Kaposi sarcoma (KS) is an angioproliferative lesion in which two main KS cell sources are currently sustained: endothelial cells (ECs) and mesenchymal/stromal cells. Our objective is to establish the tissue location, characteristics and transdifferentiation steps to the KS cells of the latter. For this purpose, we studied specimens of 49 cases of cutaneous KS using immunochemistry and confocal and electron microscopy. The results showed that delimiting CD34+ stromal cells/Telocytes (CD34+SCs/TCs) in the external layer of the pre-existing blood vessels and around skin appendages form small convergent lumens, express markers for ECs of blood and lymphatic vessels, share ultrastructural characteristics with ECs and participate in the origin of two main types of neovessels, the evolution of which gives rise to lymphangiomatous or spindle-cell patterns—the substrate of the main KS histopathological variants. Intraluminal folds and pillars (papillae) are formed in the neovessels, which suggests they increase by vessel splitting (intussusceptive angiogenesis and intussusceptive lymphangiogenesis). In conclusion, delimiting CD34+SCs/TCs are mesenchymal/stromal cells that can transdifferentiate into KS ECs, participating in the formation of two types of neovessels. The subsequent growth of the latter involves intussusceptive mechanisms, originating several KS variants. These findings are of histogenic, clinical and therapeutic interest. Kaposi sarcoma (KS) is a vascular tumor of variable behavior that can affect different organs, with a higher incidence in the skin. Since it was first described by Kaposi in 1872 ,4,5 and Among the tissue-resident stromal cells, in order to explore their capacity as precursor mesenchymal cells in KS, are CD34+ stromal cells/Telocytes (CD34+SCs/TCs), which are located in the stroma of multiple anatomical sites ,21. Thesn: 49), using conventional techniques and procedures for observation by immunochemistry, immunofluorescence and electron microscopy, we will take into account: (a) the general characteristics of the main evolutionary stages of KS; (b) the location and characteristics of the CD34+SCs/TCs in the outer layer of pre-existing blood vessels and around skin appendages, with or without perivascular inflammatory infiltrate, and with or without formation of neovessels; and (c) the findings supporting the possible role of delimiting CD34+SCs/TCs on the formation of neovessels and their behavior in the evolutionary stages of KS.Given the above, the first objective of this study on KS skin lesions is to assess whether delimiting CD34+SCs/TCs, located in the outer layer of pre-existing blood vessels and around skin appendages, are mesenchymal/stromal cells with a capacity for transdifferentiation into neovessel ECs that express markers for both blood and lymphatic vessels. A better knowledge of the type and location of resident stromal precursor cells with vasoformative capacity is of histogenic, therapeutic and experimental interest, especially for assessing their modulation during tumor development and for obtaining them for in vitro studies. Subsequently, we will follow the events of the neovessels in the evolutionary stages of KS. This information is also of clinical interest since it could be the basis for future studies in larger series of cutaneous KS cases that confirm a possible evolution toward different variants and the prognostic projection of early lesions. For these purposes, in cases of KS  in the anaplastic KS. In the cross-sections, the KS cells appear around small or varying caliber lumens with the intraluminal presence of red blood cells; the lesion adopts a honeycomb-like pattern D. When lNuclear expression for the endothelial marker ERG F and forNext, we will pay particular attention to the pre-existing blood vessels and complex structures (associated vessels and skin appendages) in KS lesions and to the newly formed vessels to establish the location, characteristics, relationship and participation of CD34+SCs/TCs in these neovessels.A close relationship between the pre-existing blood vessels, inflammatory infiltrate, and newly formed vessels was observed in KS of the skin. The pre-existing blood vessels, with or without perivascular inflammatory infiltrate, did or did not present perivascular neovessels. Therefore, the pre-existing blood vessels in early cutaneous KS lesions were (a) apparently non-affected (without perivascular inflammatory infiltrate or perivascular neovessels), (b) with perivascular inflammatory infiltrate, and (c) with perivascular neovessels, presenting perivascular inflammatory infiltrate or not. Pre-existing vessels with perivascular inflammatory infiltrate or perivascular neovessels (with or without inflammatory infiltrate) were in similar proportions and predominated over apparently unaffected pre-existing blood vessels.Double staining for CD34 and αsmooth muscle actin (αSMA) revealed three layers formed by the CD34+ ECs (intima layer), αSMA+ pericytes or vascular smooth muscle cells (media layer), and delimiting CD34+SCs/TCs  in apparently non-affected pre-existing blood vessels of the two horizontal vascular plexuses and those connecting them in the skin . DependiThe perivascular inflammatory infiltrate in the pre-existing blood vessels was observed in the space between the CD34+SCs/TCs and pericytes or vascular smooth muscle cells A. When CNeovessels were mainly observed around the pre-existing blood vessels , predomiThe precise location of the neovessels coincided with that of the previously delimiting CD34+/SCs/TCs around the blood vessels , suggestAlthough several patterns of neovessels were present in the patch and plaque stages of KS, two main types were seen (types 1 and 2) . Both tyType 1 neovessels showed lumens of differing sizes and configurations, ranging from slit-like vascular spaces to wide sacs with a lymphangiomatous appearance . GeneralType 2 neovessels were more numerous in the plaque stage and appeared located around blood vessels in the previous location of the CD34+SCs/TCs, as occurred in type 1 vessels A–D. MostIn addition to the coincidence of the neovessel EC location and that of delimiting CD34+SCs/TCs, the following findings were observed during neovessel formation. The somatic region and processes of one or more delimiting CD34+SCs/TCs folded or converged, forming occasional, isolated small vessel lumens in the early stages of KS A–C. ThesThe ECs of the initially evolved neovessels gave rise to enlarged and confluent lumens (neovessel type 1) or formed narrow cords (neovessel type 2) and showed expression for markers of both blood and lymphatic ECs. A similar expression for both types of markers was observed in the CD34+SCs/TCs interposed between the neovessels and without forming a lumen A–C. AlthSome of the transformed CD34+SCs/TCs lining the neovessels were observed as preserving one or two of their processes that extended into the surrounding tissues A1–3. TheDilated type 1 neovessels, generally with numerous intraluminal folds and pillars (papillae), were observed in the lymphangiectatic and lymphangiomatous variants of KS . Frequently, the folds in the neovessels contained pre-existing blood vessels (a vessel inside a vessel or a promontory sign). The lumen of these type 1 neovessels appeared empty or with some inflammatory cells and/or occasional red blood cells.Numerous neovessels, predominantly type 2, interposed the pre-existing blood vessels, and intra- and extraluminal red blood cells were usual components in the nodular stage of KS A–C. IntrIn the serial histologic sections with different immunomarkers in each, the following findings were specified in type 1 neovessels , includiIn KS of the skin, we contribute (a) the participation of the delimiting CD34+SCs/TCs in the external layer of pre-existing blood vessels and around skin appendages in the formation of neovessels, behaving as resident mesenchymal/stromal cells capable of transdifferentiation into neovessel ECs and (b) the formation of two main types of initial neovessels in the early stages of KS, leading to progressive sarcomatous lesions.The distinction between pre-existing blood vessels and initial neovessels was important to our observations on the evolution of delimiting CD34+SCs/TCs during their transformation into neovessel ECs in the early KS lesions. This distinction was based on the expression in their ECs of only markers for blood vessels and the presence of prominent αSMA+ mural cells (pericytes or vascular smooth muscle cells) in pre-existing blood vessels versus the expression of markers for ECs of both blood and lymphatic vessels and the absence of mural cells in the initial neovessels.The main events of CD34+SC/TC participation in the formation of neovessels and the evolution of the latter in the KS stages can be summarized as follows : (1) In The characteristics and distribution of the CD34+SCs/TCs around apparently unaffected skin vessels and appendages in early KS lesions agree with those previously described in several conditions ,29,30,31For the transitional events between the CD34+SCs/TCs and ECs, the following issues should be considered: (a) The intraluminal persistence in the neovessels of stellate CD34+SCs/TCs keeping attachment points of their processes with the lining neovessel cells is compatible with a mechanism of vessel lumen formation by separation of adjacent CD34+SCs/TCs. In addition, the intraluminal CD34+SCs/TCs and their multiple or bipolar processes can also participate in the creation of new intraluminal spaces in the neovessels, increasing neovessel numbers by vessel splitting (intussusceptive angiogenesis). Therefore, the partial separation of adjacent and contacting CD34+SCs/TCs is an additional mechanism to those previously proposed in intussusceptive angiogenesis ,63,64,65The set of events outlined above supports previous studies by other authors on the participation of mesenchymal/stromal cells in the origin of KS cells ,15,16,17The formation of two main types of initial neovessels and the predominance of one in lesions with lymphangiomatous characteristics and of the other with a sarcomatous aspect during KS evolution suggests it is related to lesion variants.Papillae in vessel lumens have been described in several tumors and pseudotumors of blood and lymphatic vessels ,70,71,72This work has a histogenic and potential clinical and therapeutic interest. Indeed, it contributes to a better knowledge of the type and location of resident stromal precursor cells with a vasoformative capacity in KS, which could make it easier to obtain precursor cells for in vitro studies and assess their modulation during tumor development. It could also be the basis for future studies that confirm the possible evolution of the early lesions toward the different variants of KS and, therefore, their prognostic projection. These studies require larger series of cutaneous KS cases to monitor histological lesions over time, patient evolution and the application of new techniques, such as histomorphometric analysis, which other authors have used on telocytes ,78.® Megalab–Hospiten Hospitals of the Canary Islands were searched for cases of KS. Specimens (paraffin blocks) were obtained from 49 cases. The patients were Caucasian: 28 males and 21 females, aged 17–88 years. All specimens were studied by conventional histologic techniques using hematoxylin–eosin staining to select blocks and more demonstrative areas in the histological sections. Of them, 30 cases were used for the immunochemistry procedures and immunofluorescence in confocal microscopy, and four cases were used for electron microscopy. A combination of different immunomarkers in the immunohistochemistry and immunofluorescence procedures and ultrastructural observations were used to establish the general characteristics of the CD34+SCs/TCs and to assess their participation in neovessel formation. Serial histological sections with different immunomarkers in each, including the observations in confocal microscopy, were undertaken to confirm the previously suggested findings. Ethical approval for this study was obtained from the Ethics Committee of La Laguna University , including the dissociation of the samples from any information that could identify the patient. The authors, therefore, had no access to identifiable patient information.The archives of Histology and Anatomical Pathology of the Departments of Basic Medical Sciences of La Laguna University, University Hospital, and EurofinsThe specimens were fixed in a buffered neutral 4% formaldehyde solution, embedded in paraffin, and cut into 3 µm-thick sections. The sections were deparaffinized, hydrated and stained with haematoxylin and eosin (H&E).Immunohistochemistry  and immunofluorescence were carried out as described elsewhere ,66. For ® 100B and JEM 1011 Akishima, Tokyo, Japan, electron microscopes.Electron microscopy for the ultrastructural study was carried out, as described previously . The spen: 30). The percentages in neovessels type 1 and type 2 are reported as a mean of each group and +/− standard deviation, and the early and advanced stages were analyzed by the Student’s t-test, considering a statistically significant p-value of less than 0.05.Quantitative analysis of the neovessels was performed in cases of KS in which immunochemistry (D2-40) was used  the transitional findings between both types of cells, including the presence of CD34+SCs/TCs expressing markers for the ECs of blood and lymphatic vessels and the replacement of CD34+SCs/TCs by the EC of neovessels; (b) the possible interrelationships of the CD34+SC/TC descendants with ECs from other sources; (c) the formation of two types of neovessels and their evolutionary phenomena, suggesting their role in the origin of the different variants of KS with lymphangiomatous or spindle-cell patterns; and (d) the subsequent neovessel growth by mechanisms of intussusception (intussusceptive angiogenesis and lymphangiogenesis), all of which are of histogenic, clinical, and therapeutic interest.


Administration of heroin results in the engagement of multiple brain regions and the rewarding and addictive effects are mediated, at least partially, through activation of the mesolimbic dopamine system. However, less is known about dopamine system function following chronic exposure to heroin. Withdrawal from chronic heroin exposure is likely to drive a state of low dopamine in the nucleus accumbens (NAc), as previously observed during withdrawal from other drug classes. Thus, we aimed to investigate alterations in NAc dopamine terminal function following chronic heroin self-administration to identify a mechanism for dopaminergic adaptations. Adult male Long Evans rats were trained to self-administer heroin  and then placed on a long access  protocol to induce escalation of intake. Following heroin self-administration, rats had decreased basal extracellular levels of dopamine and blunted dopamine response following a heroin challenge  in the NAc compared to saline controls. FSCV revealed that heroin-exposed rats exhibited reduced stimulated dopamine release during tonic-like, single-pulse stimulations, but increased phasic-like dopamine release during multi-pulse stimulation trains  in addition to an altered dynamic range of release stimulation intensities when compared to controls. Further, we found that presynaptic D3 autoreceptor and kappa-opioid receptor agonist responsivity were increased following heroin self-administration. These results reveal a marked low dopamine state following heroin exposure and suggest the combination of altered dopamine release dynamics may contribute to increased heroin seeking. The current opioid overdose epidemic reflects a dramatic increase in the prevalence of opioid use disorder (OUD) and opioid-related overdoses since the 1990s. In 2019, it was estimated that more than 130 people died each day from opioid overdose, with heroin involvement in nearly a third of those overdoses ,5. ThereTo find potential targets for OUD treatment, it is important to understand the mechanisms of action and consequences of chronic exposure to opioids. The prevailing hypothesis is that the acute reinforcing effects of exogenous opioids occur via disinhibition of dopamine neurons by activation of mu-opioid receptors (MOR) located on GABAergic neurons within the ventral tegmental area (VTA) ,10,11,12With regard to the effects of chronic exposure to opioids on dopamine function, there appears to be a general consensus that there is a downregulation of dopamine system function ,19. ThisWhile substantial evidence exists for opioid-induced adaptations in VTA dopamine neuron function and structure, considerably less is known about the impact of chronic opioid exposure on NAc dopamine terminal function or the regulators of dopamine signaling. Prior studies have shown some changes in dopamine terminals, including changes in dopamine transporter activity and phasic dopamine release ,35. In pThus, the present study aimed to identify potential mechanisms underlying the cellular and function alterations in dopamine signaling following heroin self-administration. Because hypodopaminergia promotes escalations in drug-seeking and propensity for drug relapse, pinpointing a mechanism for reduced dopamine function following heroin self-administration may lead to potential novel targets for treatments.p < 0.0001) and session (p = 0.0140). Heroin infusions within the first hour increased across sessions (p = 0.0050), and time to acquisition was negatively correlated with average responding for heroin across the last three sessions (p = 0.0499).During acquisition, rats reached acquisition criteria on average in 8.488 ± 0.9821 days (mean ± SEM) B. After 62 = 3.193, p = 0.0022), but that dopamine uptake was not significantly different between heroin-exposed animals and saline controls (p > 0.05).Rats were sacrificed for FSCV approximately 18 h following the last heroin self-administration session. We found that single-pulse stimulated dopamine release was significantly lower in heroin-exposed animals (p = 0.0423), concentration  = 267.3, p < 0.0001), and condition × concentration interaction (p = 0.0114). In addition, the IC50 for PD-128907 was significantly decreased in heroin-exposed rats . This suggests that a lower concentration of agonist was needed to reduce the signal by 50% and indicates a greater agonist potency at D3 autoreceptors in heroin self-administering animals. However, the maximal effect at the highest concentration of PD-128907 (300 nM) was not significantly different between heroin-exposed rats and saline controls . In addition, the IC50 was significantly increased in heroin-exposed rats , suggesting reduced potency of the agonist. The maximal effect of the greatest sumanirole concentration (3000 nM) was also increased , indicating an increase in agonist efficacy.To examine changes in D2 autoreceptor-mediated inhibition of dopamine release, the selective D2 receptor agonist, sumanirole, was used D. Similap = 0.0238), concentration (p < 0.0001), but no significant condition × concentration interaction (p > 0.05). While the IC50 for U50 curves were not significantly different between heroin-exposed rats and saline controls (p = 0.0913), the maximal effect of the highest U50 concentration (3000 nM) was significantly decreased in heroin-exposed rats . These results indicate that heroin-exposed rats had an increase in U50 efficacy, but no change in U50 potency.To determine the functional responsivity of KOR following heroin self-administration, the selective KOR agonist, U50,488H (U50), was used. In addition, an IC50 of agonist responsivity and the maximal effect of dopamine inhibition at the highest concentration of the curve were calculated from each slice to evaluate the potency and efficacy of U50, respectively. Concentration response curves of KOR activation by U50 suggest an increased agonist-induced responsivity of KOR in heroin-exposed rats than in saline controls A. A two-p > 0.05); however, heroin-exposed rats displayed a frequency-dependent increase in relative phasic release compared to naïve controls when dopamine release was normalized to single-pulse release (p = 0.0008), frequency (p < 0.0001), and condition × frequency interaction (p = 0.001). A Bonferroni’s post-hoc analysis revealed significant differences at 40 Hz (p = 0.0224), 60 Hz (p = 0.0067) and 100 Hz (p = 0.0103). This effect is also reflected in the calculation of phasic/tonic ratio, which compares dopamine release evoked by five pulses at 60 Hz with dopamine release evoked by a single pulse, in heroin-exposed rats . These results suggest that chronic heroin self-administration may lead to reductions in basal dopamine release while enhancing the signal-to-noise ratio of phasic release events, thereby causing dopamine dynamics to be phasic-shifted. This increase in the gain of dopamine terminal dynamics could lead to increased reinforcement of heroin self-administration and thus drive the increased escalation of intake.MOR activation results in phasic dopamine firing, which is characterized by a “burst-like” pattern of increased action potentials at higher frequencies . To exam release B. A two-p < 0.0001) and stimulation amplitude x condition interaction (p < 0.0001). A Bonferroni’s post-hoc analysis did not reveal significant differences at any of the stimulation amplitudes between groups. This effect was quantified by measuring the individual amplitude needed to elicit the greatest amount of dopamine for each slice, which showed that heroin self-administering animals required lower stimulation amplitudes to drive maximal dopamine release across the stimulation intensities tested . When measuring the amount of dopamine released at the maximal stimulation amplitude (10 V), we found heroin self-administering animals had a lower dopamine release compared to saline controls . Overall, these results suggest that the dynamic range of dopamine release across varying stimulation intensities is reduced following heroin self-administration.In addition to examining release under “phasic-like” stimulation parameters, we also measured dopamine release by altering the electrical stimulation amplitude. Heroin self-administration increased dopamine release elicited at low stimulation amplitudes and decreased dopamine release elicited by high stimulation intensities D. A two-16 = 3.052, p = 0.0076). The next morning, the flow rate was increased to 1.0 µL/min to allow more rapid (20 min) sampling times. Following the collection of six total samples where dopamine levels were stable (<10% difference and no trend up or down) for at least three collections, all rats were administered 0.1 mg/kg heroin (IV), and samples were collected every 20 min for an additional 120 min. Extracellular dopamine levels were significantly increased in saline control rats following the heroin challenges compared to heroin-exposed rats. A two-way repeated-measures ANOVA revealed the significant main effects of the drug condition (p = 0.0010), time (p = 0.0370) and condition x time interaction (p < 0.0001). A Bonferroni’s post-hoc analysis revealed significant differences at the 40 (p = 0.0293) and 80 (p = 0.0128) minute time points. Analysis of the area under the curve for the magnitude of dopamine outflow showed dopamine release was significantly lower in heroin-exposed animals than in saline controls .Following one day of recovery after cannula implantation, microdialysis probes were inserted into the NAc, and aCSF was perfused at a flow rate of 0.5 µL/min. A single 15-h baseline sample was collected and analyzed for dopamine at equilibrium between the probe and extracellular tissue, to quantify basal extracellular levels. Approximately 24 to 32 h following the last self-administration session, heroin-exposed animals had a significant decrease in basal levels of extracellular dopamine when compared to saline controls  autoreceptors modulate the release, synthesis, and the firing rate of dopamine neurons, and are expressed on the soma, dendrites, and axon terminals of dopamine neurons ,61. WhilIn this study, we isolated the effects of D2 or D3 autoreceptors and excluded postsynaptic receptors by evoking dopamine release with electrical stimulation in nucleus accumbens slices and measuring the release-inhibitory effects of D2- or D3-preferring ligands. With the lower density  but greaOther studies have reported opposing D2R and D3R changes in mesolimbic regions following chronic exposure to opioids in rodents ,69,70,71Similar to the D3 autoreceptors, there was also increased activity of another inhibitory presynaptic dopamine regulator, the dynorphin/KOR system, following heroin self-administration. Previous studies show that KORs are located on presynaptic dopamine terminals and, upon activation with their endogenous ligand, dynorphin, strongly inhibit dopamine release . Prior sPerhaps the most surprising discovery was that dopamine release under “phasic-like” electrical stimulation was increased in the NAc following heroin self-administration while tonic release was reduced. However, while heroin-exposed rats exhibited greater release than the control group at low amplitude stimulations, at high amplitudes, there was reduced release. A previous study showed that MOR activation results in presynaptic facilitation in the NAc, likely through inhibition of cholinergic interneuron activity as seen with nicotinic antagonists . This prThis idea is further supported by evidence showing that chronic nicotine exposure leads to reduced single-pulse dopamine release, increased paired-pulse ratios, and increased phasic/tonic ratios in the NAc ,99. ThisOur group recently showed that extended heroin self-administration does not lead to increased dopamine release under “burst-like” stimulations in male rats . While tOverall, this study builds support for a mechanism in which chronic exposure to heroin results in reduced dopamine system function and highlights potential targets for pharmacological interventions, including presynaptic D3 receptors and KORs. Therapeutics aimed at restoring dopamine levels after chronic opioid exposure may be beneficial in treating OUD. While the current study measured outcomes during what would commonly be considered an “early withdrawal” time point, it would be interesting to evaluate if these alterations in dopamine function persist or are even exacerbated during late withdrawal.Adult male Long Evans rats  were given ad libitum access to food and water and maintained on a 12-h modified reversed light/dark cycle . Animal care procedures were in accordance with the National Institutes of Health guidelines in the Association for Assessment and Accreditation of Laboratory Animal Care and the experimental protocol was approved by the Institutional Animal Care and Use Committee at Wake Forest School of Medicine.n = 29; Saline: n = 21) were anesthetized with ketamine  and xylazine  and implanted with a chronic indwelling jugular catheter as described previously [Intravenous catheter surgery: Rats  schedule of reinforcement using a single active lever which, when pressed, delivered an intravenous infusion of heroin (0.05 mg/kg/inf). Acquisition self-administration sessions were initiated with the extension of the active lever into the chamber, with no experimenter-delivered priming infusions, and were terminated after 20 infusions or 6 hours, whichever occurred first. During the sessions, each lever response resulted in a 20-s time-out period in which the cue light was illuminated. Acquisition criteria were set as two consecutive daily sessions during which 20 infusions were obtained. When the acquisition criteria were met, rats were placed on a long access paradigm with unlimited access to heroin (0.05 mg/kg/inf) on an FR1 schedule of reinforcement during 6-hour sessions occurring across 10 consecutive days. A separate group of age-matched rats was given access to a saline-paired lever and allowed to self-administer saline under the same parameters of long access. Saline self-administration rats received approximately 20 sessions to match the sessions needed to complete acquisition and long access for heroin self-administering rats.2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2), NaHCO3 (25), glucose (11), L-ascorbic acid (0.4). A vibrating tissue slicer  was used to prepare 400 µm thick coronal brain slices containing the NAc core. Slices were transferred to a recording chamber and submerged in a bath of oxygenated aCSF (32 °C) perfused at a rate of 1 mL/min. A carbon-fiber microelectrode  and bipolar stimulating electrode were placed in the NAc core. Endogenous dopamine release was electrically evoked by a single pulse  applied to the slice every 3 min. Extracellular dopamine was measured by applying a triangular waveform  to the CFE. After an hour of slice acclimation and when the dopamine signal was stable for at least three successive collections, slices were used for one of the following experiments listed below.Fast-scan cyclic voltammetry (FSCV) was used to examine the presynaptic NAc dopamine terminal release kinetics and dopamine autoreceptor and kappa opioid receptor (KOR) activity. Approximately 18 h following the last long access session, rats were deeply anesthetized with isoflurane and rapidly decapitated. The brain was removed and immersed in oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl (126), KCl (2.5), NaHn = 8 rats/slices; Saline n = 11 rats/slices). The responsivity of presynaptic D2 autoreceptors was evaluated using the D2 agonist, sumanirole  . KOR activity was measured using the KOR agonist, U50-488H (1–3000 nM) . Each slice was exposed to only one drug. The interstimulus interval for all concentration curves was 3 min.D2/D3 Autoreceptor and KOR Activity: To probe the responsivity of presynaptic D3 autoreceptors in the regulation of terminal release, cumulative concentrations of the D3 agonist, PD-128907 , were bath applied to separate slices . A stimulation intensity curve was collected by using a single pulse stimulation at varying intensities, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 V with an interstimulus interval of 60 s . From this dataset, the stimulation intensity needed to elicit the greatest dopamine response and the dopamine change elicited at the highest stimulation intensity (10 V) was used to quantify these results.DA Release across Varying Stimulation Parameters: The effects of two different stimulation patterns on dopamine release were tested. A frequency-response curve was obtained using five pulse stimulations at 5, 10, 20, 40, 60, and 100 Hz frequencies with 5-min interstimulus intervals  following electrical stimulation.FSCV Data Analysis: Demon Voltammetry and Analysis software  was usedn = 5; Saline n = 5). Guide cannula  were stereotaxically implanted 2 mm above the NAc , as the probe extends 2 mm past the cannula. Concentric microdialysis probes  were inserted approximately 36 h post-surgery and continuously perfused with artificial cerebrospinal fluid . For basal extracellular dopamine level experiments, overnight samples were collected at 0.5 µL/min from 1700 h to 0800 h, at which time the flow rate was increased to 1.0 µL/min. Baseline samples were collected every 20 min for 2 hours before intravenous administration of 0.1 mg/kg heroin. Following administration of heroin, 20-min duration samples were collected for another 2 hours. All dialysate samples were frozen at −80° before the determination of dopamine concentrations by high-performance liquid chromatography (HPLC) with electrochemical detection Microdialysis surgeries were performed approximately 18 h following the final long access self-administration session  by standards with known dopamine concentrations.High-Performance Liquid Chromatography. All dialysate samples were analyzed using high-performance liquid chromography (HPLC)  coupled with electrochemical detection at +220 mV using a high sensitivity analytical cell 5011 A  on a Choulechem III Electrochemical Detector . Neurotransmitters and their metabolites were separated on a Luna 100 × 3.0 mm C18 3 μm HPLC reversed-phase column . The mobile phase consisted of 75 mM NaH2PO4, 1.7 mM 1-octanesulfonic acid sodium salt, 100 µL/L triethylamine, 25 µM EDTA, 10% acetonitrile t-test. Voltammetry concentration-response curves, multi-pulse release curves, and microdialysis data were subjected to a two-way ANOVA, with group differences being tested using Bonferroni’s post-hoc tests where noted. All data are reported as mean ± SEM. All p-values of <0.05 were considered to be statistically significant.GraphPad Prism 9  was used to statistically analyze all datasets and create graphs. Behavioral data were analyzed using two-way repeated-measures ANOVA followed by Bonferroni’s post-hoc test where noted. Pearson’s correlation tests were used to measure the strength of the correlations. Baseline voltammetry release and reuptake data, IC50, maximal effect, phasic/tonic ratio, microdialysis basal, and area under the curve data were compared using a Student’s


Noise is considered severe environmental pollutant that affects human health. Using sound absorption materials to reduce noise is a way to decrease the hazards of noise pollution. Micro/nanofibers have advantages in sound absorption due to their properties such as small diameter, large specific surface area, and high porosity. Electrospinning is a technology for producing micro/nanofibers, and this technology has attracted interest in the field of sound absorption. To broaden the applications of electrospun micro/nanofibers in acoustics, the present study of electrospun micro/nano fibrous materials for sound absorption is summarized. First, the factors affecting the micro/nanofibers’ sound absorption properties in the process of electrospinning are presented. Through changing the materials, process parameters, and duration of electrospinning, the properties, morphologies, and thicknesses of electrospun micro/nanofibers can be controlled. Hence, the sound absorption characteristics of electrospun micro/nanofibers will be affected. Second, the studies on porous sound absorbers, combined with electrospun micro/nanofibers, are introduced. Then, the studies of electrospun micro/nanofibers in resonant sound absorption are concluded. Finally, the shortcomings of electrospun micro/nano fibrous sound absorption materials are discussed, and the future research is forecasted. With the development of industrial production and urban construction, noise pollution is one of the four significant pollutants currently becoming an increasingly severe issue, highly endangering human health and restricting social development ,2,3. TheLow-frequency sound waves can easily bypass obstacles and are difficult to be absorbed by traditional sound absorption materials . HoweverElectrospun micro/nanofibers possess many advantages, such as small diameter, light weight, high specific surface area, and porosity. The sound absorption materials made of electrospun micro/nanofibers are promising for noise reduction, especially in traffic ,14,15. TFibers fabricated by electrospinning have micro and nanoscale diameters, which provide high specific surface area and porosity. These features of electrospun micro/nanofibers lead to good sound absorption performance in low and medium frequencies . In thisMicro/nanofibers possess characteristics which are beneficial to the absorption of sound waves, namely small diameter, large specific surface area, and high porosity. However, micro/nanofibers usually have thin thicknesses, resulting in poor sound absorption. Micro/nanofibers are commonly combined with traditional sound absorption materials to achieve a better sound absorption effect. Electrospinning, one of the main methods of preparing micro/nanofibers, is divided into two types: solution and melt electrospinning . There aThe factors that influence the sound absorption performance of electrospun micro/nanofibers involve parameters such as materials, fiber diameter, surface density, microstructure, and thickness. Materials determine the properties of electrospun micro/nanofibers, such as piezoelectric and elastic properties. The fiber diameter, surface density, and microstructure of the fibers are related to the solution properties, process parameters, and environmental parameters of the electrospinning technology . The thiAcoustic models can be applied to theoretically evaluate the sound absorption properties of the materials, which are useful for directing the design of sound absorption materials. Recently, several acoustic models used for investigating the SAC of sound absorption materials have been published ,44. PrevThe sorts of materials determine the properties of electrospun micro/nanofibers. Modifying the materials by altering the material or adding other matter can change the physical and chemical properties of the fibers, thus affecting the sound absorption performance of the materials.Polyvinylidene fluoride (PVDF) is a material with the piezoelectric property that can convert acoustic energy into electric energy, thus facilitating the absorption of acoustic energy . Wu et aThe elasticity of the materials may influence the resonant process between electrospun micro/nanofibers and sound waves. Good elasticity of materials may promote the consumption of acoustic energy by the resonance effect, thus improving the sound absorption performance of the materials. Park et al.  preparedDiameter and surface density are the critical factors affecting the flow resistivity and natural resonant frequency of the materials. Diameter impacts the specific surface area and porosity of electrospun micro/nanofibers, while the surface density determines the fibers’ distribution. The sound absorption capability of materials can be represented by flow resistivity, which reflects the extent of curvature on the path of sound waves traveling through the materials. Materials with a low natural resonant frequency usually exhibit good sound absorption capacity at low frequency. Thus, suitable diameter and surface density for electrospun micro/nanofibers enable good sound absorption. For instance, Akasaka et al.  preparedThe microstructures of the electrospun micro/nanofibers influence the sound absorption performance of the materials. For example, the bead structure will increase the porosity, surface area, and bulkiness of the fiber, causing irregular changes in the fiber diameter. Moreover, the core-shell or hollow structure may influence the density and natural resonant frequency of the electrospun micro/nanofibers, therefore modifying their response to sound waves at different frequencies. Therefore, electrospun micro/nanofibers with different microstructures can be fabricated by adjusting the process parameters and devices of electrospinning, and their sound absorption performance will be influenced. Zhang  discusseThe thickness of electrospun micro/nanofibers is a critical factor for their acoustic performance, affecting the propagation distance of sound waves within the fibers. Thickness variation of electrospun micro/nanofibers can be achieved by adjusting the electrospinning duration. Zou  reportedPorous sound absorption materials have many internal pores, which are beneficial for sound waves to enter the interior of the materials and consume energy by friction. The sound absorption principle of porous sound absorption materials is shown in Foams are typically porous materials with good sound absorption properties, and they have been widely used for sound absorption in the field of transportation . The celTraditional foam sound absorption materials have large pore sizes and low porosity. The sound waves cannot vibrate sufficiently inside the pores, leading to the poor sound absorption performance of the foams at low frequency. Increasing the thickness of the material or setting a cavity behind the foam are common methods used to promote their sound absorption performance at low frequency. However, these methods will cause the waste of material and space. Laminating electrospun micro/nanofibers with foam materials can enhance the sound absorption performance of foams at all frequencies, especially at low frequencies . Zou et The sponge is a type of acoustic foam with many applications in noise reduction. Still, its substantial water absorption property in wet environments leads to a poor sound absorption performance. Using electrospun micro/nanofibers to modify the sponge can improve its sound absorption performance and reduce the influence of the environment on sound absorption . Xiang eFibrous structures can absorb, reflect, and transmit the incident sound waves simultaneously . AccordiComposites of electrospun micro/nanofibers and natural fibers have improved sound absorption performance while retaining the environment-friendly and biodegradable features . For insSynthetic fibers are extensively applied in sound absorption fields due to their high performance and economical price ,79. The The high porosity and specific surface area of electrospun micro/nanofibers increase the contact between the material and sound waves. However, electrospun micro/nanofibers are usually deposited as thin mats or membranes, making them perform poorly in sound absorption. Thus, they cannot be used for noise reduction individually. For this purpose, researchers have investigated the development of new sound absorption materials made of electrospun micro/nanofibers.Electrospun micro/nanofibers can be made into excellent sound absorption materials by changing their microscopic or spatial structure. Chang et al.  electrosThe sound absorption performance of electrospun micro/nanofibers can also be influenced by adding fillers in the polymer solutions. Elkasaby et al.  reportedRecently, sound absorption materials made of aerogels have been reported. Aerogels are mesoporous and open-cell solids with high porosity and low density ,89. The Resonant acoustic materials have a high absorption coefficient at low frequencies, making up for the lack of sound absorption capacity of porous acoustic materials at low and medium frequencies . HoweverTraditional resonant acoustic materials show narrow sound absorption bands. However, decorating them with electrospun micro/nanofibers can effectively broaden the absorption frequency range and improve their sound absorption coefficients. Guo et al.  preparedAlthough composites made of electrospun micro/nanofibers and traditional resonant acoustic materials have a good sound absorption performance, they still have problems such as the large size and bulky structure, causing the waste of space and limiting their applications. Using electrospun micro/nanofibers as resonant acoustic materials individually is an effective solution to save space, expanding the applications of resonant acoustic materials. The Helmholtz resonator is a structure that absorbs sound waves by resonance, and electrospun micro/nanofiber membranes can form this structure by incorporating a cavity. Therefore, the sound absorption performance of the electrospun fiber membranes at the low and medium frequencies can be promoted by setting an air layer between the fiber membrane and the rigid wall . Liu et Furthermore, nanofiber membranes can vibrate at a low frequency when forced by sound waves, and the energy of sound waves will be absorbed by the principle of membrane resonance ,96,97. KThe optimization of the electrospun micro/nanofibers’ structure is a way to enhance their resonant sound absorption ability, which can turn them into a sort of resonant sound absorption material. Xu et al.  producedThe sound absorption frequency range of traditional resonant acoustic materials can be broadened by compounding them with electrospun micro/nanofibers. Yet, their sizes are still too large for commercial applications. Resonant acoustic materials manufactured with electrospun micro/nanofibers possess less volume, and their acoustic properties can be controlled by adjusting electrospinning parameters. Therefore, it is necessary to study the resonant sound absorption materials prepared by electrospinning.This review introduces the recent research of electrospun micro/nanofibers for sound absorption. Electrospun micro/nanofibers have advantages in sound absorption owing to their small fiber diameter, high specific surface area, and high porosity. Their sound absorption properties are determined by factors such as material, diameter, surface density, microstructure, and thickness, which can be controlled by changing the materials, parameters, and duration of electrospinning. Electrospun micro/nanofibers have various forms of sound absorption. The composite of electrospun micro/nanofibers and porous materials are effective in sound absorption at medium and high frequencies. Decorating resonant sound absorption materials with electrospun micro/nanofibers can enhance their sound absorption properties at a low frequency. In addition, electrospun micro/nanofibers with a 3D or Helmholtz resonator structure can also be deemed as sound absorption materials by themselves.In the future, electrospun micro/nanofibers will surely be promising materials for the application of sound absorption in many fields. However, the application of electrospun micro/nanofibers for sound absorption faces several challenges. First, the sound absorption properties of electrospun micro/nanofibers and their composites at low frequency require additional improvement. Theoretical research can reduce the cost and pollution caused by the experimental process, and it can study the factors affecting the sound absorption performance of electrospun micro/nanofibers. The sound absorption performance of materials can be predicted by establishing a sound absorption model. The accuracy of the prediction can be increased by modifying the model according to the material and structure of the electrospun micro/nanofibers. Second, traditional electrospun micro/nanofibers have many limitations in its application under several different conditions, such as moist and high temperature environments, owing to their poor thermal stability, water resistance, and mechanical properties. Therefore, the multifunctional modification of the fibers is necessary. Electrospun micro/nanofibers can obtain properties such as good water resistance and thermal stability by modifying the materials or preparing them using an innovative process, which is beneficial for broadening their application for sound absorption. Finally, most electrospun micro/nanofibers are fabricated by solution electrospinning, which exhibits the problem of low efficiency, and the use of solvents is usually corrosive and toxic. These characteristics of solution electrospinning are not beneficial to the industrial production of electrospun micro/nanofibers and may lead to environmental concerns. Hence, it is urgent to find ways to solve the problems inherent to solution electrospinning. Using water-soluble polymers in solution electrospinning or choosing eco-friendly melt electrospinning to replace the former in the future are suggestions for solving these problems. In conclusion, further exploration of electrospun micro/nanofibers will promote their sound absorption applications in many fields.


QY value, non-radiative deactivation channels are very important in the relaxation process of the electronic excited states of different dopamine species.To understand the photochemical behaviour of the polydopamine polymer in detail, one would also need to know the behaviour of its building blocks. The electronic absorption, as well as the fluorescence emission and excitation spectra of the dopamine were experimentally and theoretically investigated considering time-resolved fluorescence spectroscopy and first-principles quantum theory methods. The shape of the experimental absorption spectra obtained for different dopamine species with standard, zwitterionic, protonated, and deprotonated geometries was interpreted by considering the advanced equation-of-motion coupled-cluster theory of DLPNO-STEOM. Dynamical properties such as fluorescence lifetimes or quantum yield were also experimentally investigated and compared with theoretically predicted transition rates based on Fermi’s Golden Rule-like equation. The results show that the photochemical behaviour of dopamine is strongly dependent on the concentration of dopamine, whereas in the case of a high concentration, the zwitterionic form significantly affects the shape of the spectrum. On the other hand, the solvent pH is also a determining factor for the absorption, but especially for the fluorescence spectrum, where at lower pH (5.5), the protonated and, at higher pH (8.0), the deprotonated forms influence the shape of the spectra. Quantum yield measurements showed that, besides the radiative deactivation mechanism characterized by a relatively small  Research on dopamine (DA) is of ongoing interest due to its wide range of applications in many fields, such as biotechnology, nanotechnology, and advanced materials science. The successive oxidation processes of dopamine lead relatively easily to a more complex chemical structure known in the literature as polydopamine (PDA) ,2,3. PDAAlthough PDA shares many basic properties with amorphous organic semiconducting polymers such as melanin , many ofThe main goal of the present investigation is to give a detailed description of the dopamine molecular structure including their light absorption and emission properties, as well as electronic excited state lifetimes using time-resolved spectroscopy and theoretical molecular modelling techniques.All reagents were commercially available and used without further purification. The buffer solutions were prepared based on the standard procedures reported for air oxidation in acidic  or basic  conditions. UV–Vis and fluorescence spectra were measured at specific dopamine concentrations as follows: concentrated −0.7 mg/mL DA and diluted −0.05 mg/mL DA. Absorption spectra were recorded after the first 5 min of the sample preparation.The UV–Vis absorption spectra were recorded using a double-beam Jasco V550 UV–VIS spectrophotometer in the 200–800 nm spectral range with a 2 nm resolution, using 1 mm-length quartz cells. Baseline corrections were run successively in air and the solvent. The fluorescence emission and excitation spectra of dopamine aqueous solutions were acquired using a Jasco FP-6500 spectrofluorometer, equipped with a Xe lamp of 150 W, 1800 lines/mm monochromator, and a 1 nm spectral resolution. The samples were measured in 3 mm optical path quartz cuvettes. The quantum yield and fluorescence spectra of dopamine solutions at pH 5.5, respectively pH 8, were acquired using an FP-850 NIR spectrofluorometer with a 1 nm resolution for both excitation and emission, equipped with a 1800 lines/mm monochromator and an 100 mm integrating sphere (ILF-835).The fluorescence lifetimes were investigated by pumping the dopamine solutions with 280 nm laser pulses. The pump was generated by frequency doubling the 560 nm second harmonic of the 1120 nm idler beam obtained using an optical parametric amplifier (OPA) . The OPA was pumped by a 1030 nm Yb:KGW pulsed laser  with a 170 fs pulse duration and an 80 kHz repetition frequency. The fluorescence kinetics was investigated using a time-correlated single photon counting (TCSPC) setup . The samples were excited with vertically polarized light, and the fluorescence emission was collected through a long-pass filter suitable for the sample emission and an emission polarizer set at the magic angle (54.7° relative to the polarization of the pump pulse). The fluorescence signal was focused on the entrance slit of a double monochromator and detected using a single-photon-sensitive photomultiplier (Becker&Hickl PMC-100-1 standard). The FWHM of the instrument response function (IRF) was 340 ps, and the IRF was measured on each day of the experiment. For the TCSPC measurements, the average power of the pump beam was 3 to 10 mW at the probe location. For fitting the kinetic curves, we employed the reconvolution method available in the Easy Tau 2.0 software from PicoQuant with both one and two exponentials in order to determine the best-fitting parameters.QY) was calculated using the FP-8600 spectrofluorometer from JASCO equipped with an ILF-835 integrating sphere accessory. The measurements were performed in a quartz cuvette of 3 nm thickness under 280 nm excitation, while the quantum yield was obtained via the spectra manager software provided by JASCO. The QY calculations are based on the following equation:ELLThe absolute quantum yield ) and Duschinsky rotations between modes of different states [The equilibrium geometries and normal mode vibrational frequencies were obtained in the framework of density functional theory (DFT) considering the B97X exchange-correlation (XC) functional  combinedam suite ,40. The am suite . The RIJam suite  designedam suite  auxiliaram suite  auxiliaram suite ,46,47,48t states . The solt states .UV–Vis absorption, as well as the fluorescence excitation and emission spectra were recorded for aqueous DA solutions with different DA concentrations see . The mosAs regards the pH dependence of UV–Vis absorption, as well as the fluorescence excitation and emission spectra, several solutions of diluted DA with pH ranking from 5 up to 12 were also prepared, and their spectra were recorded see in . As can quinone) . These nquinone)  form, whormation ,53,54,55ormation ,57. DetaAccordingly, a total of four different conformations of dopamine were considered. Namely, the standard form, which we denote as DA, the zwitterion form as DAf the DA . It was As far as the fluorescence emission spectra are concerned, it can be observed that the frequency of the maximum of the spectrum does not depend on whether the DA solution was prepared in concentrated or diluted form. For each DA geometry conformation, a very broad peak was found for the fluorescence spectrum with a maximum around 317 nm  intensity decrease for the pH 8 solution in comparison to the pH 5.5 solution. At first glance, the two absorption spectra  and diluted forms than was observed when varying the pH of the solvent. However, it should also be noted that, based on theoretical calculations, STime-correlated single-photon counting (TCSPC) was applied to determine experimentally the fluorescence lifetimes of dopamine in buffer solutions at pH 5.5 and 8, respectively. When the obtained fitting curves were analysed together with the corresponding kinetic curves of the signal from the concentrated dopamine solution, it was observed that the curve obtained with the one-exponential fitting did not cover the tail of the 322 nm kinetic curve, in contrast to the curve obtained with the two-exponential fitting. The residuals for the fitting procedure with the two-exponential function showed an improvement in the time scale corresponding to the tail of the kinetic curve. When only one exponential function was employed, the resulted fluorescence lifetime was Moving on to the dopamine solution at pH 8 see , a decreFor the dopamine solution at pH 9, the dominant fluorescence lifetime remained (i) the so-called Franck–Condon (FC) approximation, where the electronic transition is considered “vertically”, meaning that the electronic transition is most likely to occur without changes in the positions of the nuclei in the molecular entity and its environment; (ii) the Herzberg–Teller (HT) approximation, where, besides the “vertical” transition, also the vibronic coupling is taken into account; (iii) the Duschinsky (D) rotations between normal modes of different states is also considered.The theoretical estimation of different fluorescence lifetimes for the four DA conformations was performed at the ωB97X-D3/ma-def2-TZVPP level of theory by computing the vibrational normal modes both for ground and first excited states. Three different levels of approximation were considered ,60: (i) (i)–(iii) levels of approximation are collected in The fluorescence lifetimes calculated as the inverse of the radiative transition rate for the four DA species considering the three As far as the correlation between experimental and theoretical results on fluorescence lifetimes is concerned, a very good agreement in terms of the numerical value and magnitude was found. Furthermore, it is possible to make a correspondence between lifetimes and different DA geometries. Accordingly, the slightly longer As was shown especially for the DAl energy ,62. AccoQY of dopamine in buffer solutions at pH 5.5 and 8, respectively, under 280 nm excitation  were investigated considering steady-state and time-resolved spectroscopy and first-principles quantum theory methods. Different peaks found in the experimental absorption and fluorescence excitation spectra were attributed to electronic transitions occurring in different dopamine species based on DLPNO-STEOM coupled-cluster calculations. The results show that not all spectra can be attributed to only one or another dopamine species, but there are cases of concentrated and higher pH dopamine solutions where a mixture of different species of protonated, standard, and zwitterionic geometries give the final spectra. On the other hand, it was found that the fluorescence excitation spectrum of dopamine can be considered not so much pH-dependent as concentration-dependent. The fluorescence lifetimes measured for concentrated or diluted, as well as for solutions with a low or high pH using the time-correlated single-photon counting method were in the range of 1 ns, values that were confirmed by the results of theoretical calculations based on the path integral approach. The used approximation levels for the theoretical estimation of the fluorescence lifetime also proved that the fluorescence deactivation is not entirely based on Franck–Condon effects; vibronic couplings covered by Herzberg–Teller effects also have a significant contribution to the fast radiative relaxation. Furthermore, quantum yield measurements showed that, besides the radiative deactivation mechanism characterized by a relatively small


Bioinformatics has gained much attention as a fast growing interdisciplinary field. Several attempts have been conducted to explore the field of bioinformatics by bibliometric analysis, however, such works did not elucidate the role of visualization in analysis, nor focus on the relationship between sub-topics of bioinformatics.First, the hotspot of bioinformatics has moderately shifted from traditional molecular biology to omics research, and the computational method has also shifted from mathematical model to data mining and machine learning. Second, DNA-related topics are bridge topics in bioinformatics research. These topics gradually connect various sub-topics that are relatively independent at first. Third, only a small part of topics we have obtained involves a number of computational methods, and the other topics focus more on biological aspects. Fourth, the proportion of computing-related topics hit a trough in the 1980s. During this period, the use of traditional calculation methods such as mathematical model declined in a large proportion while the new calculation methods such as machine learning have not been applied in a large scale. This proportion began to increase gradually after the 1990s. Fifth, although the proportion of computing-related topics is only slightly higher than the original, the connection between other topics and computing-related topics has become closer, which means the support of computational methods is becoming increasingly important for the research of bioinformatics.The results of our analysis imply that research on bioinformatics is becoming more diversified and the ranking of computational methods in bioinformatics research is also gradually improving. Bioinformatics originated as a cross-disciplinary field because of the increasing need for computational solutions to research problems in biomedicine . Since tBibliometrics is the application of mathematics and statistical methods to evaluate the literature in different disciplines. Patra et al.  analyzedThe development of text-mining techniques provides a new perspective for bibliometric analysis. Topic model is the most prevalent approach among techniques applied for bibliometric analysis. Latent Dirichlet Allocation(LDA) is one of the most popular models applied for bibliometric analysis. Song et al.  attempteTraditional bibliometric analysis lacks thematic descriptions of bioinformatics subtopics, and the above-mentioned works filled that gap. However, LDA is a typical bag-of-words model that has two major weaknesses: it loses the ordering of the words and ignore their semantics. We choose another technical scheme: paragraph embedding , dimensiScholarly data visualization enables scientists to have a better way to represent the structure of data sets and reveal hidden patterns in the data . HoweverIn this study, we fully utilized the ability of text mining technology to extract abstract paradigms from massive data and the ability of visualization to display complex information. First, we revealed the distribution of topics in two-dimensional document space by drawing scientific maps of bioinformatics, and then checked the evolution of topic relationships with time filters. Finally, to further understand the interdisciplinary nature of bioinformatics, we combined themeriver with words co-occurrence network to elaborate the evolution of computing-related topics.Compared with previous works, present study has the following contributions: (1) A science map of bioinformatics was drawn to depict the intersection and evolution of sub-topics of bioinformatics. (2) The interdisciplinary nature of bioinformatics was explored emphatically analyzing the evolution of computing-related topics. (3) The validity and necessity of visualization in analysis were pointed out and proved.In this section, we will introduce the final visualization results. The goals of our visualization are twofold: (1) intuitively show the change in the popularity of different topics over time; and (2) clearly demonstrate the relationship between topics and how this relationship changes over timeGoal 1 can be achieved by applying themeriver , where tAs shown in Fig. Science map originated from a traditional scientific notation called “knowledge tree” . AccordiTo show the relationship between bioinformatics papers in a two-dimensional space, we need to perform dimensionality reduction again on the document vectors obtained in the previous step. This time we still chose UMAP to reduce the vectors’ dimension to 2, which will enable us to project all vectors into two-dimensional space. We choose UMAP for two reasons: first, according to Espadoto et al. ,UMAP per2Each point represents a paper, and color codes the cluster it belongs to. However, when there is a larger number of points scatter plots may cause a misleading that the greater scope covered by an area, the more is the quantity of its papers. To eliminate this misleading and make the overall structure more visible, we implemented a contour line, where color depth is proportional to the density of papers, and line spacing is inversely proportional to the density gradient. The final science map of bioinformatics is shown in Fig. In this section, we analyzed the results from three different perspectives: knowledge structure of bioinformatics, evolution of knowledge structure, and evolution of computing-related topics.As shown in Fig. Although themeriver can describe the changes in research hotspots, it cannot sketch the relationship between topics and thus cannot accomplish our goal 2. Thus, we need a science to map the whole knowledge structure of bioinformatics. By analyzing Fig. With the continuous development of bioinformatics, the relationship between topics is also changing. From 1960 to 2019, the change in the number of papers on different topics is shown in Fig. At the beginning of the development of bioinformatics, the relationship between the main topics 1, 2, 4 and 5 was relatively weak. However, from 1980 to 2000, the 2,4,5 topics began to merge to a certain extent. We selected the fusion part interactively and examined at the high-frequency MESH phrases discussed in the merged papers. The top six phrases are: escherichia coli, amino acids, binding site, plasma membrane, binding sites, and signal transduction, which are all related to biological macromolecules  has not become the main hot topic at that time, and has not produced more contact with Topic 3 and 8, but produced more fusion with topic 2, indicating that omics research began with DNA. The period from 2005 to 2010 is a period of vigorous development of bioinformatics. Topic 6, as a bridge topic and a hot topic, began to connect various parts  What is the proportion of computing-related topics in bioinformatics? Is this ratio stable? (2) What changes have taken place in the biological topics and computational methods involved in the evolution of these topics?Goal 1 can be achieved by simply applying themeriver. We will mainly introduce the solution to Goal 2.As shown in Fig. The proportion of papers that related to computation evidently changes. The proportion was close to 0.3 in the early stage of the development of bioinformatics, began to decline in the early 1980s, and only accounted for about 0.1 of the total papers in the early 1990s. The proportion then began to rise again in the middle and late 1990s, surpassing the initial proportion and occupying nearly half of the bioinformatics topics after 2005. During such changes, an obvious pattern is observed: although the proportion of computing-related topics is only a little higher than the original, the internal structure of those topics has undergone earth-shaking changes.Through further analysis of the co-occurrence word network, we conclude the following. (1) Prior to 1980, Topic 1 occupied an absolute dominant position. The computational methods involved were mainly mathematical models, and computer simulation appeared after 1970. The main biological topics discussed during this period include: active transport, cell, etc. (2) The period of 1990-2000 was a turning point for major changes in the internal structure of computing-related topics. The overall proportion began to rise, but the popularity of Topic 1 remained the same; and Topic 8 gradually replaced Topic 1 to occupy the dominant position. No new computational method was found in Topic 8, but the discussion on sequence increased significantly. (3)The period of 2000 - 2010 is an era of data and genes. The popularity of topics 3 and 8 continues to rise, but the content of the discussion has undergone great changes. The computational topics are all centered on data, data analysis, and the popularity of gene expression is the highest in biological topics while discussions on sequences began to decrease. Topic 9 has also seen a significant increase, and the computational methods involved are mainly related to data; however, biological topics focus on magnetic resonance and images. (4) In 2010 - 2019, the overall structure has not changed, and the proportion of each topic has remained relatively stable, however, the contents they discussed are different from those before. During this period, more computing methods are involved, such as machine learning, data mining, text mining, cloud computing and so on. Thus the interaction between computing methods and bioinformatics becomes increasingly closer and closer.As an interdisciplinary and fast-growing field of science, bibliometric analysis of bioinformatics has attracted the attention of many researchers. In this study, we collected 330192 bioinformatics papers and applied Doc2vec combined with clustering and dimension reduction technology to detect the knowledge structure of bioinformatics. And then we focus on the role of visual analysis in exploring this structure. Unlike previous works, we focus more on substructures’ relationship. The evolution process of computing-related topics was emphatically analyzed which is vital for understanding the interdisciplinary nature of bioinformatics. The results of our analysis imply that research on bioinformatics is becoming more diversified; the ranking of computational methods in bioinformatics research is also gradually improving. In the future, we plan to enrich and complete the knowledge structure diagram of bioinformatics by applying visualization to explore other aspects of bibliometric analysis, such as author analysis, organization analysis, citation analysis, etc.In this section, we will introduce the overall procedure of the proposed approach for visualizing the knowledge structure of bioinformatics. Figure Data were obtained from Microsoft Academic Graph (MAG) . MAG is A total of 330,192 papers were analyzed in the following discussion. We then extracted the title and abstract of these papers from the MAG to form a corpus. Detecting phrases in the corpus is crucial because the semantics of these phrases change once they are split into words. So after removing the stop words, we used phrases in Medical Subject Headings(MESH) to annotate phrases in our corpus. These phrases used to analyze bioinformatics literatures in many previous studies , 6 to deParagraph embedding is an unsupervised framework that learns continuous distributed vector representations for documents. Semantic similarity between documents can be obtained through calculating cosine similarity between vectors. Paragraph embedding is implemented in the gensim package , in which the representation vector of each word in the corpus can be obtained at the same time as the document vector by setting parameter ‘dm’ to 1, which is used to calculate the semantic similarity between words. The selection of parameters is based on the best practice given by Lau : window To have a preliminary understanding of the knowledge structure of bioinformatics, we analyzed MESH term vectors first which is also better for subsequent document classification and interpretation. We used HDBSCAN , an outsClustering the document vectors can help us to obtain the topic distribution of bioinformatics and further understand the knowledge structure. Compared with LDA, our method can flexibly select the clustering method and the number of categories according to the clustering results after obtaining the vectors. After several rounds of attempts, we chose Gaussian Mixture Model (GMM). GMM is similar to K-Means, but it can be used as soft clustering, giving the probability of data points being assigned to a category.D| is the number of clusters and To avoid high computational cost and remove noises of data, we used PCA to reduce the dimension of document vector from initial 300 to 50. To preserve data’s nonlinearity, we used the nonlinear dimensionality reduction algorithm UMAP  to furthResults are shown in Table To further investigate bioinformatics topics related to computational methods, we combine topic clustering and co-occurrence words analysis to construct a co-occurrence word network. The calculation method is as follows:The data set of each topic was divided into six time slices to show the changes in each topic over time. From 1960 to 2019, each time slice represents 10 years. We calculated the tf-idf value of MESH phrases in different time slices in each topic, and then divided top 10 phrases into computational phrases and biology phrases according to MESH classification results. A co-occurrence word network between computational phrases and biology phrases is constructed to illustrate this time slice.


CFI) gene, associated with low serum factor I (FI) levels, are strong risk factors for developing the advanced stages of age‐related macular degeneration (AMD). No studies have been undertaken on the prevalence of disease‐causing CFI mutations in patients with geographic atrophy (GA) secondary to AMD. A multicenter, cross‐sectional, noninterventional study was undertaken to identify the prevalence of pathogenic rare CFI gene variants in an unselected cohort of patients with GA and low FI levels. A genotype‐phenotype study was performed. Four hundred and sixty‐eight patients with GA secondary to AMD were recruited to the study, and 19.4% (n = 91) demonstrated a low serum FI concentration (below 15.6 μg/ml). CFI gene sequencing on these patients resulted in the detection of rare CFI variants in 4.7% (n = 22) of recruited patients. The prevalence of CFI variants in patients with low serum FI levels and GA was 25%. Of the total patients recruited, 3.2% (n = 15) expressed a CFI variant classified as pathogenic or likely pathogenic. The presence of reticular pseudodrusen was detected in all patients with pathogenic CFI gene variants. Patients with pathogenic CFI gene variants and low serum FI levels might be suitable for FI supplementation in therapeutic trials.Rare variants in the complement factor I  gene mutations in an unselected cohort of patients with geographic atrophy secondary to age‐related macular degeneration. Reticular pseudodrusen was detected in all patients with a rare  The results of this study should therefore inform what proportion of GA patients might have FI disease‐causing mutations. This would be useful information for the design of future therapeutic trials.The aims of this study were to (1) identify, for the first time, the prevalence of pathogenic 22.1This study was conducted in accordance with the Research Governance Framework for Health and Social Care (2005) and Good Clinical Practice. Ethical approval was obtained from Yorkshire & The Humber‐South Yorkshire Research Ethics Committee. This study adhered to the tenets of the Declaration of Helsinki. The University Hospital Southampton NHS Foundation Trust was the sponsor of this study, and The University of Southampton, through its Faculty of Medicine, undertook the research study. All patient samples and data were anonymized for the purpose of this study. Patient DNA and serum samples were stored for future studies. Procedures for handling, processing, and storage of patient data were in compliance with the UK Data Protection Act (1998). This study is registered on a publicly accessible database: The NIHR Clinical Research Network Portfolio ID 34996.2.2Twelve hospitals in the United Kingdom identified patients with a diagnosis of GA secondary to AMD from routine outpatient clinics, or invited patients from their local databases, to participate in this study. Informed consent was obtained from all patients before inclusion in the study. The main inclusion criterion for this study was the diagnosis of GA secondary to AMD. The exclusion criteria for this study were: (1) any other ocular disease, which could mask the condition being studied (GA secondary to AMD) due to confounding pathology; and (2) any history of neovascular (wet) AMD in either eye.2.3All patients recruited to this study with a diagnosis of GA were invited for an initial visit where their demographics and medical history were recorded. Patients provided blood samples for measurement of serum FI and C‐reactive protein (CRP) concentrations, and for potential DNA sequencing. Patient blood samples were collected and shipped in dry ice to Eurofins Central Laboratories. Kits for collection and transport of study samples to the laboratory were provided by Eurofins.2.4Serum FI concentrations were measured commercially by Eurofins Central Laboratory, using enzyme‐linked immunosorbant assay (ELISA) (Hycult Biotech).The lower limit of normal was defined as the lower 2.5th percentile of serum FI measured from 159 AMD subjects (13.1 μg/ml) plus 18% of this value to allow for assay variation, giving a final threshold of 15.6 μg/ml.2.5CFI gene was undertaken to investigate if rare CFI gene variants were present. Although there is no consensus in the literature on the minor allele frequency (MAF) of a rare genetic variant, for the purposes of this study, a rare genetic variant was described as having an MAF of less than 1%, as defined previously , The Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK, as previously described ; rs1061170 (NM_000186.4:c.1204C>A); rs10737680 (NC_000001.11:g.196710325A>C); rs1329428 (NC_000001.11:g.196733680C>T); CFI region: rs17440077 (NC_000004.12:g.109616411A>G); rs4698775 (NC_000004.12:g.109669323G>T); rs2285714 (NM_030821.5:c.345G>A); CFB region: rs429608 (NC_000006.12:g.31962685G>A); C3 region: rs2230199 (NM_000064.4:c.304C>G); ARMS2 region: rs10490924 (NM_001099667.3:c.205G>T). The expected false‐negative rate was less than 1%. Minor allele frequencies of rare CFI gene variants quoted in this study were based on population frequencies  according to the genome aggregation database (gnomAD): http://gnomad.broadinstitute.org.If patient serum FI concentrations were detected to be low (below 15.6 μg/ml), DNA sequencing of the 2.6CFI gene variant on DNA sequencing were invited back for a second hospital visit to undergo a dilated retinal examination and retinal imaging. The imaging consisted of spectral domain optical coherence tomography (SD‐OCT), fundus autofluorescence , and color fundus photographs. All patient retinal images were submitted to the University of Southampton, where they were reviewed by two ophthalmologists (AHK and GDS) masked to the results of CFI gene sequencing. A third ophthalmologist (AJL), also masked to the results of gene sequencing, reviewed specific patient retinal images in the event of a lack of agreement during the initial imaging review. The presence of reticular pseudodrusen (RPD) on retinal imaging was based on the four stages of RPD described by Querques et al.  was used for statistical analyses and graphical representation of the data obtained in this study.t test with Welch's correction was used to determine statistically significant differences in age, number of SNPs at 10 common AMD loci, and average serum FI and CRP concentrations between groups. Statistical significance was set at the p < .05 value. Single variant association studies with 2 × 2 contingency tables were used to determine statistically significant associations between factors in this study: these include the presence of CFI gene variants and association with the sex of the patient, and the presence of RPD associated with CFI gene variants. A Bonferroni correction was employed in the event of multiple analyses from the same pool of data. Both the Pearson rank and linear regression tests were employed to determine correlation and regression between variables in this study.The unpaired 33.1SD = 8.1]. Of those recruited, 36.1% (n = 169) were male and 63.9% (n = 299) were female. Subjects' serum FI concentrations ranged from 6.2 to 36 μg/ml, with a mean concentration of 19.6 μg/ml . There was minimal skew to the data. Serum CRP concentrations ranged from 0.3 to 59 mg/L, with a mean concentration of 4.2 . The demographics of the patients and key clinical findings of the study are summarized in Table n = 322) and corresponding serum FI concentration were plotted on a graph . Linear Regression testing demonstrated weak goodness of fit with R2 = .06072.A total of 468 patients with a diagnosis of GA were recruited from 12 UK hospitals to take part in this study  of total recruited patients had a serum FI concentration below 15.6 μg/ml, designated as the lower limit of normal plasma FI concentration in this study . Rare CFI gene variants, with a MAF of less than 1%, were detected in 22 patients . The prevalence of rare CFI gene variants in patients with low serum FI levels was thus 25%. All patients in whom a low serum FI level and rare CFI gene variant were both detected, described their ethnicity as “White: British”. There was no statistically significant difference in age between those patients with a CFI gene variant  and those without a CFI gene variant  using an unpaired, two‐tailed, t test (p = .5072). The percentage of patients with a rare CFI gene variant who were male was 27.3% (n = 6). The percentage of females was 72.7% (n = 16). Using a 2 × 2 contingency table, and a Fisher's exact test, there was no statistically significant association detected between the expression of a rare CFI gene variant and the sex of the patient (p = .4968).Altogether, 19.4%  and those without a CFI variant  using an unpaired t test with Welch's correction (p < .001). Patients with a rare CFI gene variant demonstrated an additional reduction in serum FI levels compared to those patients without a CFI gene variant.The serum FI concentrations of 88 patients with low serum FI levels (<15.6 μg/ml) were displayed graphically according to the detection of rare s Figure . There wCFI gene variant  and those expressing normal serum FI levels ≥15.6 μg/ml , based on an unpaired, two‐tailed t test with Welch Correction (p = .003)  and patients expressing serum FI level ≥15.6 μg/ml (p < .0001).The serum CRP concentration of patients recruited to this study with a low serum FI level and GA was also investigated. There was a statistically significant difference detected in mean serum CRP concentration between patients with a low serum FI concentration and a ) Figure . Only th3.2CFI gene variant was detected, were invited for a second visit to undergo a fundus examination and retinal imaging (one patient died before visit two). All 21 patients with CFI gene variants are listed in  guidelines, is listed with associated references. Of these 21 patients, 15 expressed a rare CFI gene variant that is pathogenic or likely pathogenic. This equates to 3.2% of total patients recruited to this study. The largest proportion of these patients (n = 10) expressed the CFI p.Gly119Arg (NM_000204.3:c.355G>A) variant. Four patients expressed the following rare pathogenic CFI gene variants: p.Pro50Ala (NM_000204.3:c.148C>G), p.Ala258Thr (NM_000204.3:c.772G>A), p.His418Leu (NM_000204.3:c.1253A>T), and p.Ala431Thr (NM_000204.3:c.1291G>A). Additionally, a fifth patient expressed a rare CFI gene variant, which is predicted to be pathogenic: p.Arg502Cys (NM_000204.3:c.1504C>T).Twenty‐one patients in this study with a low serum FI concentration, in whom a rare CFI gene variant of uncertain clinical significance: Three patients expressed the CFI p.Gly261Asp variant (NM_000204.3:c.782G>A), one patient expressed the p.Arg406Cys variant of CFI (NM_000204.3:c.1216C>T), and two patients expressed synonymous CFI gene variants: p.Thr304= (NM_000204.3:c.912A>G) and p.Gly516= (NM_000204.3:c.1548T>C).A total of 6 of 21 patients with low serum FI concentrations expressed a rare 3.3CFI gene variant were reviewed by up to three ophthalmologists and a consensus was reached on phenotypic properties. A common property was the presence of RPD in all 15 patients with GA expressing a pathogenic or likely pathogenic rare CFI gene variant described above and listed in Table CFI variant (NM_000204.3:c.355G>A). Four of the six patients expressing a CFI gene variant of uncertain clinical significance (based on bioinformatic prediction), also expressed RPD. Representative SD‐OCT images of patients with low serum FI, that display both GA and stages of RPD  and RPD using a 2 × 2 contingency table and a Fisher's exact test with a two‐sided p value (p = .0714). The comparison group was the remaining group of six patients with CFI gene variants of uncertain clinical significance.Retinal images taken of the 21 GA patients with low serum FI levels and expression of a rare s et al. ; 2013 cl3.4CFI gene variant pathogenicity. There was a statistically significant difference in mean serum FI concentration between patients expressing a pathogenic CFI gene variant  and patients expressing no CFI variants , based on an unpaired, two‐tailed t test with Welch Correction (p < .0001) . The difference in mean serum FI concentration between patients expressing a rare CFI gene variant of unknown clinical significance  and those expressing no CFI gene variants did not reach statistical significance (p = .0691).The serum FI concentrations of 88 patients with GA and low serum FI levels (<15.6 μg/ml) were displayed graphically according to rare ) Figure . Patient3.5n = 88) underwent DNA sequencing at 10 common AMD loci in addition to the sequencing of the CFI gene. Sequencing results were used to investigate: (1) an association between rare CFI gene variants and SNPs at the CFI region of chromosome 4, which could suggest linkage disequilibrium; and (2) an association between rare CFI gene variants and complement risk SNPs at common AMD loci that could suggest the influence of overall complotype on FI activity and the alternative complement pathway in GA  achieved was for the presence of SNP rs2285714 at the CFI region (PLA2G12A: c.345G>A), but this was in the absence of a rare CFI gene variant . No statistically significant associations were otherwise reached.All patients recruited to this study with GA and a serum FI concentration less than 15.6 μg/ml , but this was in the absence of a rare CFI gene variant: there were 18 cases of homozygosity of this SNP in the absence of a CFI gene variant compared to one case of SNP homozygosity in the presence of a CFI gene variant. After Bonferroni correction, this did not reach statistical significance . No other statistically significant associations were reached. Last, contingency tables were used to investigate associations between CFI gene variant expression and four different haplotypes, which each include SNPs at two common AMD loci  demonstrated a low serum FI concentration below 15.6 μg/ml (definition of threshold described in the methods section). CFI gene sequencing undertaken on all patients with a low serum FI concentration detected rare CFI gene variants in 4.7% of total recruited patients with GA. The prevalence of CFI variants in patients with low serum FI levels and GA was 25%. There was no association detected between either age or sex and the presence of CFI gene variants in these patients. Patients with low serum FI levels and a rare CFI gene variant demonstrated an additional reduction in serum FI concentration that was statistically significant compared to those patients without a CFI gene variant.The CFI study recruited 468 patients from 12 UK hospitals. The aim of the study was to identify the prevalence of patients with GA who express low systemic levels of FI, in addition to those with rare n = 15) expressed a pathogenic or likely pathogenic CFI gene variant. The largest proportion of these patients (n = 10) expressed the CFI p.Gly119Arg variant (NM_000204.3:c.355G>A), which has robust evidence of pathogenicity in previous studies  expressed the CFI p.Gly261Asp variant. Interestingly, this is in contrast to previous studies in which this variant was associated with normal serum FI levels . This could suggest influence of overall complotype on FI levels or activity in all patients with GA and a serum FI concentration less than 15.6 μg/ml (n = 88). After Bonferroni correction, no statistically significant associations were observed in these patients using single‐variant studies. Furthermore, as the risk SNPs for CFH (rs1061170) and ARMS2 (rs10490924) have a disproportionately larger AMD risk relative to other common SNPs tested, we reviewed their distribution amongst patients with CFI gene variants. Interestingly, of 22 patients with rare CFI gene variants, the CFH SNP was detected in 73% (n = 16) of them, with 27% (n = 6) being homozygous. In addition, the ARMS2 SNP was detected in 68% (n = 15) of these patients, with 9% (n = 2) being homozygous. However, there was no statistically significant association observed when assessing all GA patients with reduced serum FI concentration.This study also assessed whether there was an association between rare CFI gene variant was detected in only 22 of them. It is possible that in patients without rare CFI gene variants, low serum FI concentrations were the result of nongenetic factors, such as consumption of FI due to increased complement activity. Increased complement activity is well known in advanced AMD patients  expressing the p.Gly119Arg CFI variant. 90.5% (n = 19) of all GA patients in this study expressing low serum FI levels and any CFI gene variant displayed RPD on imaging. A statistically significant association could not be reached between pathogenic or likely pathogenic CFI gene variants and the presence of RPD, compared to those patients with rare CFI gene variants that were not classified as pathogenic . With advances in retinal imaging, clinical studies have revealed the presence of RPD to be an independent risk factor in AMD progression , have been identified in families with AMD, in line with the hypothesis that rare variants cluster in families , so that they could inform family members to undergo an ophthalmic examination under the National Health Service.The assessment of rare CFI gene variants could potentially benefit from an early FI supplementation approach. This would most likely be a cost‐effective and time‐saving method than undertaking DNA sequencing on all patients. CFI gene sequencing would cost approximately 300 GBP (400 USD) per patient, involving DNA extraction, PCR, purification, sequencing, and multiplex ligation‐dependent probe amplification, followed by analysis. This could take up to approximately 2 weeks if several patient samples have to be analyzed. In contrast, an ELISA for FI takes approximately 1 day to perform and would cost approximately 40 GBP (50 USD). The fact that this study was able to recruit 486 patients on a limited budget attests to the practicality of this approach.This study raises the prospect that patients with early features of AMD (including those with high‐risk features such as RPD) could undergo a blood test to measure serum FI levels, and those expressing low serum FI levels and pathogenic rare CFI variant detected in this study (known to result in reduced FI levels), and has a population frequency of 0.0008519  according to the genome aggregation database (gnomAD), it is estimated that approximately 235 cases of GA (0.08%) could be attributed to this gene variant in the United Kingdom. The annual number of incident cases of GA in the UK has been estimated at 43,700 ; Rashi Arora: none; Geeta Menon: none; Priya Prakash: none; Tunde Peto: none; Quresh Mohamed: Allergan (Consult), Bayer (Consult), Novartis (Consult), Roche (Consult); Winfried Amoaku: Bayer Int (Grant), Novartis (Interest), Boerhinger Ingelheim (Grant), Abbvie/Allergan (Interest), Roche (Interest); Clare Bailey: Bayer (Consult), Novartis (Consult), Roche (Consult), Alimera Sciences (Consult), David Steel: Gyroscope Therapeutics , Alcon , Bayer (Grant), Roche (Interest); Sobha Sivaprasad: Allergan , Bayer , Novartis , Boerhinger Ingelheim , Oxurion (Interest), Apellis (Interest), Heidelberg Engineering (Interest), Optos ; Paul N. Bishop: Complement Therapeutics ; Peter Charbel Issa: Gyroscope Therapeutics (Consult); Robert E. MacLaren: Gyroscope Therapeutics ; David Kavanagh: Gyroscope Therapeutics ; Andrew J. Lotery: Gyroscope Therapeutics .http://gnomad.broadinstitute.org.Genome aggregation database (gnomAD): Supplementary information.Click here for additional data file.