling down at a rate 5 °C/min.TEOS (4.0 g) was added into a water/(4 w/v%) . A diffeKPF was dissolved in ethanol at the concentrations of 80 mg/mL and 20 mg/mL, respectively. Silica nanofibers (50 mg) were added in a 5 mL KPF solution at room temperature overnight (about 15 h). The silica nanofibers were then collected by filtration and placed in a vacuum oven at room temperature to remove all the solvent. The drug loading was calculated based on thermogravimetric analysis (TGA).PLGA was dissolved in acetone at different concentrations . KPF-loaded silica nanofibers were soaked in the PLGA solutions for different times  and then taken out to be air dried in a fume cupboard. KPF was also dissolved in some PLGA solutions in order to prevent or reduce the leakage of KPF from silica nanofibers during the soaking process. KPF in PLGA coating could also provide a different release profile compared to the release from the silica nanofibers.20 mL phosphate buffer solution  was pre-heated in water bath at 37 °C for 10 min. The KPF@silica nanofibers were then added to the PBS at 37 °C with stirring. A total of 100 µL PBS was withdrawn at different time intervals and then analyzed by UV-Vis analysis. After analysis, the PBS solution was placed back into the original release medium in order to mitigate the effect of sampling.2 sorption analysis was carried out using a Micrometric 3-Flex 3500 Gas Sorption Analyzer. The drug loading and PLGA coating on silica nanofibers were determined by means of thermogravimetric analysis . The concentration of KPF in PBF solutions during the release tests was determined by UV-vis spectrophotometry .The morphology of organic and silica nanofibers was examined by scanning electron microscopy (Hitachi S4800 SEM). Silica nanofibers were also imaged using a JEOL 2100+ transmission electron microscope (TEM) operating at 200 KV. Fourier transform infrared (FTIR) spectra were collected using a Vertex 70 FTIR Spectrometer. Powder X-ray diffraction (PXRD) patterns were collected on a Bruker-AXS D8 advanced diffractometer with CuK radiation source. N−1 and the C=O stretching at 1690 cm−1 (−1 (indicative of C-N stretching) suggests that the covalent amide groups have not been formed [The FTIR spectrum of M-BTC nanofibers shows the N-H stretching approximately at 3380 cm690 cm−1 A. A lackTo produce silica nanofibers, a silica sol was firstly prepared. The M-BTC nanofibers were added and homogenized to form a stable nanofiber suspension which was then frozen in liquid nitrogen and freeze dried. To ensure a silica coating on the nanofibers instead of large pieces of silica between the fibers, the silica sol with low TEOS concentration was used. The sol–gel process at room temperature was very slow. Therefore, after freeze-drying, the materials were heated in an oven at 100 °C for 24 h to complete the sol–gel process and solidify the materials. During this preparation, CTAB of different concentrations were included in the silica sol to induce surfactant templating in the silica materials . Both th−1 and 900 cm−1, indicating the presence of Si-O-Si vibrations and Si-OH stretching, respectively . 0 and higher) indicates the presence of macropores, which may have resulted from the voids between nanofibers and the hollow nature of the nanofibers. With the induction of CTAB as a template, a type IV isotherm with an H3 hysteresis begins to appear in the range of 0.45–1.0 P/P0 [As the intended application was to upload drug molecules for controlled release, the surface area and porosity of the silica nanofibers were assessed by N1.0 P/P0 . The H3 As shown in In order to demonstrate the potential of drug uploading and prolonged release, the silica nanofibers prepared with 1.4 wt% have been selected for further assessment. This is because of their high pore volume and wide pore size distribution, which, in principle, can lead to high drug loading and prolonged constant release.The model drug KPF was uploaded into silica nanofibers by a solution soaking and solvent evaporation method. KPF loading on silica fibers was calculated via the TGA results. The initial drug test showed a fast burst release of KPF in a short period. To address this issue, it was proposed to coat the silica nanofibers with a biodegradable and biocompatible polymer PLGA. To do this, the silica nanofibers were soaked in PLGA solution (with control on soaking time and PLGA concentration) and then taken out to allow the solvent to be evaporated, forming a PLGA coating. The mass ratio of the PLGA coating to the silica nanofibers was calculated based on TGA analysis. w/v%. The concentration of KPF in PBS was determined by the UV-vis plate reader. As shown in The tests on KPF released from silica nanofibers (KPF loading 0.7 g/g) were carried out in PBF at 37 °C. In order to minimize the release of KPF during the PLGA coating process, KPF was dissolved in the PLGA solutions at the concentration of 0.7 It was found that the PLGA coating time could also affect the release behavior of KPF the from coated silica nanofibers. The results from Overall, with the use of these hollow silica nanofibers, a very high drug loading may be easily achieved. A coating of functional biopolymers on the silica nanofibers can considerably reduce the undesirable burst release. By varying the type and molecular weight of the biopolymer and the thickness/porosity of the coating, it is viable to realize a prolonged and controlled drug release system. Furthermore, these silica nanofibers can be processed to form three-dimensional networks to act as a scaffold and drug reservoir for potential tissue engineering applications ,22,23.In this study, we developed a new scalable templating method to prepare silica nanofibers with a hollow internal structure. Our method utilized the organic melamine-BTC nanofibers as templates which were formed by a simple self-assembly process under mild conditions. This preparation was solution based and could be readily scaled up. The organic nanofibers were dispersed in silica sol by homogenization. A freeze-drying and sol–gel process was subsequently employed to form silica coating on the melamine-BTC nanofibers. A calcination process was then utilized to generate hollow silica nanofibers. CTAB of different concentrations was dissolved in the silica sol in order to improve the surface area and mesoporosity of the silica nanofiber. These silica nanofibers were shown to be effective scaffolds for drug release, where a model drug KPF was uploaded with a high loading of 3.8 g/g. Importantly, a PLGA coating was formed on the KPF-loaded silica nanofibers to achieve a smaller burst release and prolonged cumulative release. The hollow silica nanofibers with mesoporosity make it easier to achieve very high drug loading and sustainable release. Forming a polymer coating adds another layer of control for prolonging drug release. This method may be extended by uploading different types of drugs, forming coatings with other desirable biopolymer and processing to produce 3D scaffolds for advanced biomedical applications.


Antimicrobial resistance (AMR) is one of the most pressing global health challenges currently. Recognised as a complex policy issue, efforts to mitigate AMR transcend national boundaries and require global coordination.Some parallels have been drawn with other complex problems such as climate change and tobacco control yet few analyses have taken a comparative and historical approach to draw lessons for AMR policy.Practical suggestions are made for improved AMR governance globally, working on the basis of shared goals but reflecting needs and priorities at a national level.Antimicrobial resistance (AMR) is a major threat to global public health.3–5Tackling AMR requires international action across multiple sectors—including human healthcare, agriculture and the environment. Such efforts include a landmark 2015 WHO Global Action Plan on AMR, followed in 2016 by a historic United Nations (UN) Declaration on AMR and establishment of the Interagency Coordination Group on AMR. The One Health Global Leaders Group (GLG) on AMR was founded in 2020 to provide leadership and maintain political momentum on the issue. The GLG is supported by the Tripartite Joint Secretariat on AMR—a shared effort to address the One Health dimensions of AMR among the UN Food and Agriculture Organisation, World Organisation for Animal Health, WHO, and more recently, UN Environment Programme.Lessons can be learnt from past experience. A comparative assessment of how AMR evolved as a policy problem alongside a similarly complex problem—climate change—and a relative success—tobacco control—can provide insight into how to better design future international AMR efforts and avoid problems encountered in other areas. Decision-makers understandably want evidence of the future harms and costs of inaction. Evidence review and consolidation has long been a prerequisite for policy-making .There have been increasing calls for the establishment of an ‘Intergovernmental Panel on Climate Change (IPCC) for AMR’.7The IPCC was designed with consensus at its core, requiring all parties to approve its published reports. This helps to ensure that IPCC outputs are widely accepted and enable policy-makers to take joint action.The system has in practice resulted in a conservative bias in the IPCC’s official reports. New or comparatively untested evidence is less likely to be included. Steps may be taken to ensure that emerging insights or alternative views are not excluded, such as publishing submissions that do not receive consensus endorsements and broadening the range of contributors.Appropriate and adequate surveillance, with good geographical coverage that spans One Health dimensions, is another powerful way to reduce uncertainty for AMR decision makers. The ability to capture and model key variables underpins much of the IPCC’s work. So too the capacity to obtain reliable data on indicators such as antibiotic consumption and resistance is prerequisite for effective international decision making for AMR.At an international level, agreement on common, evidence-based goals is critical to successful decision making. Collective action problems, such as climate change or AMR, mean that one country’s ability to tackle the issue is affected by other countries’ actions.8Access to antibiotics is also unequal. High-income countries are promoting policy measures to curb antibiotic usage, while low-income and middle-income countries are negotiating prices and availability.Climate change and tobacco control initiatives have faced difficulties in maintaining a coordinated global approach, largely due to lack of agreement on how individual country capabilities and priorities should be reflected.10Both for climate change and tobacco control, common goals may be best met through individual country targets and actions based on feasibility determined at the national level.A global approach for bringing together evidence and analysis on AMR could also draw lessons from the IPCC’s experiences regarding the type of data collected and perspectives included. Climate change was originally characterised primarily as a technical issue, stemming from concerns voiced by climate scientists beginning in the 1980s.AMR has experienced a similar trajectory. Initially treated as a technical issue, its evolution into being viewed as a social and political challenge will be important to consider in future efforts to bring together evidence on its risks and potential harms—and using it for action and investment.Social change is critical to successful policy implementation yet often considered too late in the policy-making process.Social drivers such as cooperation and conformity need to be recognised. People are more likely to do their part if they know others are doing theirs, and if their own behaviour is accountable or observable to others. Smoking bans in public places became politically attractive in part because of emerging evidence and public health campaigns highlighting the negative health impacts of passive smoking. This nurtured an appeal not to harm others, and equally a belief that others do not have the right to harm us, overriding individual objections of paternalism.The challenges of reaching a point of self-reinforcing social norms in relation to antibiotic use may be greater than for smoking which tends to be more visibleNational governments could also start valuing antimicrobials as critical infrastructure for their health systems. This approach could better integrate how to address AMR in a broader health systems context, and increase investments to ensure their availability.Tobacco control and climate change illustrate that a high level of awareness and acceptance of the available evidence is needed to effect social change and successful policy implementation.A coordinated international effort is warranted to address AMR. The GLG, UN High-Level Interactive Dialogue and recent G7 summit, among other multilateral fora, provide important opportunities to agree on how this can be done.International action requires countries to agree on shared, evidence-based goals with clear accountability and impetus to collaborate. Action can be based on shared goals but with different needs and priorities driving the course those actions take at a national level. Further consideration of how this may be operationalised to include and go beyond previously advocated models of target setting will be important.14Differences stemming from an uneven global picture need to be bridged, which may involve financial and technical assistance. Agreement is paramount; UN agencies are hampered in providing such assistance without unanimous support.There is a risk of making global architecture too complex. The GLG could serve as a platform to make supporting organisations more accountable; the GLG can advocate for Member States to more consistently and coherently monitor their own efforts and progress, and mobilise financial support. The AMR Multi Partner Trust Fund offers a financial mechanism to promote coordination within the GLG Secretariat, and provide greater support for countries in the design and implementation of One Health National Action Plans and Quadripartite Workplans. Nevertheless, the donor base consisting of the UK, Netherlands and Sweden does not represent a global effort.There is much to do. International collective action, not just advocacy, is required to avoid the catastrophic consequences that could arise from failing to address AMR. The current pandemic serves as a sobering catalyst for change.