Electrospinning is a technique that uses polymer solutions and strong electric fields to produce nano-sized fibers that have wide-ranging applications. We present here an overview of the use of electrospinning to incorporate biological products into nanofibers, including microorganisms, cells, proteins, and nucleic acids. Although the conditions used during electrospinning limit the already problematic viability/stability of such biological products, their effective incorporation into nanofibers has been shown to be feasible. Synthetic polymers have been more frequently applied to make nanofibers than natural polymers. Polymer blends are commonly used to achieve favorable physical properties of nanofibers. The majority of nanofibers that contain biological product have been designed for therapeutic applications. The incorporation of these biological products into nanofibers can promote their stability or viability, and also allow their delivery to a desired tissue or organ. Other applications include plant protection in agriculture, fermentation in the food industry, biocatalytic environmental remediation, and biosensing. Live cells that have been incorporated into nanofibers include bacteria and fungi. Nanofibers have served as scaffolds for stem cells seeded on a surface, to enable their delivery and application in tissue regeneration and wound healing. Viruses incorporated into nanofibers have been used in gene delivery, as well as in therapies against bacterial infections and cancers. Proteins  and nucleic acids (DNA and RNA) have been incorporated into nanofibers, mainly to treat diseases and enhance their stability. To summarize, incorporation of biological products into nanofibers has numerous advantages, such as providing protection and facilitating controlled delivery from a solid form with a large surface area. Future studies should address the challenge of transferring nanofibers with biological products into practical and industrial use. Nanotechnology includes the use of nanomaterials and represents one of the newest approaches in medicine, and also in science in general. Nanomaterials can have useful physicochemical properties, can be biocompatible and non-toxic, and can sense and respond to environmental stimuli . NanofibNanofibers can be produced from various polymeric materials, which can be natural, synthetic, or a combination of both. Natural polymers have better biocompatibility and lower immunogenicity. On the other hand, synthetic polymers offer greater flexibility in their synthesis and modification .Electrospinning techniques can be divided into six categories: basic electrospinning; blend electrospinning; coaxial electrospinning; emulsion electrospinning; melt electrospinning; and gas jet electrospinning . Basic eThis review will provide a brief overview of nanofibers with incorporated live cells, viruses, proteins and nucleic acids. Polymers that are used to make these nanofibers will be reviewed, as well as their applications . For othThe biological products referred to here include live cells and viruses, as well as their components, such as proteins and nucleic acids. Biological products are mainly intended for use in medicine , but can also be used in agriculture and the food industry, and for environmental protection, biosensing, and biocatalysis. They have low stability and degrade rapidly when administered to the human host. Their low viability or stability can be overcome by the use of electrospun nanofibers. Incorporation of biological products into nanofibers can protect them from environmental conditions, and allow their controlled release .1 However, therapies with biopharmaceuticals also have their drawbacks, such as limited cell viability, short half-life after administration , solution parameters , and environmental parameters .Lactobacillus plantarum into polyethylene oxide (PEO) solutions increases the conductivity due to the extracellular proteins and ions introduced with the probiotics -linked β- insulin , and forchnology .Lb. plantarum  are elongated polymers that have fiber diameters from hundreds of nanometers to a few micrometers. Together with the potential health benefits of consuming fish, these characteristics make fish sarcoplasmic proteins an attractive material for electrospinning of nanofibers. Additionally, they have been shown to inhibit the enzyme dipeptidyl peptidase-4, which is responsible for glucose metabolism and is linked to type 2 diabetes  (PCLEEP) and collagen hybrid nanofibers have been used for effective sustained delivery of neurotrophin-3 and micro-RNA (miRNA)-222 .Elastin is also present in the extracellular matrix, and maintains the elasticity of the connective tissue in the human body. Like collagen, it is of interest in medicinal applications, such as drug delivery and tissue engineering. Tropoelastin is a soluble precursor of elastin, and it is composed of hydrophobic and hydrophilic crosslinking domains. Elastin−like polypeptides are biopolymers that are composed of amino-acid sequences found in the hydrophobic domain of tropoelastin . Their tBombyx mori): sericin , and fibroin . The combination of silk fibroin and PEO electrospun nanofibers has served as a delivery system for epidermal growth factor (EGF). The ultra-fine porous structure of the nanofibers allowed oxygen permeation, promoted drainage of fluids, and inhibited exogenous microorganism invasion  (PHB) is natural polyester that belongs to the class of polyhydroxyalkanoates and is produced by a wide range of microorganisms . PHB is . LEB 18 .L-lactic acid and poly D-lactic acid). All of these have been approved by the US Food and Drug Administration as drug delivery systems or as scaffolds for tissue engineering , and poly lactic acid (PLA)  (PEG) is also a polymer of ethylene oxide, and differs from PEO only in its molecular weight. The combination of these polymers with other synthetic and natural polymers can enhance their mechanical and physical properties .Polyvinyl alcohol is another water-soluble synthetic polymer. Due to its high hydrophilicity and good solubility in water, PVA has limited applicability for electrospinning of nanofibers. To improve its mechanical properties and water resistance, PVA has to be modified by either chemical or physical crosslinking .N-vinylpyrrolidone monomers by free-radical polymerization. PVP is very hygroscopic and can absorb up to 40% water by weight  have also been used for electrospinning of nanofibers. The water solubility of these polymers is pH dependent, which allows pH-dependent drug release . The bigBacteria that have been incorporated into electrospun nanofibers usually belong to the group of probiotics. Probiotics are live microorganisms that can confer health benefits on the host when administered in adequate amounts. Their low viability and high sensitivity to environmental factors represent a hurdle for their wider application. For this reason, there is a need for new probiotic delivery systems . DiffereLactobacillus is the most common genus of probiotic bacteria that has been incorporated into nanofibers. Lactobacillus acidophilus is present in the normal vaginal flora, and it can prevent colonization and proliferation of pathogenic microorganisms. Successful incorporation of Lb. acidophilus in PVA and PVP nanofibers can enhance the viability of this bacterium for up to 90 days, and has been suggested as an easy-to-use dosage form against bacterial vaginosis , oil-palm trunks, and oil-palm fronds  , together with Lactococcus lactis were compared for their incorporation into PEO nanofibers. All of these species remained viable after their incorporation, and their viability correlated with cell hydrophobicity  . Like Lbal tract . As wellreatment . The damronments . Recentlhobicity .Bifidobacterium lactis Bb12 and Streptococcus thermophilus (TH-4). Together with Lb. paracasei 431, these bacteria remained viable after 1 week at room temperature . The oxidative enzymes of Gluconobacter oxydans allow for the oxidation of glucose to gluconic acid, and the consequent reduction of hexacyanoferrate (III). Formed hexacyanoferrate (II) is re-oxidized at an electrode and this reaction is monitored amperometrically  into PVA nanofibers increased their survival on soybean seeds, compared to the un-incorporated bacterial cultures, over the course of 30 days. The decreases in viability were ∼2 log units and ∼4 log units for incorporated and un-incorporated Pantoea agglomerans ISIB55, respectively; and ∼1.5 log units and ∼4 log units for incorporated and un-incorporated Burkholderia caribensis ISIB40. Additionally, P. agglomerans ISIB55 increased germination rates and length and dry weight of the roots, and B. caribensis ISIB40 increased the leaf numbers and dry weight of the shoots  and its biomass was used in the electrospinning. The application of PHB–hydroxyvalerate and PCL nanofibers that contained Spirulina sp. LEB 18 biomass as the bioactive component was suggested for the field of tissue regeneration. Spirulina sp. LEB 18 improved the physical characteristics of the nanofibers, and provided the nanofiber scaffold with reduced healing times and stimulated cell growth   .Stem cells can differentiate into other cell types and thus build any tissue in the body. These characteristics mean that they can have a variety of therapeutic benefits, and can be used for different purposes. However, stem cells have limited viability and are difficult to proliferate, which impedes their wider use for a variety of potential therapeutic benefits. Combinations of stem cells and electrospun nanofibers have two main advantages. First, the nanofibers can serve as a favorable scaffold for maintenance of the stem cells through alteration of the chemical properties of the nanofibers in such a way as to improve their interactions with the stem cells. Secondly, nanofibers can serve as a system for delivery of stem cells into specific tissues or organs for tissue engineering and wound healing. PLGA is the most common polymer that has been used for the production of nanofibers for stem cells. PLGA is a biocompatible polymer that supports the growth of stem cells as well as their multi-lineage differentiation . Stem-ceL-lactic acid nanofibers have been shown to be compatible with MSCs, as these cells showed high cell viability and high rates of proliferation, while their normal morphological characteristics were maintained (L-lactic acid)–co–polycaprolactone nanofiber scaffold, where they differentiated into keratinocytes and expressed keratin 10, filaggrin, and involucrin –co–polycaprolactone as the shell, and bovine serum albumin together with nerve growth factor (NGF) as the core, effectively promoted nerve regeneration in rats (in vivo (<5 h), while its encapsulation into PCLEEP nanofibers provided its sustained release over 3 months. This encapsulated NGF also retained its bioactivity, as seen by stimulation of PC12 cell differentiation into neurons (L-lactic acid-co-caprolactone)/silk fibroin blend nanofibers that contained the combination of NGF and monosialoganglioside promoted increased cell differentiation and proliferation compared to nanofibers with only NGF incorporated  and chitosan enabled full recovery and regeneration of the cellular epithelium after 7 days of treatment of dogs with oral mucositis . EGF is DL-lactide) and methyl cellulose nanofibers using emulsion electrospinning protected the structural integrity and bioactivity of lysozyme. Furthermore, its release was modified such that it showed sustained release over 2 weeks  compared to the soluble enzyme  are charged compounds with large molecular weights that have been recognized as therapeutic agents for different diseases, such as neurodegenerative diseases, cancers, and immunological disorders, among others. Although they have great therapeutic potential, their delivery still represents a challenge due to their instability in biological environments, and their immunogenicity and toxicity .Nanofibers can be used for delivery of plasmid DNA, miRNAs, and small-interfering (si)RNAs . PlasmidDL-lactide)-poly (ethylene glycol) without and with PEG or PEI. Sustained release of DNA was obtained between 6 days and 25 days, and efficient transfection of NIH3T3 cells was achieved , without and with PEI, while the sheath was made of poly -poly (ethylene glycol) nanofibers. The plasmids were released over 4 weeks, and successfully transfected into endothelial and smooth muscle cells. This resulted in the formation of a vascular network and rapid generation of mature blood vessels  can regulate expression of certain genes, such as those encoding NTF3, GAPDH, PDGFR-α, Sox6, Hes5, FoxJ3, and ZFP238, thus improving the health of the patients. miRNA-222 is a non-coding RNA that is involved in local protein synthesis and enhances axonal re-growth. The previously mentioned nanofibers that were composed of PCLEEP and collagen were used to incorporate micellar nanoparticles that contained miRNA-222. A total of 27.1% miRNA was released within the first month, followed by 2 months of steady release. The delivery of the miRNA supported axon regeneration in mice for 10 days post-implantation . miRNA-2Small-interfering RNAs are double-stranded RNA molecules, and like miRNAs, they can regulate gene expression. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is involved in glycolysis, and is often used as a model (‘housekeeping’) gene. GAPDH siRNA was encapsulated in PCL and PCL/PEG nanofibers and successfully transfected into HEK 293 cells. This siRNA was released from the nanofibers over at least 28 days, with high GAPDH silencing efficiency seen (61–81%) . SimilarElectrospinning is an established method for the formation of nanofibers; i.e. nanomaterials that appear under the microscope as a polymeric nanofiber mesh, and that macroscopically appear as a thin plastic film. Nanofibers are formed from a polymer solution, which allows straightforward incorporation of chemicals by simply mixing them into the solution. Biological products, such as live cells, proteins, and nucleic acid, are sensitive to pH, temperature, and enzymatic degradation. Therefore, their incorporation into nanofibers using electrospinning might appear counterintuitive, particularly due to the high voltage that is used during the electrospinning process. However, although still relatively rare, a considerable number of studies from the literature are reviewed here, and demonstrate that incorporation of live cells, viruses, functional proteins, and nucleic acids into nanofibers is feasible and advantageous. The rationale behind the incorporation of biological products is their protection from environmental influences (i.e. to improve their viability or stability), and their immobilization, drying and subsequent solidification for the formation of a flexible and effective form of delivery. The majority of nanofibers that contain biological products are intended as an alternative delivery vehicle for use in medical therapies, whereby they provide protection and a growth scaffold, and actively improve delivery (e.g. transfection of nucleic acids or growth of stem cells), with the added advantage of a large surface-to-volume ratio.Both natural and synthetic polymers have been used to make nanofibers. PVA, PEO, cellulose and chitosan polymers are most frequently used for incorporation of bacteria and fungi. Viruses have usually been incorporated into PCL nanofibers, and stem cells grown on PLGA nanofibers. These materials differ in their hydrophobicity and hydrophilicity, and under physiological conditions this can allow sustained or rapid release, respectively. Alternatively, pH responsive polymers (e.g. Eudragit) have been suggested for controlled release under the conditions of the gastrointestinal tract.To summarize, the literature reviewed here clearly demonstrates that incorporation of biological products into electrospun nanofibers is feasible and functional. However, most studies have focused on proof-of-principle experiments and the incorporation of model biological products. More studies are needed to consolidate the industrial applicability of electrospinning of biological products, particularly in the field of medical therapies.SS wrote the draft of the manuscript. AB wrote and revised the manuscript. SS and AB approved the submitted version.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.


Dopamine loss beyond the mesostriatal system might be relevant in pathogenic mechanisms and some clinical manifestations in PD. The primate thalamus is densely and heterogeneously innervated with dopaminergic axons, most of which express the dopamine transporter, as does the nigrostriatal system. We hypothesized that dopamine depletion may be present in the thalamus of the parkinsonian brain and set out to ascertain possible regional differences.The toxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine was administered to adult macaque monkeys using a slow intoxication protocol. The treated macaques were classified into 2 groups according to their motor status: nonsymptomatic and parkinsonian. Dopamine innervation was studied with immunohistochemistry for the dopamine transporter. Topographic maps of the dopamine transporter‐immunoreactive axon distribution were generated and the total length and length density of these axons stereologically estimated using a 3‐dimensional fractionator.Parkinsonian macaques exhibited lower dopamine transporter‐immunoreactive axon length density than controls in mediodorsal and centromedian‐parafascicular nuclei. Dopamine denervation in the mediodorsal nucleus was already noticeable in nonsymptomatic macaques and was even greater in parkinsonian macaques. Reticular nucleus dopamine transporter‐immunoreactive axon length density presented an inverse pattern, increasing progressively to the maximum density seen in parkinsonian macaques. No changes were observed in ventral thalamic nuclei. Dopamine transporter‐immunoreactive axon maps supported the quantitative findings.Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.Changes in the dopamine innervation of various thalamic nuclei are heterogeneous and start in the premotor parkinsonian stage. These changes may be involved in some poorly understood nonmotor manifestations of PD. © 2019 The Authors.  The primate thalamus is densely innervated by dopaminergic axons.Dopamine loss in PD has been reported in regions beyond the mesostriatal system, including the globus pallidus, subthalamic nucleus, and motor cortex.The thalamus is the only target of basal ganglia outflow to the cortex,Here, we explored the distribution and density of dopamine transporter‐immunoreactive (DAT‐ir) axons in the thalamus of parkinsonian macaques using a model of slow progressive dopamine depletion. DAT is the relevant dopamine marker to be analyzed in the thalamus of parkinsonian macaques: not only is it specific and expressed by the majority of dopaminergic axons in nonmidline nuclei, but the axons expressing DAT are most vulnerable in PD and MPTP‐induced parkinsonism.Macaca fascicularis previously studied to stage the degree and extent of the nigrostriatal lesion.The present study used brain tissue from our macaque monkey brain bank. We used tissue from 20 brains of young l‐dopa or dopamine agonists during the experiment. Animals were evaluated by the research team after each MPTP dose and while the parkinsonian syndrome was progressing until complete stabilization. Motor signs were assessed through the validated Kurlan motor score .Sixteen macaques aged 4–7 years were treated every 2 weeks with intravenous doses of MPTP (0.5 mg/kg).recovered.Macaques that exhibited evident parkinsonian signs after the last MPTP injection and that remained affected thereafter until sacrifice were classified as parkinsonian . Macaques that maintained normal motor features after the last MPTP dose were considered nonsymptomatic (n = 8); see Supporting Table See Supporting Appendix High‐resolution maps of DAT‐ir axons were generated through a mosaic of 20× magnification pictures taken on a Zeiss Axioskop microscope  equipped with a digital camera , using Neurolucida software . HighPass and Kodalith filters of this software were used to enhance axon‐background contrast  using the spaceball stereological approach.U test). The correlation calculation of DAT‐ir axon length density with earlier data from the same experimental subjectsP < 0.05. Data from 1 control macaque (M2) and data for the R nucleus from 1 parkinsonian macaque (M17) were excluded because of insufficient tissue sections for optimal stereological study. Outliers were included in the statistical analysis. Mean and standard error the mean are used throughout the text, and median and interquartile range are used in the box‐plot graphic representations.Statistics were performed with SPSS software  and R Studio. Because the data from our experimental groups (n = 4–8) were not normally distributed, all comparisons were made using nonparametric statistical tests  followed by the ventral nuclei (0.76 ± 0.31 m of DAT‐ir axons/mm3). CnMd‐Pf and R had low densities of DAT‐ir axons ; see Figure DAT‐ir axon length density varied in the different nuclei. The highest length density was present in MD  and by 48% in parkinsonian macaques . DAT‐ir axon length density in CnMd‐Pf was 43% lower in parkinsonian macaques compared with controls (P = 0.04) and 38% lower in parkinsonian macaques compared with nonsymptomatic macaques  and A10 ; see Figure R2 = 0.26; P = 0.01) and A10 ; see Figure Correlation analyses were performed between DAT‐ir axon length density in the thalamic nuclei, number of mesencephalic dopaminergic neurons, DAT innervation in the striatum, and motor status of the same macaques.This study provides the first demonstration of widespread changes in the thalamic dopaminergic system in slowly and progressively induced MPTP‐parkinsonism in macaques. We found a decrease in thalamic dopaminergic innervation in MD and CnMd‐Pf that was correlated with the loss of mesencephalic dopaminergic TH+ neurons. We also have shown an unexpected increase in dopaminergic axons in the R nucleus. Changes occurred in all MPTP‐treated macaques, whether nonsymptomatic or symptomatic, but were more overt and intense in the symptomatic parkinsonian macaques.The stereological method used here allows rigorous quantitative length and volume analyses that do not depend on presumptions about the shape, size, or randomness of the objects under study.DAT‐ir axon length density declines across MD in nonsymptomatic macaques, and the decline is more pronounced in parkinsonian macaques Fig. A. In addRegarding the thalamic nuclei integrated in the basal ganglia motor circuitry, only the parkinsonian macaques showed decreased DAT‐ir axon length density in CnMd‐Pf. This decline in DAT‐ir axon length density Fig. B correlaThe decrease of DAT‐ir axon length density in MD and CnMd‐Pf could be because of degeneration of DAT‐ir axons or of phenotype modifications in dysfunctional dopaminergic neurons, as previously suggested for the loss of TH and DAT axons in the striatum of PD patients.new branches on DAT‐ir axons of the R nucleus in our MPTP‐treated macaques. This is unlike the changes in dopamine axon morphology reported in the striatum of MPTP‐treated monkeys.The finding of increased DAT‐ir axon length density in R was unexpected. DAT‐ir axon length density in R does not correlate with the number of TH+ neurons,Although changes in the dopamine innervation of the thalamus in the parkinsonian brain have never been reported in as much detail as here, our findings concur with previous data in the literature on MPTP monkeys and humans with PD.Biochemical studies using chromatography in the thalamus of MPTP‐treated macaques have provided inconclusive data.The dopamine depletion in MD, although widespread, predominantly involves dorsal regions of this nucleus Figs. A,B,D–H. The CnMd‐Pf complex has been implicated in PD pathophysiology and suggested as a possible target for functional neurosurgery.Changes in dopamine innervation of R deserve comment. R is an inhibitory nucleus that receives nonreciprocal projections from the cortex and subcortical structures and is reciprocally connected with all thalamic nuclei; each region of R is connected with a set of thalamic nuclei.Full understanding of changes in thalamic dopaminergic transmission in PD and PD models requires additional studies on potential changes in dopamine receptors and dopamine synaptic function, as well as on their impact on the thalamocortical circuits involved.This study gives evidence of well‐defined and specific changes in thalamic dopamine innervation in nonsymptomatic and symptomatic MPTP‐treated macaques. Interestingly, MD and CnMd‐Pf nuclei sustain marked dopamine denervation, whereas R exhibits hyperinnervation. Thus, the present findings reveal yet another nonstriatal site, the thalamus, in which dopamine changes may affect brain function in the presymptomatic and symptomatic phases of parkinsonism. We speculate that changes in thalamic dopamine innervation may contribute to the pathophysiology of some ill‐understood manifestations of the parkinsonian state such as attention and executive deficits and sleep impairment.M.H.G.M.: 1A–C, 2A–C, 3A,B.J.B.: 1A–C, 2C, 3B.M.A.G.C.: 1A–C, 2C, 3B.J.A.O.: 1A,B, 2C, 3B.C.C.: 1A–C, 2C, 3B.1) Research project: A. conception, B. organization, C. execution; 2) statistical analysis: A. design, B. execution, C. review and critique; 3) manuscript: A. writing of the first draft, B. review and critique.M.H.G.M. is an employee of the Autonomous University of Madrid and HM‐Puerta del Sur University Hospital. J.B. is currently funded by grant S2017/BMD‐3700 (NEUROMETAB‐CM) from Comunidad de Madrid co‐financed with the Structural Funds of the European Union, Fundación BBVA and Fundación Tatiana Pérez de Guzmán el Bueno. M.Á.G.C. is an employee of the Neural Systems Laboratory, Department of Health Sciences, Boston University. J.A.O. is funded from the Ministry of Science and Education of Spain, Fundación La Caixa and CIBERNED; has received honorarium for lecturing in meetings organized by BIAL, Zambon, and Boston Scientific in Spain, as member of Insightec's advisory board, and as member of the Adverse Event Committee for the Biogen randomized immunotherapy trial. C.C. is an employee of the Autonomous University of Madrid and is funded by the Chair UAM‐Fundación Tatiana Pérez de Guzmán el Bueno.TABLE S1. Macaque monkey features, number of MPTP injections, parkinsonian scores, and survival after MPTP.TABLE S2. Stereological estimations. Total DAT+ axon length, nucleus volume, and DAT+ axon length density of each macaque and nucleus analyzed.FIG. S1. DAT‐ir axon map generation and stereological method. High‐resolution maps of DAT‐ir axons were generated through a mosaic of 20× magnification pictures (A). HighPass (B) and Kodalith (C) filters were used to enhance axon‐background contrast. Stereological images . The hemispherical probe used as a method of length estimation (D). The gray arrow represents the thickness of the section, the red arrow represents the radius of the hemisphere, and the green arrow, the upper guard area. Axons are depicted in brown (D). Microphotographs of a sampling point (E) from top to bottom. The distance between each image is 1 μm on the Z axis, from the top (1) to bottom (14) of the section. The white circles represent consecutive perimeters of the hemisphere. The red arrowhead  indicates the intersection of a DAT‐ir axon with the hemisphere. The calibration bar applies to all images.FIG. S2 Correlation matrix. Correlation matrix between motor scale, DAT‐ir axon length density in mediodorsal (MD DAT+), centromedian‐parafascicular (CnMd‐Pf DAT+), ventral ,and reticular (R DAT+) nuclei, as well as the dopaminergic innervation of the striatum analyzed by optical density (Str DAT OD) and the numbers of mesencephalic dopaminergic neurons . The color code is on the bar to the right, which gives the correlation scores from ‐1 to 1.Appendix 1. Material and Methods. Brain ProcessingAfter being stable for several weeks in the corresponding motor state, the macaques were deeply anesthetized with intraperitoneal sodium pentobarbital (10 mg/kg). Saline was perfused through the ascending aorta, followed by 4% paraformaldehyde in phosphate buffer (PB) and a series of PB sucrose solutions of increasing concentrations (5%‐10%‐20%). One hemisphere of each brain was stereotaxically blocked in the sagittal plane. Brain blocks were cryoprotected in 30% sucrose for about two weeks under gentle movement at 4° C until sunk. Then, 40 μm parasagittal sections were obtained using a freezing microtome.Appendix 2. Results. Correlation analysesP = 0.006), optical density in the striatum , and number of TH+ neurons in mesencephalic dopaminergic groups A8, A9, and A10 . DAT‐ir axon length density in the CnMd‐Pf complex correlated with motor scale , and DAT optical density in the striatum . In addition, significant correlations were found between DAT‐ir axon length density in CnMd‐Pf and the numbers of TH+ neurons in A9 and A10 , but not with the number of TH+ neurons in A8. In R nucleus, DAT‐ir axon length density did not correlate with motor scale score or DAT optical density in the striatum, but it was correlated with the number of TH+ neurons in group A8 .In MD, DAT‐ir axon length density significantly correlated with motor scale (rho = ‐0.62, Click here for additional data file.


A recent analysis from Stanford University suggested that without any changes in currently available treatment, prevention, and public health approaches, we should expect to have 510,000 deaths from prescription opioids and street heroin from 2016 to 2025 in the US. In a recent review, Mayo Clinic Proceedings (October 2019), Gold and colleagues at Mayo Clinic reviewed the available medications used in opioid use disorders and concluded that in private and community practice adherence is more important as a limiting factor to retention, relapse, and repeat overdose. It is agreed that the primary utilization of known opioid agonists like methadone, buprenorphine and naloxone combinations, while useful as a way of reducing societal harm, is limited by 50% of more discontinuing treatment within 6 months, their diversion, and addiction liability. Opioid agonists may have other unintended consequences, like continuing the down regulation of dopamine systems. While naltrexone would be expected to have opposite effects, adherence is also low even after detoxification and long acting naltrexone injections. Recent studies have shown Naltrexone is beneficial by attenuation of craving via “psychological extinction” and reducing relapse. Buprenorphine is the MAT of choice currently but injectable Naltrexone plus an agent to improve dopaminergic function and tone may renew interest amongst addiction physicians and patients. Understanding this dilemma there is increasing movement to opt for the non-addicting narcotic antagonist Naltrexone. Even with extended injectable option there is still poor compliance. As such, we describe an open label investigation in humans showing improvement of naltrexone compliance and outcomes with dopamine augmentation with the pro- dopamine regulator KB220 (262 days) compared to naltrexone alone (37days). This well studied complex consists of amino-acid neurotransmitter precursors and enkephalinase inhibitor therapy compared to treatment as usual. Consideration of this novel paradigm shift may assist in not only addressing the current opioid epidemic but the broader question of reward deficiency in general. In response to the devastating and unimaginable death toll of hundreds of thousands of people dying from overdose of opioid/ opiate narcotics throwing many communities in economic trouble, the addiction medicine field is in a panic . SpecifiMoreover, the epicenter of the second but worst opioid epidemic driven in-part by Big Pharma (now being fined) with disastrous deaths due to overdose is so overwhelming the total societal cost is reaching an unimaginable amount north of one –trillion. This epidemic has crippled so many communities across America with dismal outcomes in spite of utilization of MAT such as buprenorphine combinations. There is argument that one reason for failure is underutilization, in-part due to high addiction liability. Moreover, simply the idea of treating one narcotic with another narcotic, even with some special properties including partial agonism at Mu receptors, seems counter intuitive .Understanding the nature of addiction liability has led to the increasing utilization of narcotic antagonism. One –major problem is compliance and as such the long-acting naltrexone injectable (e. g. Vivitrol®) has been developed with varying results. One issue is the misbelief that naltrexone molecules actually block opioid craving behavior via direct neurobiological mechanisms. This fallacy has led to false claims of the benefits of narcotic antagonism. We hereby point out that in fact the primary benefit is simply “psychological extinction.” Understanding the psychopharmacological profile mandates the continued search for better treatments including the induction of genetically guided (GARS) precision pro-dopamine regulation and subsequent potential induction of dopamine homeostasis . The latThe history of MAT goes back to the 60’s when Dole & Nyswander  suggesteet al. [It is to be noted that the antagonistic approach is represented by naltrexone. In 1985, the oral version was approved by the FDA. A major advantage is that while naltrexone occupies the opioid type receptors especially mu, it does not produce euphoria or reward. In terms of the pharmacokinetics, the oral version requires daily or three times weekly administration, but patients can relapse simply by stopping the medication for 48 hours. As such the oral version has had only limited success. However, an extended release version of naltrexone has become available which may prevent relapse up to 30 days. While some patients find it worthwhile and convenient to return monthly for an injection rather than to take a daily medication, many do not. In favor of the injectable form sold under the name of Vivitrol® (XR-NTX), McDonald It is noteworthy to know that naltrexone is a relatively weak antagonist of κ- and δ-receptors and a potent μ-receptor antagonist. Dosages of naltrexone that effectively reduce opioid and alcohol consumption also strongly block μ-receptors, but down-regulates meso-limbic dopamine release. While these studies show benefit especially in the short term there is ongoing evidence that the retention and compliance on Vivitrol® is not sufficient to characterize adherence as high ,21 SpeciWe now provide a detailed analysis of a previous hypothesis type article showing some dramatic and clear evidence that by coupling a known highly researched pro-dopamine regulstor, KB220, a complex of amino-acid neurotransmitter precursors and enkephalinase inhibitor therapeutic to long-term methadone addicts rapidly detoxed with naltrexone  improved compliance and outcomes .“rapid detoxification method” is to provide the patient with a pure narcotic antagonist to eliminate by blocking the opiate induced pleasurable effects. However, while the addiction medicine or recovery space embraces this approach, it is rift with poor compliance and still significantly high recidivism rate [et al., [It is noteworthy, that Against Medical Advice (AMA) rate  among hardcore addicts even today approaches as high as 90%. The basic concept of ism rate ,21. One ism rate  is that [et al.,  followinJ.T. Payte MD, PA Clinic, San Antonio, Texas. Inclusion criteria for study entry included both genders with a history of up to 30 years abusing psychoactive chemicals including opioids. Prior to entry each patient was diagnosed as hardcore addicts using the DSM-1V criteria for opioid dependence/heroin. The total number of study participants was 1012 mixed gender divided into two unbalanced groups consisting of Group BC (baseline control) and Group ENTX . Group BC consisted of 1000 patients  and Group ENTX consisted of 12 patients . The average age of the total population was 49 whereby the age range was from 40–70 years. The trial received IRB approval from not only the San Antonio Methadone Clinic but from PATH Medical Foundation (registration #IRB00002334). In addition each study participant signed an IRB approved consent form prior to their entry into the investigation  alone and in combination with the Pro-dopamine regulator KB220 with heavy addicted long-term methadone patients, was accomplished at the In this investigation our rapid detoxification method was consistent with every participant, whereby, each subject (N=1012), was pre-evaluated by first administering an injection of 0.4 to 0.8 mg of naloxone (Narcan®) and if they passed this first test they were subsequently delivered an oral dose of 2.5 mg of naltrexone (Trexan®). Following the naltrexone dose each subject was re-evaluated for withdrawal symptoms over a 90 minute period. Finally, if they passed this second test they were then provided with 50 mg of oral naltrexone. The 1012 patients were given 50 mg of naltrexone at the clinical site daily until the patient relapsed. Of cause following the initial pre-evaluation the 12 patients placed in the ENTX group that were selected, had been maintained on methadone on the average of 18–30 years.The basic formula for this study conducted in the early 90’s primarily consisted of varying amounts of L-phenylalanine (precursor to dopamine synthesis in brain); -Tyrosine (rate limiting molecule for dopamine synthesis), L-Tryptophane (precursor for serotonin synthesis in brain); chromium salt (increases gut to brain tryptophane for serotonin synthesis in brain); L-Glutamine (precursor for GABA synthesis in brain), D-phenylalanine  and pyridoxine-5 –phosphate . The research code name is KB220/KB220Z and to date there are at least 41 published studies including pre-clinical and human  and group ENTX (N=12). We also used the Satterthwaite approximation which is away to account for two different sample variations to correct for unequal variances. We used the following formula:Given the complexity of monitoring 1,012 patients daily for approximately 365 days (study truncated), the staff directed by Dr. J.T. Payte (now deceased), albeit a few failures, carefully documented the results of the investigation. As displayed in The coupling of amino-acid therapy and enkephalinase inhibition, while blocking the delta-receptors with a narcotic antagonist even if weak, may be a quite promising novel method to not only induce rapid detox in chronic methadone patients but as a frontline modality to treat OUD. This may also have important ramifications in the treatment of both opiate and alcohol-dependent individuals; enhanced compliance with Vivitrol® (as an extended release injectable) and especially as a relapse prevention tool.It may also be interesting to further test this hypothesis both in a more substantial cohort and with the sublingual combination of the partial opiate mu receptor agonist buprenorphine. In terms of buprenorphine and dopaminergic function, acute doses increase dopamine release, whereas, chronic administration leads to reduced dopamine release. However, with naltrexone it was found that in human’s dopamine release increased over an 8-day period but dissipated over time. In animal studies the opioid antagonist naltrexone has been shown to attenuate the subjective effects of amphetamine. However, the mechanisms behind this modulatory effect were unknown up until April 2017, when Nitya Jayaram-Lindström and associates  hypothesCollectively, these data suggest that the opioid system becomes engaged during the more chronic phase of drug use, evidenced by the modulatory effect of naltrexone on dopamine release following chronic amphetamine administration. The importance of opioid-dopamine interactions in the reinforcing and addictive effects of amphetamine is highlighted by these findings and may help to facilitate medication development in the field of drug dependence especially as it also relates to buprenorphine /naloxone combinations.It is a fact that most of the FDA approved drugs work by favoring dopamine blockade and subsequent extinction of substance seeking behavior with full or partial agonistic activity (e.g. Methadone & Buprenorphine). We also know that thses approved FDA MAT have high addiction liability and in the case of methadone even cardiovascular adverse effcts. However, if we could find novel ways to improve both the compliance and outcomes with the use of naltrexone, in the injectable form, as we show herein, the recovery cimunity may be well served.et al. [Along these lines, we are not surprised about our dramatic findings with KB220. Resting fMRI data analysis in the heroin users after KB220 and placebo clearly reveal that KB220 induced an increase in BOLD activation in caudate-accumbens-dopaminergic pathways compared to placebo following 1-hour acute administration. Furthermore, KB220 also reduced resting-state activity in the cerebellum of abstinent heroin addicts suggesting an induction of dopamine homeostasis. In the second phase of this pilot study of all 10 abstinent heroin-dependent subjects, Blum et al.  observedet al. .et al. [in vivo, as is proposed by the NIDA ABCD study [As pointed out by Kunøe et al.  naltrexoCD study . This isCD study , 30. MorTo be clear there may be other promising modalities other than MAT such as repetitive transcranial magnetic stimulation (rTMS),  exerciseNaltrexone holds some promise in the short term as a psychologically induced deterrent therapeutic modality. Moreover, by adding a pro-dopamine regulator to help balance dopaminergic function especially important reward circuitry sites, has heuristic value. Therefore, these findings presented herein should be embraced by the clinical community challenged with early harm reduction in active OUD patients.only then will we be able to consistently and effectively address not only the opioid epidemic but the broader question of addiction as a whole”. Similarly for a review of the area the reader is encouraged to see a number of earlier published works on narcotic antagonism and buprenorphine [Furthermore, we must be reminded that drug seeking behavior is indeed a chronic enduring illness that has genetic antecedents and there is no real quick fix . Insteadnorphine ,34–46 Wi


The antioxidant activities, including scavenging ability for hydroxyl radicals and DPPH free radicals, chelation ability for Fe2+ ions, and reducing ability of CSP increased with decreased molecular weight of CSPs within 6.0 to 124 kDa. However, antioxidant activity weakened when the molecular weight of CSPs reached 3.5 and 2 kDa. CSP3 with a molecular weight of 6.0 kDa exhibited the strongest antioxidant activity. After protection with 60 μg/mL CSPs, the viability of human renal proximal tubular epithelial cells (HK-2) damaged by nano-COM crystals increased, the level of reactive oxygen species decreased, and the amount of COM crystal adhered onto the cell surface decreased. The ability of CSPs to protect cells from CaOx crystal damage was consistent with their antioxidant activity. CSPs can specifically combine with CaOx crystal to inhibit the conversion of calcium oxalate dihydrate crystal to calcium oxalate monohydrate crystal. All these results showed that the activity of CSPs was closely correlated with molecular weight. A very high or low molecular weight of CSPs was not conducive to their activity. CSPs, especially CSP3 with a molecular weight of 6.0 kDa, can be used as a potential antistone drug.Corn silk polysaccharide  was degraded by ultrasonication to obtain five degraded polysaccharides, namely, CSP1, CSP2, CSP3, CSP4, and CSP5, with molecular weights of 26.1, 12.2, 6.0, 3.5, and 2.0 kDa, respectively. The structures of these polysaccharides were characterized by FT-IR,  Kidney stone is a common disease that affects 10% to 15% of the population and has a high recurrence rate .The formation of kidney stones is closely correlated to the heterogeneous nucleation, growth, and aggregation of minerals and the adhesion of crystals onto renal epithelial cells caused by urine supersaturation . Renal ePlant polysaccharides can also regulate the growth of CaOx crystals and inhibit the adhesion of crystals onto renal epithelial cells . Gomes ePlant polysaccharides have biocompatibility, biodegradability, nontoxicity, and some specific therapeutic activities , 11. The2O2-induced oxidative damage. Zhao et al. . The mixture was incubated at 50°C for 30 min. 2.5 mL 10% trichloroacetic acid was added to the mixture which was then centrifuged for 10 min at 3000 r/min. The supernatant (2.5 mL) was mixed with 0.5 mL FeCl3·6H2O  solution and 5 mL distilled water. The solution was mixed fully and left to stand for 10 min. Then, the absorbance was measured at 700 nm.ng Power . 2.5 mL Ferrous Ion-Chelating Capacity , 38υas blue shift continuously. In the fingerprint region, the absorption bands of COD crystals were at 912 and 668 cm−1, which were obviously different from 951, 887, and 518 cm−1 of COM, of which 887 and 783 cm−1 belonged to the COM C–C stretching vibration and O–C–O in-plane bending vibration, respectively /[Ox2-] ratio in local areas, and increases the energy state of calcium. Both high-energy interface and high-calcium energy state can promote thermodynamic metastable COD formation [The –COOH groups in CSP can complex free Caormation .2+ in solution, CSP3 had the best inhibitory effect on CaOx crystals and the highest percentage of induced COD  has protective effects on KBrO3-induced renal injury in rats. Protection of LBP-4a can increase the activity of antioxidant enzymes in rat kidney tissue, decrease mitochondrial membrane potential, and reduce DNA damage in renal cells. Li et al. [Li et al.  reportedi et al.  reported2+ in the solution, reduce the quality of CaOx crystals, inhibit COD to COM conversion, increase the absolute value of zeta potential of the crystal surface, and reduce the degree of crystal aggregation and COM crystal size. These CSPs induced no cytotoxicity on HK-2 cells, protected HK-2 cells from damage by nano-COM crystals and increased cell vitality, and reduced the level of ROS and the adhesion of crystals onto cells. The activity of CSPs was closely correlated with the molecular weight, i.e., very high or very low molecular weight of CSPs was not conducive to CSP activity. CSP3 with a moderate molecular weight had the best antioxidant effect and the best cell protection effect. Overall, our results suggested that each CSPi, especially CSP3, may be potential antistone drugs.CSP0 with a molecular weight of 124 kDa was degraded by ultrasonication, and five degraded polysaccharides with molecular weights of 26.1, 12.2, 6.0, 3.5, and 2.0 kDa were obtained. The structure was characterized by FT-IR and NMR analyses. These CSPs exhibited scavenging activity on hydroxyl and DPPH, as well as reducing power and chelation ability for ferrous ions. Each CSPi can increase the concentration of soluble Ca


This has not only furthered our understanding in the mechanisms of spatiotemporal cell biology and toxicology, but also given us insight into the dynamical patterns of spatial transcriptomics during the course of disease occurrence and development.3o sections (Figure Spatiotemporal molecular pathology is a central part of spatiotemporal molecular imaging to link clinical images and molecular phenotypes. The spatiotemporal molecular imaging requests the morphological match between clinical phenomes and images, between clinical detections and microscopic structures, and between pathology and molecular omics Figure . Asp et s Figure , in whics Figure . The com4Spatial temporalization of clinical images has been developed and evidenced as a critical approach in clinical and translational practices, e.g., radiomics which was used to detect the alternations in the microenvironmental circulation and vascularization in solid tumors and to predict the prognosis of patients.5Spatiotemporal clinical images contribute to the routine clinical practice of diagnosis and therapy and are ready to welcome further development of organ‐wide molecular construction. Four‐dimensional ultrasound images could spatiotemporally define and characterize the qualification and visualization of protein deposition, mineral distribution, cell positioning, tissue property, and microenvironmental events, highly dependent upon ultrasound transducers, probes, and programs. Ultrasound‐oriented spatiotemporal delivery of drugs could increase the cell sensitivity to drugs and the specificity of drug‐acted locations.6The process from organ anatomy to spatiotemporal molecular images is developing through various phases of spatiotemporal clinical images, tissue images, and image integration Figure . The metIn conclusion, spatiotemporal molecular imaging is a critical and indispensable part of spatiotemporal molecular medicine by integrating spatiotemporal clinical images with molecular pathology. Spatiotemporal molecular pathology includes tissue section images and positioning of gene expression and cell phenotypes by visualization and modeling to bridge clinical images and molecular profiles together Figure . Althoug


I feel part of a massive effort to understand what is wrong with motor systems in the brain relating to Parkinson’s disease. Today, the symptoms of the disease can be modified slightly, but dopamine neurons still die; the disease progression continues inexorably. Maybe the next research phase will bring the power of modern genetics to bear on halting, or better, preventing cell death. The arrival of accessible human neuron assemblies in organoids perhaps will provide a better access to the processes underlying neuronal demise. My interest in neuroscience started in the company of inquisitive psychiatrists wondering about the origins of schizophrenia. The ideas frequently discussed with my fellow worker and teammate Tim Crow were about the role of dopamine in the type I symptoms of schizophrenia, which are characterized by delusions, hallucinations, and erratic and disorganized speech and thinking. We were confident that dopamine had something to do with the disease; the problem was that the dopamine receptor blockers used to aid schizophrenic symptoms could result in a condition similar to Parkinson’s disease (PD) ,2.Intrigued about dopamine, I departed, thanks to the Welcome Trust, for a postdoctoral year in Stockholm to participate in the development of the first animal model of PD. That happened more than a decade before an addictive drug contaminant (MPTP) turned out to be a potent neurotoxin of dopamine cells and caused Parkinsonism . In SwedThe paper of Garcia-Munoz, et al.  was one Cali Ingham, an electron microscopist, joined Edinburgh just as she completed her PhD in Oxford. She was interested in the formation of new synapses in the adult brain as described by Raisman and Field . Then, iWhile we were involved in these details, one of the great advances in the dopamine field came. Two output pathways from striatum, with different dopamine receptors on each, were proposed ,27. The Then, we began to count dendritic spines again, this time differentiating between the two pathways from striatum. Since that original paper had interested others, by the time we had a possible answer, the world had joined us and we published together. The article included results from four laboratories concluding that out of the D1- and D2-expressing striatal medium spiny neurons, the D2-medium spiny neurons lost spines first . Today, When Jeff Wickens visited my laboratory in Edinburgh, he suggested we take a look at the actions of dopamine in striatum from a different point of view. His theory that dopamine would act specifically on recently active cortical inputs to the striatum resulted in a series of experiments involving intracellular recordings from rat brain slices. Cortical stimulation produced long-term depression (LTD), and application of dopamine along with the stimulation converted LTD to a long-term potentiation (LTP) . This leOne of the most important consequences of the two output systems was the advent of deep brain stimulation (DBS) for PD. The model of PD proposed that the D2 cells of the output pathway were highly active, so they would inhibit globus pallidus which would in turn, disinhibit the subthalamic nucleus (STN). This increased activity of the excitatory STN was expected to result in the bradykinesia. Therefore, removing the STN should reduce the symptoms. Indeed, it worked in monkeys, although it turned out to be a very risky surgery in humans . This suFollowing the suggestion that overactivation of STN should silence the neurons, it was proposed that its stimulation in patients should improve their condition at least by alleviating the worst of the motor symptoms . That woFurther, while we were building up the account of the basal ganglia loops, we found their final return to cortex from the thalamic area . The axoMeanwhile, the idea of the two functionally separated striatal output systems was in various kinds of trouble. Firstly, Costa , Cui et The simple story that D1 cells say ”go” and D2 cells ”stop” is clearly very oversimplified. When we trained mice to reach for a chocolate pellet with one paw, interruption of the action of D1 cells, although it disturbed the early part of the reach, did not change much its initiation. In contrast, activation of D2 cells led animals to miss the target, but the movement stopped just fine . This isWe have now begun to look beyond the cortical consequences of dopamine loss, particularly considering that animal models only address the end-stage of the disease. Bilateral lesions kill animals very quickly  unless tI had earlier resisted the genetic path of investigation because, although many genes are implicated in familial PD, only about 10% of patients have a clearly established inherited link. The newer Genome Wide Association Studies suggest that many genes associated with the disease might have been missed since some genes might have strong associations with the disease ,77 and hIn Okinawa Institute for Science and Technology (OIST), we had developed methods to build cortico-striatal cultures of mouse neurons . These hFor the future, the hope is that these model organoids will be more closely matched to the normal human brain and so will provide a better platform on which to test therapeutics for neurological diseases. It could be that the current failures in therapeutic developments are due to the tests having been done on rodent brains or in isolated cells, lacking the complexity of an interactive network, another characteristic of human brain cells. In principle, organoids grown from induced pluripotent human stem cells (iPSCs) expressing various genetic causes of the disease could provide the much-needed detailed pathology and, perhaps, a final common path. It is not at all a straightforward task: for example, it seemed that LRRK2 gain of function mutations were a cause of alpha-synuclein accumulation  and thatThe fact that since 1969, PD patients’ life expectancy has shifted from 5 to 20 years is some justification for what we have been involved with. All of the pharmacology, anatomy, and neurophysiology that have been my life so far have contributed to treatment strategies, from L-DOPA itself (still the first line treatment), to surgical lesions, and deep brain stimulation, which have increased survival and reduced symptoms for patients. The idea that the brain compensated for injury was new in 1973, but now we are beginning to understand just how widespread it is, and how much of a mixed blessing it can be. Raisman’s experiments, which pioneered the idea that the brain did rewire after injury, had the synapses that were missing replaced by cells not normally making synapses at that site. Not so much a repair as a rewiring that did not have obvious behavioral consequences. Now we have ample evidence that the brain normally remodels itself, even during the formation of memories for instance ,92. As tAs we put 2020 behind us, 2021 might become a bright new year, not only for the responses to the COVID-19 pandemic, but for the generation of new targets for therapeutic interventions to save, or better to rescue, dopamine cells from their death, and patients, from the Parkinsonism that ruins their quality of life, until it finally ends it.


Introduction: In premature neonates, the persistence of hemodynamically significant ductus arteriosus (hsPDA) can be associated with short- and long-term consequences, impairing their outcome. The correct strategy of management for such condition is under debate, especially regarding contraindications and/or side effects. In recent years, metabolomics was applied to several perinatal, pediatric, and adult conditions to investigate potential biomarkers of disease, which have become useful for early diagnosis and/or therapeutic management.Aim of the Study: The main purpose of our exploratory study was to asses, through 1H-NMR metabolomics analysis of urinary samples at birth, possible metabolic pathways differentiating, with a significant predictive power, those preterm neonates who will subsequently develop hsPDA and neonates of comparable gestational age (GA) who will undergo spontaneous ductal closure or the persistence of an irrelevant PDA (no-hsPDA). Moreover, we investigated potential prenatal or perinatal clinical factors potentially influencing the development of hsPDA.Materials and Methods: We enrolled n = 35 preterm neonates with GA between 24 and 32 weeks; urinary samples were collected within the first 12 h of life. Patients were closely monitored regarding intensive care, respiratory support, fluid balance and administered drugs; an echocardiogram was performed at 48–72 h.Results: Our results reported a significant correlation between lower GA at birth and the development of hsPDA. Moreover, neonates with GA ≤ 30w developing hsPDA were characterized by lower Apgar scores at 1′ and 5′, higher rates of perinatal asphyxia, higher need of delivery room resuscitation and subsequent surfactant administration. Interestingly, metabolomics analysis at birth detected a clear separation between the 1H-NMR urinary spectra of subjects GA ≤ 30w not developing hsPDA (n = 19) and those of subjects born at GA ≤ 30w in which hsPDA was confirmed at 48–72 h of life (n = 5).Conclusions: This is the first study applying metabolomics to investigate the PDA condition. Although preliminary and conducted on a limited sample, our results reveal that metabolomics could be a promising tool in the early identification of hsPDA, potentially superior to the clinical or laboratory predictive tools explored to date and even to the clinical observations and correlations in our sample, through the detection of specific urinary metabolites. Ductus arteriosus (DA) or ductus Botalli, one of the fetal shunts allowing oxygenation during intrauterine life , 2, physvia increasing prostaglandin levels  and increased sensitivity to prostaglandins and nitric oxide (NO) compared to full term neonates . A low-gn levels , 6.The risk of developing a persistence of ductal patency (PDA) increases with decreasing gestational age (GA), with relevant short- and long-term consequences in term of morbidity, especially in case of hemodynamically significant PDA (hsPDA) , 7, 8.PDA closure can be performed pharmacologically or, in case of failure or contraindication, with surgical ligation or transcatheter closure –18.However, all these strategies can have side effects and potential risks and, as evidenced by current literature, the ideal strategy of management is still under debate, especially regarding if, when and how to treat or not PDA.Several predisposing factors have been associated to PDA condition, sometimes with contrasting results, such as genetic factors –21, mateObjects of study have also been the factors that can influence therapy response, as reported for male gender , polymorThe full comprehension of all the molecular and genetic mechanisms underlying ductal closure or patency maintenance could help in identifying serum or urinary biomarkers potentially associated with PDA condition, especially hsPDA, supporting or anticipating ultrasound or clinical diagnosis.HsPDA determines ductal diastolic steal, leading to systemic hypoperfusion  and the subsequent hypoxia can affect tissues and endothelium with variable impact on hemodynamics. Identifying early markers of this condition could help in the precocious identification of those newborns at high risk of hsPDA, orienting clinical strategy, and avoiding overtreatment of subjects who would undergo spontaneous ductal closure, improving diagnosis and treatment.Up to now, several literature studies have tried to associate different biomarkers to hsPDA, such as serum B-type Natriuretic Peptide (BNP), segment of the amino terminal B-type Natriuretic Peptide (NT-proBNP) , 50–59, Systemic inflammation –78 and oFinding biomarkers that are able to predict therapy response could be useful to avoid unnecessary treatment and potentially perform a targeted therapy, choosing and treating only those infants that could respond to the treatment.Metabolomics is one of the “omics” technologies that, in the last years, emerged as a promising tool to be applied in several fields of neonatology and pediatrics to characterize and describe pathophysiological processes , 82.Due to its ability in dynamically detecting, in a non-invasive way, the whole set of low molecular weight molecules  in cells, tissues and biological fluids, it can help in describing, in a highly individualized way, the exact response of an organism to environmental stimuli and pathophysiological conditions, including drugs, nutrition, lifestyle, diseases and others, as recently reviewed by our group .Metabolomics biomarkers, if accurately identified and confirmed through repeated studies and validation tests, could be investigated to obtain, in a precocious and non-invasive way, information regarding disease onset, progression and, potentially, therapy response.Our research group has been interested in metabolomics for many years, carrying many promising studies on several prenatal, perinatal, infant, and adult conditions and diseases, with the aim of achieving a personalized medicine. More details can be found in our reviews on the topic , 82.Even if, to date, the exact meaning of each metabolite variation and its pathophysiological implication was not completely defined, we strongly believe that the clinical interpretation of the promising results obtained could be completed by creating a specific and updated atlas.Due to these reasons, we hypothesize that metabolomics could provide useful biomarkers even in the condition of PDA. Thus, we decided to perform an exploratory study analyzing urinary samples at birth in a cohort of preterm neonates, to assess, among the complete overview of possible prenatal or perinatal factors taken into account, if some metabolites could result as predictive of hsPDA, highlighting substantial differences among those neonates who subsequently will develop hsPDA and those who will undergo spontaneous closure, or in which PDA will not determine hemodynamic consequences.To the best of our knowledge, this is the first metabolomics study in the investigation of PDA disease. A previous preliminary report by our group was performed few years ago  and a prOur hypothesis was to identify, through potential early metabolic perturbations in the group of newborns that will develop hsPDA, different urinary pathways compared to those newborns in which the duct will undergo spontaneous closure or minimal patency without impact on hemodynamics.1H-NMR metabolomics analysis of urinary samples at birth regarding the development of hsPDA. In fact, we investigated possible metabolic differences between urinary pathways of those preterm neonates who will subsequently develop hsPDA confirmed at 48–72 h of life, rather than neonates of comparable GA who will undergo spontaneous closure or the persistence of irrelevant PDA at the same time point (no-hsPDA).Thus, the principal aim of the exploratory study was to investigate, for the first time, the possible predictive role of We also aimed to evaluate whether the predictive role of urinary metabolomics may be superior to the clinical criteria or biomarkers considered up to now. Therefore, we investigated potential prenatal or perinatal clinical factors potentially influencing the development of hsPDA in our sample group.Finally, we tried to find a potential link correlating metabolic variation in the two groups and pathogenesis of hsPDA; in our opinion, the early identification of specific metabolites suggestive of PDA could be a fundamental and complementary tool to be used, alongside the clinic, for applying a personalized approach to the treatment of PDA.n = 35 preterm neonates admitted to Neonatal Intensive Care Unit of Duilio Casula Hospital, University of Cagliari, with GA included between 24 and 32 weeks, was enrolled between October 2019 and June 2020.A population of In all the newborns, a urinary sample of 3 mL was collected until 12 h after birth, through the non-invasive technique of the cotton wad.All the subjects underwent cardiologic evaluation performed by the neonatal cardiologist of our neonatal intensive care unit (NICU) within the first 24 h and successively at 48–72 h and 120 h after birth. Echocardiography and Color-doppler aimed to evaluate cardiac anatomy and functionality and to assess the presence and the hemodynamic features of PDA. In subjects with a persistent PDA or hsPDA, an echocardiography was performed in the subsequent days, according to the clinical indication.Each echocardiography aimed to asses PDA diameter (indexed for body weight expressed in kilograms), left atrium/Aortic Root Ratio (LA/Ao), patent ductus arteriosus/left branch of pulmonary artery ratio (PDA/LPA), systolic and diastolic velocity of duct flow to assess the restrictive or non-restrictive pattern. Additional cardiological evaluations were performed according to clinical necessities. PDA was considered hemodynamically significant in case of a PDA diameter/neonatal weight ≥ 1.5 mm/Kg and LA/Ao > 1.4 in addition to an eventual PDA/LPA > 0.8 and/or a peak diastolic velocity lower than 50% of the peak systolic velocity measured at ductal level 48–72 h after birth.Echocardiography was performed using Esaote MyLab™Twice EVO 13.0/13.0 m, and the images' acquisition was obtained with 8 MHz two-dimensional probe.During hospitalization, all the newborns underwent instrumental exams  and hematic exams , eventually repeated according to the clinical needs.All demographic data and the information regarding pregnancy, birth, neonatal management in delivery room, assistance in NICU, invasive and non-invasive ventilation, oxygen needs, surfactant administration, fluid balance, nutrition, and drugs were recorded.The Ethics Committee of University of Cagliari gave its approval for this study (Protocol NP/2017/470) and the parents signed an informed consent and gave their approval for this study.1H-NMR analysis  containing sodium 3-trimethylsilyl-propionate-2,2,3,3,-d4 (TSP) as an internal standard . The mixture was vortexed and an aliquot of 650 μL was transferred to 5-mm NMR glass tubes for analysis .1H-NMR spectra were recorded at 300 K using a Varian UNITY INOVA 500 spectrometer operating at 499.839 MHz for proton and equipped with a 5 mm double resonance probe . The acquisition parameters of the 1H-NMR spectra are reported in our previous article  was removed. The ACD Labs intelligent bucketing method was used for spectral integration . For statistical analysis, Student's A Chi-Square-test was used for testing relationships on categorical variables and to evaluate tests of independence.Principal component analysis (PCA), Partial Least-Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least-Squares Discriminant Analysis (OPLS-DA) were used for multivariate statistical analyses of NMR data . Principp-value < 0.05 was considered statistically significant. The Benjamini-Hochberg method was subsequently applied to the obtained p-values to acquire the level of significance for multiple testing  for the AUC curves were also calculated.GraphPad Prism software  was used to perform the univariate statistical analysis. The potential significant metabolites identified were quantified using Chenomx NMR Suite 7.1  . The sta testing . Finallyn = 35 newborns with GA included between 24+2 and 32+4 weeks. Mean GA was 29.92 ± 2.06 weeks. Mean birth weight was 1,346 ± 367 g and n = 23/35 subjects were males.Our study population was composed by All the information regarding pregnancy characteristics, complications and outcome are reported in n = 4/35 were born by vaginal delivery (VD) and n = 31/35 by urgent cesarean section (UCS). On a total of n = 29 pregnancies, n = 3 deliveries were vaginal, while n = 26 were UCS. The causes of preterm birth through UCS have been represented by intrauterine growth restriction (IUGR) and flux alteration (23%), cardiotocography (CTG) alterations/neonatal bradycardia (23%), placental abruption (19.2%), preterm labor/other maternal causes (19.2%), preeclampsia (8%), twin-to-twin transfusion (3.8%), and fetal tachycardia (3.8%).In our sample of preterm neonates, n = 5/35 neonates (14.3%) hsPDA was confirmed at echocardiographic examination at 48–72 h of life. All the newborns developing hsPDA were born at GA ≤ 30weeks. Thus, to make the sample more homogeneous, we decided to report our results excluding those neonates of GA > 30 weeks, since hsPDA resulted as being highly dependent on GA at birth and was absent in neonates > 30 weeks. However, all the results obtained by the analysis of the whole population can be found in All the information regarding perinatal characteristics of our group of newborns have been reported in n = 24 neonates. All the information on pregnancy, perinatal characteristics and neonatal early outcome regarding this subpopulation of newborns were analyzed and statistical comparisons were made between those newborns with GA ≤ 30weeks subsequently undergoing spontaneous closure of PDA or maintaining an hemodynamically irrelevant PDA (no-hsPDA) and those neonates developing hsPDA  included n = 5/24 neonates, p < 0.01.The first group, (≤30 w no-hsPDA) was composed by Maternal/pregnancy complications were analyzed, including chorioamnionitis, placental abruption, hypertension in treatment, premature rupture of membranes (PROM > 18 h), and not statistically significant differences occurred in the two groups ≤30 w no-hsPDA vs. ≤30 w hsPDA .n = 15/24 mothers, n = 12/19 in the group ≤30 w no-hsPDA vs. n = 3/5 in the group ≤30 w hsPDA, p = 0.9.Maternal administration of steroids occurred in Information regarding maternal glucose load was available only for a few patients, since such a test is performed between 26 and 28 GA and many of the women delivering preterm were not able to perform it. Thus, statistical evaluations were not performed regarding eventual alteration in glucose tolerance or pregnancy induced diabetes mellitus.n = 14/24 were males.In the group of neonates ≤30 w, mean GA was 29 ± 1.85 weeks and mean BW was 1239.92 ± 329 g; n = 9/24; twins are not even in number, since one of them was excluded by the study because we could not collect the urinary sample.Twins were p < 0.001 and even BW was higher in the fist group 1,302 ± 261 g vs. 1,004 ± 476 g, without statistical significance (p = 0.069).GA was significantly higher in the ≤30 w no-hsPDA group (29.63 ± 1.03) vs. ≤30 w hsPDA (26.62 ± 2.46), with statisticalsignificance n = 9/19 vs. ≤30 w hsPDA n = 0/5, p < 0.05.Twins were more frequent in the group ≤30 w no-hsPDA p = 0.75). Moreover, even delivery mode in terms of VD or UCS was not statistically different in the two groups (p = 0.6).Information regarding length, weight, and head circumference percentiles at birth were not statistically significant in terms of number of patients large for GA (LGA), appropriate (AGA) or small for GA (SGA) (p < 0.01 and at 5 min of life (5′) (7.48 ± 1.21) vs. (6 ± 2.45), p < 0.05. The incidence of perinatal asphyxia was higher in the ≤30 w hsPDA group than ≤30 w no-hsPDA group (p < 0.05); in detail, severe asphyxia was more frequent in the first group (p < 0.01).Significant differences occurred, taking into account the Apgar score in the two groups of patients. Apgar score was significantly higher in the ≤30 w no-hsPDA group (6.58 ± 1.74) vs. ≤30 w hsPDA (3.8 ± 3.11), at the first minute of life (1′), with statistical significance p < 0.05), especially regarding the needs of endotracheal intubation, chest compressions, or adrenaline, occurring in n = 2/19 (10.53%) in the ≤30 w no-hsPDA group, vs. 3/5 (60%) in the ≤30 w hsPDA group (p < 0.05).The needs for delivery room resuscitation was higher in the ≤30 w hsPDA group (n = 24/24 (100%).RDS occurred in the whole sample of newborns ≤30 w n = 10/19 in the ≤30 w no-hsPDA group and n = 5/5 in the ≤30 w hsPDA group (p < 0.05).The need for one or more doses of surfactant was n = 4/5 (80%) were pharmacologically treated for hsPDA closure.In the group ≤30 w hsPDA group, n = 4 (20%) it occurred with treatment, while n = 1/5 underwent spontaneous closure (20%).HsPDA closure was achieved in the whole ≤30 w hsPDA group. In n = 2/19 among the ≤30 w no-hsPDA neonates vs. n = 2/5 among the ≤30 w hsPDA (p = 0.11); late onset sepsis (LOS) occurred in n = 5/19 vs. n = 1/5 (p = 0.77) and the co-occurrence of EOS + LOS in 2/19 and 2/5 (p = 0.11) in the same groups.In our sample, early onset sepsis (EOS) was detected through blood culture in n = 5/19 among neonates ≤30 w no-hsPDA vs. n = 2/5 in the ≤30 w hsPDA group (p = 0.5), not statistically significant. Serum creatinine values at 3–5 days of life were 0.57 ± 0.21 mg/dl in the ≤30 w no-hsPDA vs. 0.65 ± 0.21 mg/dl in the ≤30 w hsPDA group (p = 0.46).Early aminoglicosydes administration to treat EOS was administered in None of our patients died.1H-NMR spectroscopy coupled with multivariate data analysis. Metabolites were identified on the basis of literature information (http://www.hmdb.ca) and the 500 MHz library from Chenomx NMR suite 7.1. Major NMR resonances were originated from free amino acids, organic acids, sugars, small organic compounds and osmolytes. A first unsupervised PCA analysis was conducted to identify potential outliers (outside the 95% confidence limit). The result of the PCA analysis (data not shown) shows a rather homogeneous cohort of samples and no NMR spectrum was excluded from subsequent analyses. To remove potential information not related to the pathology of interest and highlight possible differences in metabolomics urine profile between No-hsPDA (≤ and >30 w) and hsPDA (≤30 w) subjects, a PLS-DA analysis was subsequently conducted on the same dataset.The metabolomics profile of urine samples was investigated using ormation  and by up(corr) > 0.5 was selected as a significance level. The spectral regions identified in the S-line plot characterized by the superimposition of NMR signals corresponding to the different metabolites present in urine, were quantified with Chenomx NMR Suite 7.1. and subjected to the Mann-Whitney U-test to identify significant changes in the of metabolites concentration in the two groups. The result of the univariate statistical analysis showed as 3-Methylxanthine, betaine, glucose, glycylproline, lactate, trimethylamine N-oxide (TMAO), tryptophan, myoinositol and 4-Hydroxyproline changed significantly in No-hsPDA (≤30 w) compared to hsPDA (≤30 w)  and the area under the curve of ROC analysis (The PLS-DA scores plot  shows a  (≤30 w) . The Figanalysis  was founp < 0.001). BW, although lower in the ≤30 w hsPDA group, did not reach a statistical significance (p = 0.069).Our group confirmed that prematurity influences the occurrence of hsPDA, which resulted as more frequent at a lower GA  and at 5′ (p < 0.05); therefore, the incidence of perinatal asphyxia was higher in this group (p < 0.05), especially severe asphyxia (p < 0.01). The association between lower values of the Apgar score and hsPDA have also been observed in the studies of Pourarian et al. and Lee et al. .Due to their worse condition at birth, those neonates subsequently developing hsPDA, also compatible with their aforementioned lower GA, more frequently required delivery room resuscitation . Such a result seems in agreement with some recent literature results  and as evidenced in n = 5/35, red circles) and neonates no-hsPDA . Moreover, this urinary analysis also shows a clear dependence on GA, which increases in the newborns moving from the top to bottom of the plot: in fact, as showed in n = 19/35), while those represented in green all show GA > 30 w.In detail, analyzing the whole number of urinary samples (n = 5/35) and therefore well-separated in the plot from the whole sample of the neonates no-hsPDA  and even located in the upper half area of the plot with neonates GA ≤ 30w. In fact, the lower half of the plot is almost exclusively populated by neonates with GA > 30 w and the lower half of the plot is occupied by neonates of GA ≤ 30w  with the aforementioned clear separation between ≤ 30 no-hsPDA (blue) and ≤30 w hsPDA.From this analysis, it can be deduced that urinary samples at birth are strongly different in neonates subsequently developing hsPDA, all showing GA ≤ 30w  and no-hsPDA  can be also observed in the plot of The clear separation between urinary profiles of neonates hsPDA  seem mildly distant from the other samples. Interestingly, they showed some unique clinical features, represented by a congenital viral infection of the TORCH group in one subject and early pulmonary hypertension treated with NO immediately after birth in the second.Moreover, in the plot, Additionally, we decided to quantify the most characterizing metabolites responsible for sample separation. As reported in the results, the ≤30 w hsPDA group was characterized by lower levels of 3-methylxanthine, betaine, glycylproline, TMAO, tryptophan, myoinositol and 4-hydroxyproline, and higher levels of glucose and lactate.Firstly, we considered the potential interference of drugs in our group of neonates, by evaluating all the molecules and excipients administered . Timing and modality of sampling were standardized and all the newborns underwent the same nutrition regimen.Then, we tried to give a meaning to metabolomics variations, taking into account that there are not similar studies in literature.The significantly higher levels of urinary lactate observed in hsPDA group could be determined by the increase in anaerobic glycolysis following oxygen reduction and ischemia, with lactate being the end product of anaerobic metabolism , 98. We Lactate could also represent a compensatory and protective metabolite facing the increase in oxidative stress induced by reperfusion after ischemic damage, potentially lowering the levels of reactive oxygen species and superoxide anion , 98.via the effects of acidosis and oxidant mechanisms linked to reperfusion on vasodilation and ductal relaxation .Tryptophan is mostly metabolized through the kynurenine pathway (90%); the 3% is converted into serotonin (5-OH-tryptamine) and the remaining quote undergoes intestinal microbiota degradation with production of indole and its derivatives .Alterations in one of these processes could determine several effects in neuronal metabolic pathways.Tryptophan and kynurenine could pass the blood-brain barrier and determine anti-inflammatory, immunosuppressive and antioxidant functions with neuroprotective actions .We should also underline that tryptophan is reduced in neuroinflammatory diseases and neuroblastoma , 125, AlIn our sample, we speculated that reduced levels of such metabolite in the urines of hsPDA neonates could be related to cerebral hypoperfusion; the resulting oxidative/ischemic or inflammatory damage could determine an increase in tryptophan transport to the brain, with a reduced excretion of such metabolite, to improve neuroprotective effects. However, such compensatory mechanisms still need to be verified and eventually clarified.Finally, the reduction observed in tryptophan level could also be related to microbial alterations, potentially modulating brain function.It is a major constituent of collagen, contributing to its stabilization, and elastin. It is a non-essential aminoacid derived by hydroxylation of proline.The increase in hydroxyproline levels (serum or urine) can be related to muscle damage  and collThis metabolite is a dipeptide composed by glycine and proline; it is involved in collagen metabolism. It can be mostly found in cytoplasm and it was correlated with Alzheimer's disease and inborn metabolic disorders too .It is one of the xanthine purine derivatives compounds; it can be mostly found in cytoplasm .In humans, 3-methylxanthine, adenosine agonist, is a metabolite of theophylline  and caffIn our sample, urinary 3-methylxantine can derive from the metabolism of caffeine administered to prevent prematurity-related apnea.This metabolite, although not representing the most significant among our findings, was reduced in urinary samples of hsPDA neonates.The newborns of the two groups (no-hsPDA and hsPDA) assumed the same dose of caffeine, thus the reason of such different excretions is not clear.According to literature data, caffeine and theophylline metabolism are influenced by hepathic and renal function, in addition to some cytochrome polymorphisms .The exact meaning of the decrease in 4-hydroxyproline, glycyproline and 3-methylxanthine in our sample of hsPDA neonates still requires clarification.p < 0.01). According to our study, we can affirm that the increase in glucose and the decrease in tryptophan and TMAO resulted the best predictors of hsPDA.In conclusion, the early evaluation of neonatal urine to predict at birth the subsequent development of hsPDA should include the three “top metabolites” , which resulted the most significant in our preliminary results  to assess if biochemical changes in the brain agree with the urinary variations reported.1H-NMR evaluation.According to our results, metabolomics seems to have the unique ability to predict, in a sensible and specific way, those preterm newborns who will undergo physiological closure of DA or will maintain a no-hsPDA, instead of those neonates who will develop a hsPDA confirmed at 48–72 h of life, only through a non-invasive collection of a small urine sample at birth undergoing We underline that our analysis was performed on the urine of the first 12 h of life and the metabolomics results can be considered a portrait of the metabolic basal status of each newborn. The role in predicting the subsequent development of hsPDA, if confirmed in future studies on a larger cohort of patients, could represent a unique peculiarity of metabolomics, able to individuate those newborns more predisposed to hsPDA before the occurrence of lung hyperflow clinical signs.Such metabolomics approach could have the promising role to help in predicting those neonates who could develop hsPDA, preventing delay in therapeutic treatment, at the same time avoiding treatment to those who probably would not require it due to the spontaneous ductal closure.This is the first time that metabolomics was applied in the early detection of hsPDA, and we decided to evaluate urinary samples due to the non-invasiveness of urine collection, being performed in the first hours after birth, and even taking into account the experience of our research group on urinary metabolomics in neonatology , 137–140In our opinion, this kind of experimental study opens the way for future studies on a greater number of samples, possibly also multicenter studies, and the results, if confirmed, could lead to the individualization of clinical approaches in such field.The analysis of seriate urinary samples before, during and after treatment, in subjects developing hsPDA, could help in detecting potential biomarkers predicting therapy response or drug toxicity, allowing physicians to choose in a personalized way the drug to administer, and potentially reducing side effects and improving treatment efficacy.Thus, we strongly believe that, in the near future, such preliminary results could acquire a clinical and therapeutic meaning in improving hsPDA treatment. Such an innovative approach would result very differently from generalized therapeutic prophylaxis, that can determine side effects of complications even in those subjects who will have spontaneously achieved ductal closure.The exact meaning of the metabolomics variation in our sample still requires a full comprehension since we only made some hypotheses and clinical speculation on the basis of what is currently known oh hsPDA pathophysiology.In fact, this is the first available study on such a topic and our results, although promising, are preliminary reports on a limited number of samples that should be confirmed on larger groups.However, despite these limitations, we believe that metabolomics is highly promising in the field of hsPDA, providing a combination of specific metabolites, also considering that, despite years of study on this topic, sensible and specific markers of such a condition are still lacking. In our opinion, the predictive power of metabolomics could result as potentially superior to the clinical or laboratory predictive tools explored to date.If our preliminary data will be confirmed, in the future, a simple and non-invasive urinary stick at birth could gave relevant and predictive information on the spontaneous closure or persistence of ductus arteriosus. This could be highly promising, given the great impact of such condition on immediate and long-term health, even taking into account the concept of perinatal programming. Metabolomics approach could help the clinicians to orient their therapeutic strategies in the management of such vulnerable newborns, early identifying those who could develop a hsPDA and therefore avoiding delaying treatment in such subjects, as well as, at the same time, identifying groups of newborns in which this condition is unlikely to occur and the therapy would therefore result in an overtreatment, not exempt from toxicity and side effects.In our opinion, it could be useful to perform a subsequent study to confirm these data on larger samples and even to evaluate urinary profiles in relation to the kind, timing, and response to the treatment.If these results will be confirmed, metabolomics could become a good tool in the early detection of hsPSA neonates, showing a stronger predictive power than clinical findings and laboratory parameters investigated to date, to optimize their management thanks to specific and holistic biomarkers in a field currently full of controversies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethic Committee University of Cagliari. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.FB selected the patients, collected urinary samples, reviewed literature, and wrote the manuscript. CP performed metabolomics experiments, metabolomics data analysis, and wrote the manuscript. PN and AA contributed in writing the manuscript and performed echocardiograms. VF supervised the whole experiment and critically revised the manuscript. All authors contributed to the article and approved the submitted version.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.