The spinal cord also expresses all dopamine receptors; however, how the specific receptors regulate spinal network output in mammals is poorly understood. We explore the receptor-specific mechanisms that underlie dopaminergic control of spinal network output of neonatal mice during changes in spinal network excitability. During spontaneous activity, which is a characteristic of developing spinal networks operating in a low excitability state, we found that dopamine is primarily inhibitory. We uncover an excitatory D1-mediated effect of dopamine on motoneurons and network output that also involves co-activation with D2 receptors. Critically, these excitatory actions require higher concentrations of dopamine; however, analysis of dopamine concentrations of neonates indicates that endogenous levels of spinal dopamine are low. Because endogenous levels of spinal dopamine are low, this excitatory dopaminergic pathway is likely physiologically-silent at this stage in development. In contrast, the inhibitory effect of dopamine, at low physiological concentrations is mediated by parallel activation of D2, D3, D4 and α2 receptors which is reproduced when endogenous dopamine levels are increased by blocking dopamine reuptake and metabolism. We provide evidence in support of dedicated spinal network components that are controlled by excitatory D1 and inhibitory D2 receptors that is reminiscent of the classic dopaminergic indirect and direct pathway within the striatum. These results indicate that network state is an important factor that dictates receptor-specific and therefore dose-dependent control of neuromodulators on spinal network output and advances our understanding of how neuromodulators regulate neural networks under dynamically changing excitability.Dopamine is well known to regulate movement through the differential control of direct and indirect pathways in the striatum that express D Dopamine is a monoamine neuromodulator that is well known for action selection in vertebrates through the control of direct and indirect circuits of the basal ganglia that express excitatory D1 and inhibitory D2 receptors respectively . Dopamine is also important for the regulation of spinal motor networks that control rhythmic movements including but not limited to locomotion . In larval zebrafish, phasic and tonic firing patterns of descending neurons that provide the primary source of spinal dopamine correlates with locomotor episodes and quiescence, respectively9. These different firing patterns can impact the cellular release of dopamine and as a consequence, the receptor subtypes it activates10. For example, high levels of dopamine released during phasic cell firing activate lower affinity excitatory D1 receptors and promote locomotor activity, and lower levels of dopamine released during tonic activity activate higher affinity inhibitory D2 receptors and suppress motor output11. Similarly, dopamine has dose-dependent effects on locomotor circuits in precocial species with functional swim networks12.Neuromodulators are critical for central nervous system function and diversify circuit outputs by altering synaptic and intrinsic properties13 which is consistent with work in invertebrates15. This is important in developing spinal motor networks which produce a wide repertoire of patterned outputs at birth, including locomotor activity, as a consequence of dynamically fluctuating network excitability. That being said, neonatal rodents rarely produce coordinated bouts of locomotion and in vitro preparations of isolated spinal cord require pharmacological or electrical stimulation to drive the network into a high excitability state to produce fictive walking patterns. Instead, most of the movements observed in neonatal mice are ataxic. These movements correlate with spontaneous network activity, which can be observed in vitro19. Nevertheless, the vast majority of what we know about neuromodulation of developing mammalian spinal networks has been derived from studies on fictive locomotor activities with dopamine being predominantly excitatory when spinal networks are operating in this state31.We have recently demonstrated that neuromodulation of developing mammalian spinal circuits is state-dependent32 with similar mRNA distribution as early as day 4 . However, prior to this time data on dopamine receptor expression is sparse. Changes in intrinsic properties and receptor expression can therefore lead to robust changes in neuromodulatory control of spinal circuits (Reviewed by Refs.34). Based on our previous work, we hypothesized that receptor actions, and therefore concentration-dependent control of network output by dopamine, is linked to the underlying network excitability state13. As a result, more complex receptor-dependent actions may have been masked during high excitability states of fictive locomotion. We therefore examined how dopamine modulates spinal output at the network and cellular level of neonatal mouse spinal cords in vitro during a low excitability state characterized by spontaneous activity17. Portions of these data were presented in abstract and preprint form36.mRNA for all 5 dopamine receptor subtypes is expressed in the mouse spinal cord and putative motoneurons by postnatal day 1431, we recorded spontaneous motor activity with extracellular suction electrodes from single ventral roots of the second or fifth lumbar (L2/L5) segments simultaneously from two or four spinal cord preparations sharing the same chamber. Each preparation in these series of experiments was naïve to dopamine exposure and received only a single dose of its respective concentration. This configuration ensured consistent experimental conditions across several preparations exposed to different concentrations of dopamine. Low concentrations of dopamine (1–30 µM) consistently suppressed spontaneous motor activity, whereas higher concentrations (100–300 µM) excited spontaneous motor activity, evoking episodic and continuous rhythmic patterns  = 10.5. p < 0.001). A detailed description of the rhythmic activities generated by higher concentrations of dopamine 30–300 µM were analyzed using spectral analyses on data obtained from preparations included in this study and have been previously published13.We focused on the modulation of perinatal spontaneous activity patterns to investigate how dopamine modulates spinal network output during a physiologically relevant low excitability state. Using the same experimental set-up as Sharples et al.2 , D2/D3 , D3 , D4  or D1/D5 receptors  and L-745870 , response ratios indicated that the inhibitory effect of 10 µM dopamine was attenuated, compared with dopamine alone  = 7.3, p < 0.001; Tukey post hoc p = 0.09), to a level where it was not significantly different from a time-matched vehicle control  = 7.3, p < 0.001; Tukey post hoc, p = 0.98). Burst analysis revealed no difference in the number or amplitude of episodes when antagonists were present, compared with baseline  = 9.5, p < 0.0001; two-way ANOVA for amplitude: F = 3.4, p = 0.023). Together these data suggest that the inhibitory actions of dopamine depend on parallel action of D2, D3 and D4 receptors.To delineate receptor contributions to dopamine’s bidirectional effects on endogenous spontaneous activity in isolated spinal cord preparations, we used antagonists selective for the family of dopamine receptors. At low concentrations of dopamine, we observed a negative response ratio Fig. A,D1, whi2-adrenergic receptors28. The remaining minor inhibitory effect of dopamine at 10 µM was blocked by antagonizing the α2-adrenergic receptors with yohimbine (4 µM) in the presence of D2/D3 and D4 receptor antagonists. We observed significantly different response ratios, compared with 10 µM dopamine alone  = 7.3, p < 0.001; Tukey post hoc, p = 0.006). Low dopamine concentrations did not change the number or amplitude of episodes in the presence of these antagonists, compared with baseline 40. Thus, we first tested the role of the lower affinity D1 receptor system and then examined whether the D2 receptor system has a cooperative role in the control of spontaneous activity. With the addition of dopamine at higher concentrations , spontaneous activity patterns became rhythmic, often producing a slow rhythm with episodes of high frequency rhythmic activity. Moreover, the presence of the D1-like receptor antagonist, SCH-23390, reduced this effect  = 11.4, p < 0.001); fast rhythm power, F = 12.6, p < 0.001; slow rhythm power, H(4) = 12.8, p = 0.013; Fig.  = 11.4, p < 0.001; fast rhythm power, F = 12.6, p < 0.001; slow rhythm power, H(4) = 12.8, p = 0.013, Dunn’s post hoc: p = 0.03; one way ANOVA used in tests). These results suggest that the excitatory effects of dopamine are primarily mediated by the D1-like receptor family.Dopamine binding to D2 receptors have been previously reported to interact with D1 receptors leading to excitatory responses40. We tested this idea by administering D2-like antagonists (sulpiride + L745870) and found that the power of the fast rhythm elicited by dopamine at 100 µM was reduced  = 12.6, p < 0.001) to the same extent as the D1-antagonist, with no effect on the power of the slow rhythm  = 12.8, p = 0.013; Dunn’s post hoc, p = 1.0). This suggests that D2 receptors may contribute to the excitatory effects of dopamine.D1 and D2 receptors in isolated neonatal spinal cords, we performed co-immunoprecipitation for D1 and D2 receptors and used agonists to activate both receptor subtypes. After immunoprecipitating D2 receptors from neonatal spinal cord lysates, we used an antibody to probe for D1 receptors. We detected D1 receptor protein within the D2 receptor immunoprecipitates  and the D2 agonist quinpirole (50 µM) elicited a more robust depolarization of the ventral root DC potential, compared with 50 µM of the D1 agonist alone  = 5.2, p = 0.01; Tukey post hoc: p = 0.02). We observed no difference in the amount of spontaneous network activity evoked with co-application of a D2 agonist, compared with application of the D1 agonist alone, as indicated by the response ratio  = 12.0, p < 0.001; Tukey post hoc, p = 0.5). In contrast, lower concentrations of the same agonists (10 µM) produced no effects  = 0.73, p = 0.24; response ratio, t(6) = 0.9, p = 0.19). Thus, consistent with previous reports for striatal neurons40, we found a dose-dependent effect of dopamine agonists wherein co-applying high doses, but not low doses, of D1 and D2 receptor agonists, produced more robust depolarization than a D1 agonist alone.In support of this idea, co-application of the D1 and D2 agonists, we tested co-activating D1 and D2-like receptors with the D1/D2 co-agonist SKF 83959 (50 µM)41. As predicted, the co-agonist elicited a more robust depolarization of the ventral root DC potential, compared with the D1 agonist, when applied alone  = 5.2, p = 0.01; Tukey post hoc, p = 0.03). Interestingly, the co-agonist also robustly facilitated superimposed spontaneous activity, as indicated by a larger response ratio than co-application of the D1 and D2 agonists produced  = 12.0, p < 0.001; Tukey post hoc, p = 0.004). These data suggest that under certain conditions, D2 receptors that are typically inhibitory can play an excitatory role and may interact with D1 receptors to contribute to lumbar motor network excitation in the neonatal mouse spinal cord.In addition to co-applying separate D = 18.0, p < 0.001) and a reduced number of spontaneous episodes  = 11.8, p = 0.0004), with no change in amplitude  = 1.3, p = 0.3). We questioned whether extracellular dopamine metabolism may have dampened the effect of the reuptake blocker, thus diminishing the predicted increase in endogenous dopamine levels. Therefore, we repeated this experiment in the presence of bifemelane, a monoamine oxidase A and B inhibitor. Under these conditions, we found further reductions in spontaneous activity when DAT was blocked, as indicated by a significantly reduced response ratio  = 18.0, p < 0.001). Burst analysis revealed significantly fewer episodes  = 11.8, p = 0.0004) with no change in amplitude  = 1.3, p = 0.3).We next examined how the endogenous dopamine system regulates perinatal spinal network function. Endogenous levels of dopamine were increased by blocking the dopamine transporter (DAT) with an antagonist GBR 12909 (10 µM). Blocking DAT (n = 8 preparations) produced a modest but significant reduction in spontaneous activity compared to time-matched vehicle controls, reflected by a reduced response ratio . Thus, our in vitro experiments indicated that low levels of endogenous dopamine play a role in D2-mediated inhibition.We followed these experiments up with high-performance liquid chromatography (HPLC) to verify endogenous levels of dopamine. In P3 lumbar spinal cords (n = 11) we detected low levels of dopamine; in P60 adults (n = 17) we detected a threefold increase in dopamine levels compared to at P3 . Some of these motoneurons  were filled with fluorescein-conjugated dextran amine  and verified post hoc using immunohistochemistry for the presence of choline acetyltransferase , input resistance , resting membrane potential  and bias current  for the 75 motoneurons included in this study are equivalent to those reported in the literature46. Motoneurons recorded from animals age P3–4 days old had a significantly higher capacitance than those recorded from animals P0–2 days old  = 2.4, p = 0.02); but did not differ in input resistance  = 1.3, p = 0.2). Motoneurons also display characteristic time-dependent changes in repetitive firing frequency during sustained depolarizing current injections49. We found that all but two cells demonstrated a time-dependent reduction in firing rate throughout the duration of a 0.5 s depolarizing current injection, as indicated by the ratio between maximum steady-state and first spike interval frequencies. This phenomenon is known as spike frequency adaptation . In addition, doublet firing occurred at the onset of repetitive firing in 5 cells (8%).In the next set of experiments, we were interested in determining the cellular mechanisms that mediate dopamine’s complex modulatory effects on spinal network output. As integrators of premotor network activity that generate many of the rhythmic outputs of the spinal cord, motoneurons are ideally suited to amplify spontaneous activity and respond to dopaminergic modulation. Given that they not only serve as the final output for spinal networks, but they also participate in the generation of rhythmic activity51 and with our network recordings in this report, 100 µM of dopamine increased motoneuron excitability ; we reproduced this effect with the D1 agonist SKF 81297 . Cells were held at -75 mV in voltage clamp  = 18.9, p < 0.001), increased the amount of bias current required to maintain membrane potential at − 75 mV  = 18.9, p = 0.001) increased the input resistance  = 16.0, p < 0.001), and decreased rheobase  = 5.0, p = 0.016) beyond that of the time-matched vehicle control. We found no change in spike rise time  = 1.0, p = 0.4) or half width  = 0.8, p = 0.5). The D1 agonist reduced the amplitude of the afterhyperpolarization (AHP) to a greater extent than the time-matched vehicle control. 100 µM dopamine also reduced AHP amplitude compared to baseline, however, the change was not greater than that of the time-matched vehicle control  = 7.7, p = 0.003). Frequency–current (FI) relationships were measured during the first spike interval and steady-state firing in response to a series of depolarizing current pulses  = 9.6, p = 0.002). Dopamine (100 µM) and the D1 agonist increased the slope of the exponential region of the FI relationship for the first spike interval  = 8.4, p = 0.002) and reduced the slope of the steady-state FI relationship  = 9.4, p = 0.009). The reduction in steady-state slope was due to a leftward shift in steady-state FI relationship  = 14, p < 0.001) with no change in the maximum steady-state firing rate (H(2) = 1.4, p = 0.5). These results indicate that activation of D1 receptors elicit consistent effects as high concentrations of dopamine on motoneuron excitability and is a likely mechanism contributing to dopaminergic excitation of motor output.Consistent with previous reports from our laboratoryamp Fig. B1 while amp Fig. B2. Both 2 agonist quinpirole  or quinpirole . These results suggest that the inhibitory actions of dopamine on spinal network output are not due to D2-receptor inhibition of intrinsic motoneuron excitability; instead, dopamine may be acting on premotor interneurons.We next set out to determine the cellular mechanisms that mediate the inhibitory effects of dopamine on spinal network output with motoneurons as our first target. In contrast to our network recordings, neither low concentrations of dopamine  = 26.1, p < 0.0001) and a transient hyperpolarization of membrane potential in 10% of interneurons  that persisted for 209 ± 108 s before returning to baseline levels. The change in resting membrane potential was greater in the responders compared to non-responders  = 7.8, p < 0.0001). Quinpirole reduced the input resistance  = 4.1, p = 0.025) and increased the spike rise time  = 5.2, p = 0.01; Dunn post hoc test, p = 0.02) but not the half width  = 2.6, p = 0.086) in the group of responding interneurons (“responders”). For both responders and non-responders, quinpirole did not alter rheobase  = 0.5, p = 0.6), action potential threshold  = 0.3, p = 0.7), AHP amplitude  = 0.04, p = 0.96), duration  = 0.8, p = 0.45), threshold for repetitive firing  = 0.01, p = 0.98), steady-state FI slope  = 0.9, p = 0.4) or maximum steady-state firing rate  = 0.8, p = 0.5 ). The capacitance  = 4.2, p = 0.02) and holding current  = 4.9, p = 0.02) were higher in responders than in non-responders  and non-responders  = 0.9, p = 0.35) were obtained.Many of the premotor interneurons that produce the rhythmic activities generated by the spinal cord are distributed across lamina’s VII–X of the ventral lumbar spinal cord. We next recorded from ventral interneurons located in lamina VII–X to determine a cellular locus for D54, display intrinsic burst properties55 and are rhythmically-active during neurochemically-evoked fictive locomotion56. dCINs were retrogradely labelled with tetramethylrhodamine-conjugated dextran amine (molecular weight (MW) 3000; Molecular Probes, Inc.) inserted into the ventrolateral funiculus at the L4 segment . There was no correlation between baseline FI slope and changes in FI slope in response to quinpirole .We next set out to determine the type of interneurons that were hyperpolarized by quinpirole. Given that many of the responding cells were located medially, we next targeted descending commissural interneurons  = 2.5, p = 0.06); however, did reach significance when the cells that responded with a transient hyperpolarization were included in the analysis (paired t-test: t(6) = 2.7, p = 0.03). Consistent with our global interneuron survey the change in resting membrane potential elicited by quinpirole was significantly greater in responding  compared to non-responding  V3 interneurons (unpaired t-test: t(16) = 3.8, p = 0.002).Consistent with our global interneuron survey, quinpirole produced a sustained hyperpolarization of the resting membrane potential in a proportion of V3 interneurons  that we recorded from  and transient hyperpolarization in 3 13%) V3 interneurons. The magnitude of the response in the 5 cells that responded with a sustained hyperpolarization was variable and approached, but did not reach significance  but is probably best known in vertebrates for the control of dedicated circuits in the basal ganglia that control action selection (reviewed by Ref.64 ). Work in small circuits of invertebrates has established that circuit connectomes define the constraints on which networks operate and that neuromodulators diversify outputs by altering intrinsic and synaptic properties of the neurons that compose the circuit (for reviews see Refs.3). In line with this, the distribution of receptors within circuits constrain the effect of neuromodulators on circuit output. For example, dopamine is exclusively inhibitory in spinal circuits of Xenopus tadpoles prior to free-swimming stages65 due to expression of D2 but not D1 receptors. We show that dopamine has bidirectional concentration-dependent effects on spinal network output in neonatal mice where all dopamine receptor types are expressed32 which is consistent with what has been reported in tadpoles at free swimming-stages12. Our data highlights that neuromodulator concentration is also important because receptors have varying ligand affinities which underlie concentration-dependent actions of modulators which is consistent with other work demonstrating opposite effects on spinal network output through activation of opposing receptor subtypes (Reviewed by Ref.34). This includes opposing actions of serotonergic 5-HT7/5HT2a and 5HT1 receptors66 , noradrenergic α-1 and α-2 receptors67 and cholinergic M2 and M3 receptors68. Although dopamine predominantly inhibits spinal output in neonatal mice, similar to pre-free swimming tadpoles and larval zebrafish69, it is primarily due to the concentration of spinal dopamine, not the distribution of receptors. Our previous work has shown that neuromodulation of mammalian spinal networks is dependent on network excitability state13 which is consistent with findings from invertebrates15. Our current work builds upon ours and extends that of others that demonstrate bi-directional control of spinal networks by noradrenaline67, serotonin66 and acetylcholine68 and shows that receptor mechanisms and concentration-dependent control of spinal network output is also state-dependent. This is important because receptor expression, modulator concentration and network excitability are not fixed and fluctuate dynamically71. Therefore, these three factors need to be considered if we wish to understand how networks create diverse neuromodulator-dependent outputs . One caveat is that we may have underestimated the cellular targets that underlie dopamine’s robust inhibitory effect on the network given that in this series of experiments we looked at the activation of D2 receptors alone. Alternatively, D2 receptors may modulate synaptic transmission in presynaptic contacts from premotor to motoneuron synapses similar to observations in the striatum89. Our previous work suggests a presynaptic role for D2 receptors in depressing recurrent collateral mediated excitation29, and others have also established a dopamine-mediated depression of the monosynaptic reflex90. Given our current data showing little effects of D2 receptors agonists on intrinsic properties together with previous work, this supports a D2 receptors mediated presynaptic role.The inhibitory D18 and has been reported to decrease over the first postnatal week as spinal networks mature16. The reduction in spontaneous network activity is paralleled by differential maturation of flexor and extensor motoneurons during the first 6 postnatal days in the rat91 and reduced passive excitability and diversification of mouse motoneuron intrinsic properties93 leading into the second postnatal week as weight-bearing locomotion begins to emerge. Consistent with this, we find an increase in whole cell capacitance of motoneurons when comparing cells obtained from animals age P0-2 days old to animals age P3–4 days old. Intrinsic properties of different classes of genetically-defined interneurons also undergo intrinsic property diversification during this time . A robust inhibitory endogenous dopaminergic system may act as a brake on premotor network activity during perinatal development when passive excitability of motoneurons is relatively high93 and chloride-mediated synaptic transmission still causes partial depolarization96. An inhibitory brake would prevent runaway excitation of spinal network activity during this critical period of development. A robust background inhibitory system mediated by the parallel inhibitory action of D2, D3, D4, and α2-adrenergic receptors activated by low levels of endogenous dopamine would counteract depolarizing chloride-mediated transmission96.Our analysis of dopaminergic regulation of spontaneous network activity and spinal neuron excitability spanned an age range from postnatal days 0 to 4. Spontaneous network activity generated during embryonic development is critical for maturation of spinal networksXenopus tadpoles65 and the larval zebrafish97. Signaling through D2-like receptors may also play a role in driving the maturation of spinal networks. In larval zebrafish, D4 receptors drive the maturation of spinal locomotor network organization98 and function leading to changes in locomotor behaviour99. Similar processes may also occur perinatally in rodents, in that the preferential activation of the D2 receptor system may favour intracellular signaling that results in network reorganization. Serotonin receptors have been found to shape network function and inhibitory synaptic transmission during early postnatal days of rodents101. Dopamine could, therefore, act analogously via the D2-system during perinatal development. Our results provide insight into the functional expression of dopamine receptors in spinal neurons during this critical stage in postnatal development. Our results indicated that D2 receptors are distributed across ventral interneurons and D1 but not D2 receptors are functionally expressed in motoneurons at this stage in development. However, we did not explore D1 regulation of ventral interneuron excitability, and indeed high concentrations of dopamine have been reported to augment rhythmicity in glutamatergic Hb9 interneurons50. Future work exploring expression and function of D1 receptors in ventral interneurons may provide insight into populations that underlie the diverse rhythms and modulation of network output by dopamine in vitro31. These works will serve as a foundation to explore modulation of spinal circuits during later stages of postnatal development into adulthood, approaching freely behaving stages around the third postnatal week and when descending dopamine systems are fully mature by P21102.The dopamine receptor expression profile amongst different populations of spinal neurons is also likely to change during postnatal development, and indeed this is the case in 103. Consistent with this notion, we demonstrate that the inhibitory action of low dopamine is mediated in part by α2 noradrenergic receptors, therefore conversion to noradrenaline or activation of endogenous sources of noradrenaline cannot be excluded. While we did not test the contribution of non-dopamine receptors to the excitatory effect of high dopamine, it remains a possibility. In addition many of the agonists/antagonists we employed have known affinities for non-dopamine receptors. For example, the D1-agonist we employed has known affinities as a partial agonist for the 5HT2c receptor106 and antagonist for the 5HT2a receptor107. That said, collectively, our results are consistent with our hypotheses—agonists reproduce the effects of low and high dopamine and their effects can be blocked by respective antagonists. In all likelihood, neuromodulatory systems of the spinal cord do not operate in isolation, but in parallel to globally regulate spinal network function. There is evidence for this in the stomatogastric nervous system of the crab/lobster therefore, cooperation amongst neuromodulatory systems may be more important than their individual actions. In light of our previous work13, cooperation amongst neuromodulatory systems may provide degeneracy with respect to the mechanisms for moving the network through various excitability states.A caveat of our results is that many of our experimental approaches rely on pharmacological manipulations of target receptors. Indeed dopamine itself has been demonstrated in other regions of the brain to be cross-reactive with non-dopamine receptors and in some instances, to be converted to serotonin or noradrenaline by presynaptic neurons and can alter the activity of other monoamine-releasing neurons1-mediated pathway that acts through excitation of motoneurons, and possibly recurrent excitatory collaterals to CPG neurons, however given that endogenous levels of dopamine are low, we propose that this pathway is physiologically silent at the developmental stage studied here (P0–4). These data advance our understanding of how neuromodulators regulate network output in light of dynamically changing modulator concentrations and levels of network excitability.Here we present evidence for an inhibitory physiological role of dopamine in the regulation of developing mammalian spinal networks. We also demonstrate an excitatory D2/5% CO2) aCSF . We exposed the spinal cord with a ventral laminectomy and isolated it by cutting the nerve roots that connected it to the vertebral column. The isolated spinal cord was then transferred to a recording chamber, perfused with carbogenated aCSF, and placed ventral side up. The bath temperature was gradually increased to 27 °C, to be closer to the physiological temperature in vivo, and to ensure that the preparation temperature was locked at a level above fluctuating room temperature30. We let the spinal cords stabilize for 1 h before performing experiments.We anesthetized all animals by hypothermia. Pups were decapitated and eviscerated to expose the vertebral column and rib cage. The isolated vertebrae and rib cage were transferred to a dish lined with a silicone elastomer  and perfused with room-temperature (21–23 °C) carbogenated . Using a vibratome  we cut 250-µm-thick lumbar slices, collected and transferred them to a recovery chamber containing regular carbogenated aCSF (see Tissue Preparation) heated to 32 °C for one hour, then maintained them at room temperature for at least 30 min before transferring them to a recording chamber.Following isolation, spinal cords were transected above the tenth thoracic (T10) and below the first sacral (S1) segments and transferred to a slicing chamber. Pre-warmed liquefied 20% gelatin was used to secure cords to an agar (3%) block that was glued with cyanoacrylate to the base of a cutting chamber and immersed in ice-cold, carbogenated, high-sucrose slicing aCSF  into a cut in the ventrolateral funiculus at the L4 segment. Spinal cords recovered for 4 h to allow retrograde uptake of the fluorescent dye. Fluorescently labelled cells were visualized with epifluorescent illumination.Extracellular neurograms were recorded by drawing ventral roots of the second (L2) and fifth (L5) lumbar segments into tight-fitting suction electrodes fashioned from polyethylene tubing (PE50). Signals were amplified 1000× in total via 10× pre-amplification and 100× second-stage amplification . Amplified signals were band-pass filtered (0.1–1000 Hz) and digitized at 2.5 kHz . Data were acquired in Clampex 10.4/10.7 software (Molecular Devices) and saved on a Dell computer for offline analysis. All experiments were performed on spinal cords naïve to drugs and experimental treatment.2, 1.0 MgSO4, 0.5 Na2HPO4, 21 NaHCO3, 30 d-glucose; approximately 310 mOsm) and stabilized in the recording dish with a stainless-steel harp. We gradually heated aCSF to 27 °C. Slices were visualized  under 5× magnification and putative motoneurons identified using a 40× objective with infrared differential interference contrast (IR-DIC) illumination. We identified putative motoneurons based on their location in the ventrolateral spinal cord and a soma diameter of greater than 20 µm. A cohort of motoneurons was passively filled with fluorescein dextran amine  added to the intracellular solution to visualize and localize the recorded cells, for 20–60 min and motoneuron identity was verified post hoc by performing immunohistochemistry for choline acetyltransferase (ChAT)45. To verify motoneuron identity in subsequent experiments, basal biophysical properties of putative motoneurons were compared with a cohort (n = 8) of FITC-filled ChAT-positive cells and are included in Supp. Fig. Spinal cord slices were gently transferred to a recording chamber perfused with room-temperature carbogenated recording aCSF . Intracellular solutions were balanced to a pH of 7.3 with 10 M KOH and osmolality was cross-checked to fall within the range of 275–290 mOsm. Data were acquired at 10 kHz using Clampex software .Recording electrodes were pulled from borosilicate capillary tubes  using a Flaming/Brown Model P-97 micropipette puller . Pipettes pulled to record from motoneurons, and interneurons were within the range of 3–5 MΩ and 6–9 MΩ, respectively. Pipettes were backfilled with intracellular solution , using the ionic composition of our extracellular and intracellular solutions. Protocols for the examination of intrinsic properties have been described elsewhere 1-like receptors ; quinpirole for D2-like receptors ; and the D1/D2 receptor co-agonist SKF 83959 . For dopamine receptor antagonists we used the D1-like antagonist SCH-23390 ; the D2-like antagonists sulpiride (20 µM) and L-741626 (12 µM); the selective D3 receptor antagonist SB 27701A ; the selective D4 receptor antagonist L-745870 . We also used the α2 adrenergic receptor antagonist, yohimbine . Endogenous dopamine levels were manipulated with the DAT inhibitor GBR-12909  and the monoamine oxidase A and B inhibitor bifemelane .Dopamine hydrochloride  was bath applied in separate experiments at 1 µM, 3 µM, 10 µM, 30 µM, 100 µM, and 300 µM to determine dose-dependent effects on motor activity. The receptor-selective agonists we used included SKF 81297 for DSpinal cords were dissected from neonatal mice (age P0–4) in ice-cold (4–8 °C) aCSF and homogenized in lysis buffer containing 50 mM TrisHCl, 150 mM NaCl, 10 mM EDTA, 0.1% Triton-X, and 5% Glycerol. Lysis buffer contained protease inhibitors (Sigma) and phosphatase inhibitors (GBiosciences). We homogenized three spinal cords in 100 µL of buffer and incubated them on ice for 1 h before centrifuging them at 10,000 rpm for 30 min at 4 °C. Lysates were then extracted and stored at − 20 °C.2 receptors . Anti-rabbit Ig IP beads were added, and samples were incubated overnight at 4 °C with gentle agitation. Immunoprecipitates were washed with lysis buffer, heated in loading buffer  to 95 °C for 10 min, electrophoresed on a precast SDS gel , and transferred onto a nitrocellulose membrane. After blocking, the membranes were incubated with guinea pig antibody to D1 receptors  and rabbit antibody to D2 receptors , washed, incubated for 2 h at room temperature in fluorophore-conjugated secondary antibodies , and visualized via antibody fluorescence at 680 or 800 nm . Full length unprocessed images can be found in Supp.Fig. To reduce nonspecific binding, we first incubated lysates in anti-rabbit Ig agarose beads  for 30 min, on ice and in the absence of primary antibody. We then removed the supernatant and incubated the lysates on ice for 1 h with rabbit antibody to DFollowing the completion of experimental protocols, we post-fixed slices overnight in 4% paraformaldehyde (PFA) at 4 °C, washed them the next morning in phosphate-buffered saline (PBS) for three 20-min intervals, and stored them at 4 °C. On the day we performed immunohistochemistry, we first washed the slices for 30 min (3 × 10 min) in PBS with 0.5% Triton-X 100 (PBST) then incubated them at room temperature (21–23 °C) for 6 h in blocking solution containing 10% donkey serum in 0.5% PBST. Primary antibodies for choline acetyltransferase (ChAT)  and the fluorescence marker  were diluted in blocking solution and the slices incubated with them for at least 24 h at room temperature. Slices were then washed in PBST (0.5%) for 80 min (4 × 20 min) at room temperature before secondary antibodies  were applied for 4–8 h at room temperature, then washed for 80 min in PBST (0.5%). Slices were mounted and cover slipped with fluorescent mounting medium ; coverslips were separated from slides by 500 μm spacers to prevent crushing.+ microscope  operating in confocal mode with a 16 × water-immersion objective lens . Image acquisition used a z-step of 1 µm and averaged two frames with a resolution of 2048 × 2048. Pixel dwell time was 2.5 ms and exposure settings were maintained for all sections. We used NIS-Elements software (Nikon) for image acquisition and ImageJ to perform maximum intensity projections of z-stacks.All sections processed via immunohistochemistry were imaged on a Nikon A1R MP108. Tissue was homogenized in ice-cold 0.1 M perchloric acid containing EDTA (10 mg%) and ascorbic acid (50 µM). We centrifuged the homogenate and used 10 μl of supernatant in the assay. The HPLC system consisted of a Waters Alliance 2695 XE Separations Module equipped with a Waters Atlantis dC18 3 µm (3.0 × 100 mm) analytical column and a Waters 2465 electrochemical detector with an applied potential set at 0.64 V. The mobile phase was composed of 55 mM monosodium phosphate, 850 µM sodium octyl sulfate, 470 µM EDTA, 8% acetonitrile and 2 mM sodium chloride in water, with the pH adjusted to 2.9. The mobile phase flow rate was 0.6 ml/min. Retention times (in min) were 8.6 (NE), 16.2 (DA) and 39.2 (5-HT).Monoamine content of neonatal and adult spinal cords was measured using high-performance liquid chromatography (HPLC). We dissected spinal cords from neonatal  C57BL/6 mice in aCSF as described above and extracted adult spinal cords  with a pressure ejection method. Tissue was then flash-frozen with liquid nitrogen, stored at − 80 °C, and analyzed for biogenic amines with a modified version of our previously-published HPLC method31: we calculated a response ratio from single ventral root neurograms between the root mean square of 5 min of basal spontaneous activity and 5 min of activity recorded 20 min after adding the drug. We subtracted 1 from the response ratio so that positive values reflect excitation and negative values reflect inhibition. The response ratio was used as a high throughput assay to detect global changes in network activity. Neurogram data were analyzed with Spike2 software. Bursts of spontaneous activity were analyzed using Clampfit (Molecular Devices) to determine how episode number and amplitude contributed to changes in response ratio. Spectral analyses were conducted using Spinalcore software109 whenever we detected excitatory changes or observed rhythmicity.We determined the relative inhibitory or excitatory effects of dopamine and dopamine receptor agonists on spontaneous motor network activity using methods similar to those in our previous work109, consistent with Sharples and Whelan13.Patterns of rhythmic motor activity recorded from single ventral roots were analyzed with autowavelet spectral analysis. We created and analyzed frequency–power spectrograms by selecting regions of interest around frequency ranges that coincided with those in the raw neurogram. The spectrograms revealed two high power regions that reflect distinct rhythms evoked by dopamine at high concentrations: a slow 0.01–0.04 Hz rhythm and a fast 0.8–1.2 Hz rhythm. Regions of interest were selected within these frequency ranges and analyzed over the time course of each experiment. Frequency power within selected regions of interest that corresponded to the fast and slow rhythms were used as a measure of rhythm robustness. Data were segmented into 30 s bins and averaged over 5-min intervals for statistical analysis. We used tools available in Spinalcore for all analyses of rhythmic motor activity92, with the exception of repetitive firing analyses, which examined the instantaneous firing rate for the first spike interval and steady-state firing separately.Motoneuron and interneuron intrinsic properties measured during whole-cell patch-clamp experiments were analyzed as in our previous workp < 0.05 were further examined with post hoc analyses. We used Holm-Sidak post hoc tests to compare all treatment conditions to the appropriate normalized time-matched vehicle control. Data that violated assumptions of normality (Shapiro–Wilk test) or equal variance (Brown–Forsythe test) were analyzed via nonparametric Mann–Whitney U (if two groups) or Kruskal–Wallis (if more than two groups) tests.All experiments were repeated measures. We tested for differences in the magnitude of effects between conditions with one-way ANOVAs, focusing on comparisons to time-matched vehicle controls. Two-way ANOVAs compared baseline to multiple post-drug conditions. All effects surpassing a significance threshold of Sim1Cre/+; Rosafloxstop26TdTom which were used to visualize and record from V3 interneurons. All methods were carried out in accordance with relevant guidelines and regulations by the University of Calgary Health Sciences Animal Care Committee and the University Committee on Laboratory Animals at Dalhousie University. Also, all procedures performed were approved by the University of Calgary Health Sciences Animal Care Committee and the University Committee on Laboratory Animals at Dalhousie University.Experiments were performed on male and female neonatal  C57BL/6 mice. High performance liquid chromatography (HPLC) was also performed on a cohort of adult  C57BL/6 mice. A subset of experiments were performed on spinal cord slices obtained from neonatal  Supplementary Figures.Supplementary Video.


Enterobacteriaceae family, and, in this paper, we show their shared geometrical and electrostatic characteristics. Then, we used metadynamics simulations to reconstruct the free energy for permeation of selected diazobicyclooctans through OmpF. We demonstrate how porins features are coupled to those of the translocating species, modulating their passive permeation. In particular, we show that the minimal projection area of a molecule is a better descriptor than its molecular mass or the volume. Together with the magnitude and orientation of the electric dipole moment, these are the crucial parameters to gain an efficient compensation between the entropic and enthalpic contributions to the free energy barrier required for permeation. Our results confirm the possibility to predict the permeability of molecules through porins by using a few molecular parameters and bolster the general model according to which the free energy increase is mostly due to the decrease of conformational entropy, and this can be compensated by a favorable alignment of the electric dipole with respect to the channel intrinsic electric field.Modern medicine relies upon antibiotics, but we have arrived to the point where our inability to come up with new effective molecules against resistant pathogens, together with the declining private investment, is resulting in the number of untreatable infections increasing worldwide at worrying pace. Among other pathogens, widely recognized institutions have indicated Gram-negative bacteria as particularly challenging, due to the presence of the outer membrane. The very first step in the action of every antibiotic or adjuvant is the permeation through this membrane, with small hydrophilic drugs usually crossing through protein channels. Thus, a detailed understanding of their properties at a molecular level is crucial. By making use of Molecular Dynamics simulations, we compared the two main porins of four members of the  Antibiotics have been regarded as magic bullets to treat infections for almost the entire 20th century. Modern medicine relies upon them, but due to their spread misuse, we have arrived to the point where almost all antibacterial drugs developed so far have been followed by detection of resistance ,2. ResisMany fundamental gaps need to be filled in our knowledge about antibiotics. The mode of action, the penetration pathway, and the mechanisms adopted by the pathogen to degrade or pump drugs out are just a few general examples. High-throughput screening protocols are looked with great hope as a way to identify novel drug scaffolds, but these protocols need to be fed with proper selection rules, otherwise the risk is high that they can be biased ,5. AlongAmong other pathogenic microorganisms, which certainly deserve much attention, widely recognized institutions have indicated Gram-negative bacteria as particularly challenging ,11,12,13Pseudomonas aeruginosa and Acinetobacter baummanii are characterized by a variety of rather small porins that are specific for the uptake of the different nutrients [Enterobacteriaceae express two principal trimeric porins. These are classified as nonspecific general porins, since they allow the passage of ions and metabolites up to 600 Da with no clear specificity [Escherichia coli is usually taken as prototypical example for enteric Gram-negative bacteria, the literature is more extended than about other species. In addition, in order to find possible general rules for protein channels permeation, we preferred to focus on general porins, leaving the specific ones to a next stage. In the case of E. coli, the major porins are OmpF and OmpC. They share remarkable similarities in their amino acid sequence and three-dimensional structure, with OmpC being narrower than OmpF. Moreover, the level of expression of these two porins differs with varying growth conditions, leading to the conclusion that pore size is key to channel permeability [Porins are typically arranged either as homotrimers or monomers, with a basic β-barrel folding that forms a transmembrane water-filled pore. While species like utrients ,24,25, tcificity . Since Eeability . Howevereability ,29. Expeeability ,28,30,31eability ,36,37,38eability .OmpF and OmpC are homotrimers formed by 16-strand β-barrels. The size of the lumen is determined by the loop L3, which folds into the channel and forms a restricted portion almost halfway through the pore. This is usually referred to as the constriction region (CR), which separates the extracellular vestibule from the periplasmic one  in an overall hourglass shape. The loop L3 is characterized by several negatively charged residues facing a series of aligned basic residues of the β-barrel, the so-called basic ladder, which results in a remarkable transversal electrostatic potential gradient ,41. The E. coli resistant clinical strains isolated from a patient treated over two years with cephalosporins, carbapenems, and fluoroquinolones [Recently, we performed an atom-level computational comparative study  on severinolones ,44. Inteinolones . More reinolones . A serieinolones ,46,47,48Enterobacteriaceae family, namely, E. coli, Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, in order to verify whether the electrostatic fingerprint of OmpF and OmpC can be actually taken as a model for other species of the same family. Then, we used biased Metadynamics simulations (metaMD) to reconstruct the translocation free energy of selected β-lactamase inhibitors through OmpF. All the selected inhibitors are avibactam derivatives, belonging to the class of diazobicyclooctans. Thus, they are not β-lactams, and as it has been recently shown by using other scaffolds, [In this work, by making use of Molecular Dynamics (MD) simulations, we first performed the structural comparison of the two main porins of four different members of the affolds, ,50 they E. coli at atomic level was published in 1992 [Enterobacteriaceae: Omp35/Omp36, OmpE35/OmpE36, and OmpK35/OmpK36, respectively, from E. aerogenes, E. cloacae, and K. pneumoniae [The first three-dimensional structure of OmpF from  in 1992 , OmpC in in 1992 . OmpF aneumoniae . We compThe structures obtained by X-ray diffraction show the same trimeric quaternary structure and share the same β-barrel architecture of each monomer, comprising 16 strands linked by short turns on the intracellular side and longer loops on the extracellular one . The looThe alignment of all structures is shown in In a previous work , the strz coordinate, here chosen to be aligned with the main axis of the channel. The calculation was performed on the last 300 ns of our MD trajectories (400 ns), and results are reported as time averages. The main energy barrier for substrate translocation is expected to be of steric nature; thus, a cross-section profile of the protein channel is of primary importance. We calculated the average radius of the lumen as a function of the All the investigated porins are characterized by a net negative charge and have been reported to be slightly cation selective . The selysiology  are repoz-coordinate and compared with that of the bulk solution outside the channel through the following equation:From a general point of view, the slightly larger negative net charge of OmpC and its orthologs correlates with the higher selectivity for cations than OmpF and its orthologs. However, in order to obtain additional details about structural differences, we calculated the free energy profile of ion permeation from MD simulations. The density of ions was derived as the time average over the three monomers as a function of the +, the profile for Cl− is characterized by a wide barrier over almost the entire channel axis, which accounts for the cation selectivity. In OmpF and its orthologs, a minimum is present in the POR, right above the CR, such that, overall, the energy cost for translocation is larger in the case of OmpC and its orthologs. The different profile is attributable to the abovementioned K80W and R167L mutations . These observations account for the slightly larger cation selectivity of OmpC and its orthologs and bolster the importance of the POR in differentiating the two groups of general porins investigated.In all of these general porins, while free energy oscillates around zero for Kz > +10 Å; +11 Å > z > +5 Å; +6 Å > z > 0 Å; +1 Å > z > −5 Å; and −4 Å > z > −10 Å. For each section, the xy-distribution of positively and negatively charged residues was obtained from the coordinates recorded along the MD trajectory and their difference calculated. Enterobacteriaceae. The presented results complement the already reported EF calculations for the same general channels [We complemented this analysis and the reported structural alignment with the inspection of the distribution of charged residues along the channel. The channel was divided into five cross-sections along the main axis, namely +16 Å > channels  and conf− permeation is characterized by a broad energy barrier extending over the entire channel axis , it is restricted to a specific section of the channel, while Cl− experiences an energy cost all along the translocation process. The detailed analysis of the simulation showed favorable interactions at the POR, as well as deeper in the CR. The minimum energy translocation path of avibactam is characterized by its dipole aligned to the transverse EF of OmpF and, at the same time, direct interactions with both the positive residues of the basic ladder through the sulfate group and with the negatively charged residues of the loop L3 through the amino group. According to our model where the steric barrier to translocation can be compensated by favorable interactions, avibactam showed a remarkably good condition since its relatively small size allows it to fit into the CR with an orientation of its electric dipole well aligned to channel’s EF. In this work, we wanted to extend the investigation to some avibactam derivatives of larger size  and the corresponding average radius (MPA=πR2)  similar MPA=πR2) . Thus, tAll of the investigated avibactam analogues are characterized by a predominant axis of inertia. The electric dipole is mainly due to the presence of the negatively charged sulfate group and a positively charged amino group on the amide substituent. The electric dipole is almost perfectly aligned with the predominant molecular axis of inertia. If the molecule crossed the CR by staying aligned with the channel axis, entropic barrier would be minimum but the electric dipole would be poorly aligned with the channel electric field . On the The FES obtained for the permeation of the selected diazobicyclooctans β-lactamase inhibitors through OmpF are shown in z-coordinate” of the center of mass  and the “orientation Φ” of molecule’s dipole moment with respect to the z-axis were selected as collective variables. Values of Φ smaller than 90° correspond to the electric dipole pointing upwards , values of Φ larger than 90° correspond to the electric dipole pointing downwards . The region of z-coordinate between 10 Å and −10 Å is the most important one inside the pore as far as the permeation is concerned. This is rich of energy minima, forming the minimum free energy path to cross the central energy barrier at the CR. Not surprisingly, all the inhibitors show the highest free energy values in the most restricted region of the channel, as reported in the literature for a number of antibiotics [The “ibiotics . The eneibiotics , but theibiotics , such asThe other point of attention is the dipole moment. Zidebactam shows an average larger dipole moment that relebactam, suggesting a larger compensation of the steric free energy. The smallest values of the minimal projection area are also characterized by a larger dipole moment of zidebactam with respect to relebactam, as shown by the two-dimension distribution plot B. In facIn order to obtain a more detailed description of the interactions between the inhibitors and the protein channel, the conformers were extracted from the energy minima and the saddle point along the minimum energy path on the FES for 10 Å > In summary, in the case of zidebactam, the alignment of the large electric dipole to the channel’s EF represents an effective enthalpic compensation to the entropic barrier, which is allowed by its flexibility. Differently, the smaller electric dipole, together with the considerable rigidity of relebactam, make its FES the one with the largest barrier among the inhibitors investigated in this work.z position only. While the height of the maximum of the one-dimensional free energies is comparable for the three inhibitors, with the only exception of relebactam, the profile in the shaded region of z ~5 Å. This corresponds to the POR suggesting that zwitterionic molecules have a greater affinity for the POR, which is the gate to the more restricted CR. Then, among the zwitterionic molecules, the strength of the electric dipole makes the difference in driving the molecule into the favored orientation with respect to the channel’s EF lines, as discussed above [Additional information can be obtained by comparing the one-dimensional FES shown in ed above . The deped above . Insteaded above . The fre formula ,55. At a formula . The two formula , from EVThe results presented in this work confirm those already reported for avibactam: Ref.  The heigz-axis, hence z positive values refer to the extracellular vestibule (EV), and z negative values refer to the periplasmic vestibule (PV). Using the NAMD software version 2.12, USA [z only. The system was solvated with ~17,000 water molecules, and the total number of atoms was ~100 K in a box with size 11 × 11 × 9 nm. A suitable number of potassium and chloride ions were added to reach the desired concentration. An excess of K+ ions was required to neutralize the total negative charge of the trimer (−33 e). After releasing the constraints, an equilibration stage of 4 ns in the NPT ensemble at 1.0 bar and 300 K was performed. Finally, 400 ns MD simulations were performed in the NVT ensemble without restraints. Only the last 300 ns were used for structural analysis. Production run in the NVT ensemble was performed by using the ACEMD code [X-ray structures of OmpF (PDB ID 2OMF), Omp35 (PDB ID 5O78), OmpE35 (PDB ID 6ENE), OmpK35 (PDB ID 5O77), OmpC (PDB ID 2J1N), Omp36 (PDB ID 5O9C), OmpE36 (PDB ID 5FVN), and OmpK36 (PDB ID 5O79) were used as starting coordinates for molecular dynamics (MD) simulations. All the amino acid residues were simulated in their ionization state at neutral pH, except for E296 (OmpF), D299 (OmpC), E102 (Omp35), D307 (Omp36), D235 (OmpE35), D295 (OmpE36), E102 (OmpK35), and D297 (OmpK36), which were protonated (net charge 0), in all the three monomers for each trimer, as suggested for OmpF by Varma et al . Each tr.12, USA  the systEMD code , runningEMD code . The Lanhttp://www.dsf.unica.it/~gmalloci/abdb/ [The Gaff-force field parameters were obtained by following the protocols adopted in ci/abdb/ . We usedci/abdb/  to draw ci/abdb/  was usedci/abdb/  at the Hci/abdb/ , which aEach inhibitor was placed above the first monomer in the EV, about 20 Å away from the constriction region (CR), using the final configuration from the protein simulation.Because the typical time for a substrate to permeate through such a pore is of the order of several hundreds of microseconds, a bias method is necessary to simulate such a process. Substrates permeation was investigated by using well-tempered metadynamics simulations (WT-METAD)  with PluA first step of normal metadynamics simulation of inhibitor permeation was performed until the first effective translocation through the protein constriction region (CR) was observed. Then, four configurations were selected, two with the inhibitor located in the extracellular vestibule (EV), and two in the periplasmic vestibule (PV). Correspondingly, we performed well-tempered metadynamics with four replicas  , each coz”, defined as the difference of the z coordinate between the center of mass (com) of the substrate and that of the porin first monomer ; (ii) the “orientation Φ”, defined as the z component of the substrate’s dipole moment. These two geometric variables allow the substrate to sample more easily all positions inside the pore along the axis of diffusion z, by exploring also different orientations.We defined two collective variables to be able to follow the permeation of substrates through the pore: (i) the substrates “position Each walker was run for at least 450 ns, corresponding to a minimum total simulation time of 1.8 µs. During the metadynamics, the energy biases were added every 5.0 ps to each CV . Well-tempered ∆T was 4800 K. The FES of the translocation process is reconstructed by the integration and sign inversion of the Gaussian bias functions added during a simulationEnterobacteriaceae species share common geometrical and electrostatic properties. These properties can modulate the passive permeation of polar molecules, coupling with the size, the flexibility, and the electrostatic properties of the diffusing substrates. In particular, considering a series of avibactam derivatives, all under investigation as potential beta-lactamase inhibitors, we showed that the size of the molecules is better described, in the present context of permeation, by its minimal projection area instead of the molecular mass or volume, taking into consideration its distribution. Furthermore, the exact location of the charged groups in the molecular scaffold, which is described by the strength and direction of the electric dipole moment, is crucial to have an efficient compensation of the free energy barrier due to the decreased size of the pore. Thus, general molecular parameters, such as the minimal projection area and its distribution, the charge, and dipole moment, more than the presence or absence of particular chemical groups, are key to determine the permeation through hourglass-shaped pores such as the main general porins in Gram-negative bacteria. Interestingly, the molecular size and the dipole moment are not independent parameters. This still confirms the possibility to predict the permeability of molecules through porins by using a few molecular parameters, when properly considered.We demonstrated with all-atom molecular dynamics simulations that the OmpF/OmpC orthologs from


This essay considers how scholarly approaches to the development of molecular biology have too often narrowed the historical aperture to genes, overlooking the ways in which other objects and processes contributed to the molecularization of life. From structural and dynamic studies of biomolecules to cellular membranes and organelles to metabolism and nutrition, new work by historians, philosophers, and STS scholars of the life sciences has revitalized older issues, such as the relationship of life to matter, or of physicochemical inquiries to biology. This scholarship points to a novel molecular vista that opens up a pluralist view of molecularizations in the twentieth century and considers their relevance to current science. Molecular biology is not merely the description of biology in terms of molecules—if this were the case, it would not only include biochemistry, but also all those nineteenth-century studies in chemistry or in physiology, that led to the characterization of biological molecules. With such a broad definition, even Pasteur would have been a molecular biologist! Morange , p. 2.Michel Morange once remarked on the absurdity of trying to define molecular biology in abstract or transhistorical terms:longue durée view of molecularization enables scholars to better situate the molecular gene as part of a broader endeavor to transform the material basis of biological research into large, portable data sets  of proteins, viruses, or muscle fibers, chemistry was clearly a leading concern, although rarely trumpeted. In fact, John Kendrew, Max Perutz, Fred Sanger, Linus Pauling, and Gunther Stent were all chemists of some sort  are key genetic signatures of cancer cells, making cytogenetics especially valuable in oncology and cancer research. Her work suggests that scientific fields do not simply operate as political regimes that replace each other; more often, specialties continue on parallel tracks, and the public visibility of one discipline need not spell the demise of others .While this focus on structure has provided scholars with a useful vantage point, there are other avenues into the lesser-visited corners of the history of twentieth-century biology: these include studies of the prolific use of metaphors in the life sciences (besides the gene-as-code-metaphor); the history of lower-status fields, such as, nutrition science; and analyses of biological phenomena such as symbiotic relationships. The study of metaphorical language illuminates how historical actors understood and communicated conceptualizations of life as well as its experimentally known underpinnings Keller . The mosNeglected approaches to “molecules” and “life” also come into view when historians survey low-status areas of research. Nutrition is especially important in this regard—the association of life with chemical transformation and metabolism runs from nineteenth-century animal economy right through the industrialization of food production in the twentieth century  that advocates for greater attention to key molecules as regulators. Aligned with these calls for diversification of which objects count as historically relevant agents, Sarah E. Tracy has examined research that aimed to measure the effects of the flavor chemical monosodium glutamate (MSG). While glutamates are naturally occurring compounds, they are also common food additives, and their effects have been a source of controversy since the late-1960s. Tracy’s paper pointed to the striking compartmentalization of research on MSG. On the one hand, diabetes researchers have relied upon the obesogenic effect of large doses of MSG upon newborn mice. On the other hand, food scientists have pursued the potentially advantageous appetitive and digestive effects of MSG, based on findings from adult rodent models . Key connections between the flavor industry’s objectives and the risk of metabolic disorder due to food additives have been overlooked as a result of the compartmentalization of research and the complexity  of glutamate’s bioactivity  data, computing, or even machine learning in biology have achieved a significant degree of abstraction and independence from the material problems that historically occupied molecular biologists. As Soraya de Chadarevian asked off-handedly during the session, “Is this because genomics is no longer a chemistry-adjacent science?” Additional questions raised by the audience highlighted interests and concerns about the intersections between molecular biology and industry. We believe these types of questions and discussions are neither features limited to our panel nor circumstantial, but indicate broader shifts afoot.or educational backgrounds, and from underrepresented groups, genders, and ethnicities in the doing of science, including molecular biology. The foregrounding of marginalized actors, be they women, persons of color, or scientists from the Global South, can help diversify the curricula that is used to teach about the history and philosophy of the life sciences to new generations and thus impact whose histories are included and how those histories are remembered (Spanier The new attention to lower-status fields can also help make visible scientists of different socioeconomic  Spanier .13 Atten Spanier , p. 501  Spanier , it is n Spanier  to technSince the development of high-throughput genome sequencing in 2006, scientists have been increasingly doing cutting-edge biology in silico, relegating the bench to background labor Stevens . Looking


Despite improvements in prevention and treatment, cervical cancer (CC) still poses a serious threat to women’s health. CHMP4C (chromatin modified protein 4C) is a subunit of the endosomal sorting complex required for transport, which is expressed in both nucleus and cytoplasm. Here, we examined the effect of CHMP4C on the biological behavior of CC cells and the underlying mechanisms. We report that CHMP4C expression is higher in CC tissues, and high CHMP4C expression is associated with lower survival. Up‐regulation of CHMP4C in C‐33A cells accelerates cell proliferation, migration and invasion, whereas down‐regulation of CHMP4C in Ca Ski cells had the opposite effect. Moreover, overexpression of CHMP4C induced activation of the epithelial–mesenchymal transition pathway, whereas depletion of CHMP4C inhibited activation. Our results suggest that CHMP4C contributes to the viability and motility of CC cells by modulating epithelial–mesenchymal transition and may facilitate the identification of novel biomarkers for CC therapy. Chromatin modified protein 4C is highly expressed in cervical cancer (CC) tissues and cell lines, and contributes to the proliferation, invasion and migration of CC cells by modulating the epithelial–mesenchymal transition. Our findings may facilitate the identification of novel biomarkers for CC therapy. CCcervical cancerCCK‐8Cell Counting Kit‐8CHMP4Cchromatin modified protein 4CEMTepithelial–mesenchymal transitionHPVhuman papillomavirusqRT‐PCRquantitative real‐time PCRSDstandard deviationCervical cancer (CC) is one of the most common gynecological malignancies , 2. Everet al. [Chromatin modified protein 4C (CHMP4C) belongs to the CHMP (chromatin modified protein) family. It is a subunit of the endosomal sorting complex required for transport, which is expressed in both nucleus and cytoplasm , 8. Endoet al.  found thet al. , ovarianet al. , prostatet al. , among oThis work aimed to verify the relevancy between the expression of CHMP4C and survival rate of patients with CC, and to explore whether the modulation of CHMP4C expression would impact the biological behavior of CC cells, as well as the regulatory pathways, hoping to discover a novel biomarker that can predict the development of CC.https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), including tumor group (n = 306) and normal group (n = 3). These data were used to analyze the differential expression of CHMP4C in CC tissues and the correlation between CHMP4C expression and outcomes of patients with CC.The expression data of CC were downloaded from The Cancer Genome Atlas database  and normal control cells (Ect1/E6E7) were obtained from the cell bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences. Cells were routinely cultured in Roswell Park Memorial Institute‐1640 medium with 10% FBS, 100 U·mLCHMP4C#1: 5′‐CCAAGAAATCTCAGAAG‐3′; si‐CHMP4C#2: 5′‐GATGGCACACTTTCTAC‐3′ and si‐con: 5′‐CGAACUCACUGGUCUGACC‐3′) were synthesized from Shanghai GenePharma Co., Ltd. , which were used for knockdown of CHMP4C. Simultaneously, for overexpression of CHMP4C in CC cell lines, pcDNA3.1‐CHMP4C and corresponding control vector were also purchased from Shanghai GenePharma Co., Ltd. Lipofectamine 2000 Reagent  was used for transfection, according to the manufacturer’s protocol.siRNA sequences  were obtained from GENEWIZ . Glyceraldehyde‐3 phosphate dehydrogenase was used as internal reference with sequences , and the expression of CHMP4C was calculated with the 2−ΔΔCt method.The total RNA of cells was extracted using the TRIzol reagent  based on the manufacturer’s protocol. After reverse transcription to form cDNA, quantitative real‐time PCR (qRT‐PCR) was carried out to detect the expression of CHMP4C using SYBR Green I (Invitrogen), according to the supplier’s standards. The The radioimmunoprecipitation assay lysate (containing protease inhibitor) was used to crack the cells, and M‐PER Mammalian® Protein Extraction Reagent  was used to extract the proteins. The protein concentration was detected using bicinchoninic acid kit. About 20 μg protein was added to each well in the vertical electrophoresis tank, and then electrophoresis was performed with 10% SDS/PAGE. The protein on the gel was transferred to poly(vinylidene fluoride) membrane, followed by blocking with 5% defatted milk powder for 1 h. Then the membrane was incubated overnight at 4 °C with primary antibodies as follows: CHMP4C , E‐cadherin , N‐cadherin , Vimentin , Snail  and glyceraldehyde‐3 phosphate dehydrogenase . The next day, the membrane was incubated with horseradish peroxidase–conjugated secondary antibody  for 1 h at room temperature. After rinsing, electrochemiluminescence developer was added to develop the images. Glyceraldehyde‐3 phosphate dehydrogenase was used as a control to evaluate the relative protein levels.2. We assessed the cell viability at 24, 48 and 72 h, following the standard of the CCK‐8 kit . The absorbance at 450 nm was measured using a microplate reader .For Cell Counting Kit‐8 (CCK‐8) assay, cells were seeded into 96‐well plates (1000 cells per well) and cultured with CO2 at 37 °C. When clones were visible to the naked eye, the cells were fixed with 4% paraformaldehyde and dyed with 0.1% crystal violet dye. Ultimately, numbers of colony were counted.The colony formation assay was performed using the methods described in previous studies ; 400 cel5 cells were added to the upper chamber with serum‐free medium, and 500 mL serum medium used as the chemical attractant was put to the lower chamber. After incubation, the remaining cells in the upper chamber were erased using a cotton swab. The bottom membrane with invaded cells was fixed with 4% paraformaldehyde and dyed with 0.1% crystal violet. Then the randomly selected fields of vision were photographed for counting.Cell invasion was measured by cell‐penetrating matrix gel‐coated membranes. The invasion assay was conducted as described previously . After tThe migration assay was similar to the invasion assay, but it was evaluated by the penetration of cells into the plain membranes.t‐test, and one‐way ANOVA and Dunnett posttest were used to compare the multiple groups. The relationship between gene expression and clinical features was assessed using chi‐square test. Kaplan–Meier analysis was used to plot the survival curve, and the difference between groups was measured using log‐rank test. P < 0.05 was considered statistically significant.SPSS22.0 statistical analysis software  was used to analyze the experimental data. All the assays were repeated three times, and data were presented as the mean ± standard deviation (SD). The discrepancy between two groups was compared with Student’s n = 3) and CC tissues (n = 306) were downloaded from The Cancer Genome Atlas database to evaluate the differential expression of CHMP4C. It can be seen from Fig. P < 0.01). Subsequently, qRT‐PCR assay was performed to detect the expression of CHMP4C in CC cell lines. The data indicated that the expression of CHMP4C was up‐regulated in different degrees in the CC cell lines  compared with normal Ect1/E6E7 cells . Furthermore, C‐33A cells with the highest relative expression were selected for knockdown experiments, whereas Ca Ski cells were chosen for overexpression experiments, which showed relatively low expression.CHMP4C has been reported to be imbalanced in many cancers, but whether CHMP4C expression is associated with CC is still unknown. First, the data of control tissues (P = 0.039).To further analyze whether high CHMP4C expression leads to unoptimistic prognosis, we divided the patients into high‐CHMP4C‐expression and low‐CHMP4C‐expression groups based on the median level, and analyzed the survival rate of the high‐ and low‐expression groups with Kaplan–Meier survival analysis. The results indicated that the higher the CHMP4C expression, the shorter the survival time , and we can clearly observe that patients with age older than 60 years have higher ratio of overexpressed CHMP4C.Then the relationship between the expression of CHMP4C gene and clinical features was evaluated using chi‐square test. The statistical results in Table Moreover, we conducted a Cox regression analysis to investigate whether CHMP4C can be used as an independent predictor for CC prognosis Table . UnivariP < 0.01). Meanwhile, as presented in Fig. P < 0.01).To achieve knockdown and overexpression of CHMP4C, we transfected si‐ CHMP4C#1 and si‐CHMP4C#2 into C‐33A cells, respectively, and transfected pcDNA3.1‐CHMP4C into Ca Ski cells. After 24 h, the mRNA and protein levels of CHMP4C in the transfected cells were detected using qRT‐PCR and western blot assay. It can be seen from Fig. P < 0.01), whereas the viability of Ca Ski cells transfected with pcDNA3.1‐CHMP4C was markedly enhanced compared with that transfected with vector . The similar results can be seen from the colony formation assay. The colony number was obviously decreased in the si‐CHMP4C group compared with that in the si‐con group , whereas up‐regulation of CHMP4C in Ca Ski cells can promote the capability to form colonies .Next, we conducted functional experiments to measure whether CHMP4C contributes to the malignant biological behavior of CC cells. From the CCK‐8 assay, we can see that down‐regulation of CHMP4C can decrease the viability of C‐33A cells compared with control , whereas overexpression of CHMP4C in Ca Ski cells showed opposite results . Taken together, these results illustrated that CHMP4C functioned as an oncogene in the malignant biological behavior of CC cells, thus possibly being involved in the development of CC tumors.Transwell assay was performed to detect the influence of knockdown or overexpression of CHMP4C on cell migration and invasion. It can be seen from Fig. P < 0.01). Meanwhile, overexpression of CHMP4C in Ca Ski cells presented the opposite outcomes . Because the absence of E‐cadherin is considered to be a marker of EMT, these results illustrated that inhibition of CHMP4C can inhibit the EMT of CC cells. These data prompted a possibility that CHMP4C may contribute to the regulation of CC malignant progression through regulating the EMT pathway.Epithelial–mesenchymal transition (EMT) is a key event that promotes the migration and invasion of resting tumor cells . Thus, wThe occurrence and development of CC is a complex process of multifactor regulation . Currentet al. [et al. [The function of CHMP4C in cell dynamics and endocytogenesis has been widely reported , 21, 22.et al.  found foet al. . In addi [et al.  demonstr [et al. . Inhibit [et al. . BecauseEMT is considered to be the key factor to accelerate tumor invasion and migration , which cIn summary, our study found for the first time that CHMP4C was highly expressed in CC, and its high expression caused poor outcomes. Furthermore, CHMP4C may promote cell proliferation, invasion and migration by activating the EMT pathway. These findings prompted a novel strategy for CC therapy; however, the potential application in clinical treatment still needs further exploration.The authors declare no conflict of interest.S‐LL, MW and QL performed the experiments. S‐LL and MW analyzed the data. S‐LL and Q‐QC wrote the manuscript. S‐LL, Q‐QC and QL reviewed and edited the manuscript. All authors read and approved the final manuscript.


Kaposi’s Sarcoma (KS), first reported by Dr. Moritz Kaposi in 1872, is the most common vascular tumor in patients with AIDS. This disease is characterized by the appearance of violet lesions in the skin, mucosa, and gastrointestinal tract and may also affect other organs. KS is a multicentric angioproliferative spindle cell tumor originating from human herpes virus type 8 (HHV-8)-infected endothelial cells from lymphoid tissue [Most patients have painless skin lesions, which may have macular, papular, nodular, or plaque appearance. The lesions can have a range of colors from pink to red or purple and range in size from several millimeters to large confluent areas. Lesions are typically located in the oral cavity, face, and lower extremities, but may involve other sites. Visceral disease sometimes occurs in the absence of cutaneous lesions. Oral lesions can lead to ulceration, dysphagia, and secondary infection .In the immunocompetent host, the clinical course of HHV-8 infection is generally asymptomatic. In immunocompromised hosts, such as post-transplant patients and HIV-infected patients, HHV-8 develops into a variety of lymphoproliferative disorders .While HHV-8 is highly seroprevalent in sub-Saharan Africa >50%), it is quite rare in most European countries, Asia, and the United States (<10%) . The ser0%, it isClassical : cutaneoEndemic : An aggrIatrogenic : relatedEpidemic/associated with acquired human immunodeficiency virus (HIV) infection : more frNon-epidemic Kaposi : fifth aThere are currently five clinical variants of KS that have been described:KS associated with AIDS exhibits a broad spectrum of clinical presentations. The prognosis relies on the stage of KS, the level of immunosuppression, and the response to highly active antiretroviral therapy (HAART) .HIV-induced immunosuppression is a crucial factor in inducing the development of KS. The decrease in absolute CD4 T cell counts and the decrease in HHV-8-specific T cell immunity are associated with KS . In addiThe initial treatment for AIDS-related KS is with HAART , which, A male, 35 years old, with no known comorbidities was admitted to the emergency department, referred by the Basic Health Unit, due to edema and non-painful violet lesions in lower limbs without clear etiology, in addition to losing more than 10 kg in 2 months.The patient reported that the clinical picture started 12 months ago, initially with edema of the left lower limb, accompanied by a longitudinal erythematous cord that extended from the thigh to the ipsilateral foot, in addition to a single verruciform lesion located in the plantar region of the second toe . The patOn physical examination, the patient presented with pale mucous membranes and was emaciated. Upon inspection of the lower limbs, edema 4+/4+ without locker, in addition to evident hyperchromic lesions and hyperpigmented plaques with irregular edges and verruciform lesions, especially in the toes of both feet . In addiLaboratory examinations of the patient’s arrival were performed. It was observed that the patient had anemia and leukopenia with lymphocytopenia. In addition, he was diagnosed with HIV on his arrival at the hospital. He had a CD4 of 31 cells/µL, a CD8 of 301 cells/µL, a CD4/CD8 ratio of 0.10, and a viral load of 474,071 copies. Skin biopsies of the lesions were performed, which resulted in a suggestive aspect of KS. Computed tomography (CT) scan of the chest indicated numerous irregular opacities with ill-defined limits accompanied by small peribroncovascular pulmonary nodules, located in both lungs. The abdominal CT scan showed a liver with usual contours and dimensions, observing two small hypoattenuating images in the right lobe, both measuring 0.7 cm, undetermined. Fibrobronchoscopy revealed a trachea with two violet spots on the anterior wall which were biopsied, resulting in a lesion suggestive of KS. Upper digestive endoscopy identified the stomach with the presence of diffuse violet lesions of nodular shapes, without determining obstruction . In coloAfter the diagnosis, the patient started follow-up with the Infectious Diseases and Oncology Departments, starting HAART and liposomal doxorubicin as a treatment for KS.Considering that the patient did not initially present the characteristic violaceous lesions of KS, several erroneous diagnoses were made during the 12 months of the beginning of the first symptoms, and numerous wrong treatments were prescribed to the patient, contributing to the delay in his diagnosis and impairment of the patient’s prognosis. When the patient was admitted, the lesions were already presented in a typical form, with violet coloration. In addition, the oral cavity, trachea, lungs, stomach, and colon had already been affected.The main interest of this report is to emphasize the importance of early diagnosis. Despite the fact that the incidence of KS is decreasing with the emergence of HAART, this neoplasm still can indicate an unknown HIV status and significantly alter the prognosis and the survival of patients as the diagnosis is delayed.


Nanofibrous biomaterials have huge potential for drug delivery, due to their structural features and functions that are similar to the native extracellular matrix (ECM). A wide range of natural and polymeric materials can be employed to produce nanofibrous biomaterials. This review introduces the major natural and synthetic biomaterials for production of nanofibers that are biocompatible and biodegradable. Different technologies and their corresponding advantages and disadvantages for manufacturing nanofibrous biomaterials for drug delivery were also reported. The morphologies and structures of nanofibers can be tailor-designed and processed by carefully selecting suitable biomaterials and fabrication methods, while the functionality of nanofibrous biomaterials can be improved by modifying the surface. The loading and releasing of drug molecules, which play a significant role in the effectiveness of drug delivery, are also surveyed. This review provides insight into the fabrication of functional polymeric nanofibers for drug delivery. Nanofibers are a significant kind of biomaterial that could be used for biomedical applications, due to their special structure and properties such as high surface area , superioco-glycolic) acid (PLGA) and Poly(N-vinylpyrrolidone) (PVP) are usually utilized to form composites with natural polymers for nanofiber fabrication and for sustainable and controlled drug release [Nanofibrous biomaterials can be prepared from a wide range of polymers for drug delivery . Polymer release .Due to the superior properties of nanofibers, various nanofiber production technologies have been studied and utilized by many studies, including electrospinning, centrifugal spinning, solution blowing, phase separation, and self-assembly. Recently, electrospinning has been one of the major methods for nanofiber production, because of its numerous advantages, such as simple principles and equipment, broad material choice, and fabrication of nanofibers with versatile and uniform morphologies ,13,14. OMorphology and structure of nanofibrous biomaterials also significantly influence the function and effectiveness of drug delivery . The morThe drug loading methods and drug release rate significantly influence the effect of drug delivery. Drug loading methods can be divided into chemical and physical adsorptions. Drug release rate from nanofibers is determined by various factors, including drug diffusion, fiber erosion and biodegradation. This review introduces the current state of development in the field of drug loading molecules on nanofibers for drug delivery. It will be followed by discussion and comparison of various nanofiber production technologies. The current challenges and perspectives of nanofiber scaffolds for drug delivery are presented, and the future research directions of the field are also highlighted.Over 200 polymers can be utilized to spin nanofibers; however, only those that are biocompatible and biodegradable have been utilized as biomaterials to load drugs for tissue engineering . Table 1N,N-dimethylacetamide (DMA), dimethylformamide (DMF) and dichloromethane (DCM) (cellulose is quite difficult to dissolve) [Cellulose is a popular polysaccharide and exists throughout the world. Due to their excellent biodegradability and chemical resistance, cellulose nanofibers show a great potential for biomedical applications ,47. The issolve) ,49,50,51issolve) . Celluloissolve)  and to lissolve) . CA nanoissolve) . Ethyl cissolve) . In addiissolve) ,56,57.N-acetyl-d-glucosamine residues, which can be obtained from seafood wastes and invertebrate skeletons [Chitin is a linear 1, 4-linked polymer composed of keletons . Chitin keletons . It is akeletons . Chitin keletons . Therefod-glucosamine units, which is derived from N-deacetylation of chitin. As a significant derivative of chitin, chitosan is also biodegradable, biocompatible and nontoxic. Moreover, due to its antibacterial and antifungal properties, chitosan has great potential for tissue engineering applications. Recently, chitosan-based nanofibers have been utilized as matrix molecules for drug delivery [Chitosan is a linear polymer composed of β (1-4) linked delivery . Their adelivery . As a vedelivery , ocular delivery , intestidelivery , and nasdelivery . The spidelivery . Chitosadelivery . In addidelivery .Collagen is not only the primary structural element of ECM, but also the most abundant protein of the human body . CollageOther natural polymeric biomaterials, such as silk fibroin, keratin, alginate, and chondroitin, are also broadly studied for drug delivery and tissue engineering. For example, silk fibroin is derived from cocoons, which is a promising biopolymer due to its excellent biocompatibility and low biodegradation rate in the human body . Ang et However, the disadvantages of natural biopolymers include inconsistent compositions and weak mechanical strength . Additioco-glycolic acid (PLGA) is a co-polymer material from poly lactic acid (PLA) and poly glycolic acid (PGA) with different ratios, such as 75:25, 65:35, 50:50 and 25:75 [Poly lactic-nd 25:75 . The melnd 25:75 . The mecnd 25:75 . PLGA isnd 25:75 . PLGA isnd 25:75 ,37, DMF nd 25:75 ,34, THF nd 25:75 , chlorofPolycaprolactone (PCL) has been widely explored due to its excellent properties  and semi-crystallinity) and low cost. In addition, PCL can be dissolved into many common solvents, such as HFIP, chloroform, acetic acid, methanol and dichloromethane ,82,83. D2CH2)n-OH. Compared with other water-soluble synthetic polymeric biomaterials, PEO is unique in its linear structure. This special linear structure represents an excellent polymer–solvent interaction in water. For nanofiber production, the molecular weight of PEO is usually between 300,000–7,000,000. PEO is a particularly effective synthetic polymer for protein resistance because of its hydrophilicity [Polyethylene oxide (PEO) is a crystalline synthetic polymer with thermoplastic properties. It is a water-soluble polymer with a simple chemical formula, H-(OCHhilicity . Moreovehilicity . Qu et ahilicity . Additiohilicity . Sadri ehilicity .N-vinylpyrrolidone) (PVP) is also a water-soluble polymer with low toxicity and chemical stability. PVP has been employed as a blood plasma substitute in drug delivery systems, due to its blood compatibility and physiological inactivity [Other biocompatible and biodegradable synthetic polymers are also extensively studied for drug delivery in tissue engineering applications, such as PVA and PVP. Polyvinyl alcohol (PVA) is a water-soluble polymer and widely utilized by blending with other biopolymers. It has been combined with chitosan to enhance cell attachment and biocompatibility of the composite nanofibers . Poly is generated between the polymer solution and a grounded collection plate by connecting the needle of the spinneret with a high voltage power, and connecting the collector with the ground . When a Currently, the electrospinning system is mainly divided into two categories: needle electrospinning and needleless electrospinning. Needle electrospinning involves single-needle electrospinning , multi-needle electrospinning and multiaxial electrospinning. The production rate of single-needle electrospinning is only around 0.1 mL/h; therefore, a straightforward method to improve the productivity is to increase the number of needles , and a wA needleless electrospinning system dramatically improves the productivity of the electrospinning method, compared with needle electrospinning. In a needleless electrospinning system, Taylor cones are created on the surface of the polymer solution which cover the fiber generator . For thiElectrospinning is the most popular nanotechnology. However, the low production rate (needle electrospinning) and high energy consumption limit the extensive range of commercial applications of nanofibers to take advantage of the unique properties. For these reasons, it is highly necessary to develop more effective approaches to produce nanofibers.Centrifugal spinning is an alternative technological method to fabricate polymer nanofibers at a high production rate but a low energy consumption, even though it is not a novel technology. This technique was evolved from a fabrication technology for cotton candy invented in 1897 . CentrifCentrifugal spinning is an emerging nanofiber fabrication method because it not only has a high production rate with a low cost, but can also produce multiaxial nanofibers to improve the functions of nanofibers ,105. MulSolution blowing is a simple alternative method for nanofiber fabrication, due to the setup of this technology being simple, as shown in Self-assembly is a process by which nanofibers are manufactured by holding molecules without external guidance or management. The technology of self-assembly can be divided into two types, intramolecular and intermolecular self-assembly, respectively ,110. In Phase separation is another technique to fabricate polymeric nanofibers. The process of phase separation includes polymer dissolution, gelation, phase separation, solvent removal, and drying . First oThe native ECM has a dynamic and 3D porous structure with a diameter at the range of 50–500 nm. Therefore, the morphologies of artificial nanofibers for tissue engineering and drug delivery should be similar to native ECM, so as to provide an ideal microenvironment for cell adhesion and proliferation. The morphologies of nanofibers mainly involve fiber orientation and cross-section shape.The fiber orientation can be divided into isotropic and anisotropic fibers, as shown in The cross-sectional shapes of nanofibers are various, including single nanofibers and multiaxial nanofibers , as shown in Multiaxial nanofibers are able to encapsulate the drug into the nanofiber core, so as to provide protection from the surrounding environment and control drug release . Many coChemical adsorption: drugs and bioactive agents are modified onto the surface of nanofibers through various functional groups, including carboxyl groups, amine groups, hydroxyl groups and hydrophilic linkers, as shown in Carboxyl groups have been frequently used to immobilize bioactive agents onto the surface of nanofibers. Carboxyl groups, for example, were successfully grafted onto the surface of PLGA, PLLA and PGA nanofiber scaffolds for conjugation with collagen, to improve cell adhesion and proliferation . CarboxyHowever, direct modification of drug molecules on the surface of nanofibers might exhibit some limitations for cell attachment and proliferation. For example, cells cannot easily recognize the modified bioactive agents, as these agents are not entirely exposed to cells. Therefore, hydrophilic linkers are introduced to combine bioactive agents and nanofibers for promotion of cellular recognition. For example, EGF has been chemically modified onto the surface of amine-terminated block polymer composite nanofibers via polyethylene glycol (PEG) linkers .Currently, there are mainly three methods for physical adsorption of drugs in nanofibers or on the surface of nanofibers: simple physical adsorption, nanoparticle assembly, and multilayer assembly, as shown in Simple physical adsorption: nanofibers have an extremely high porosity and surface area, allowing for a great number of drugs to be adsorbed on the surface of nanofibers. This is the simplest method for the delivery of drug via nanofibers, which can be used for burst release of drugs from the surface of nanofibers. PCL-gum tragacanth–curcumin nanofibers have been produced via dissolving curcumin into PCL-gum tragacanth solution, for preventing bacterial infection within a few hours during wound healing . HeparinNanoparticles assembly: drugs are encapsulated into polymer nanoparticles, before these nanoparticles are adsorbed on the nanofibers. This method can effectively promote the drug loading capacity . PVP nanMultilayer assembly: coaxial and triaxial micro–nano fibers with unique features (including reinforced core and hollow structure) can be utilized to sequester stimulants in different compartments and control the release of various drugs through changing drug positions and thickness of layers . This drDrugs or bioactive molecules are transported through the drug delivery system, which is controlled by the random movement of drug molecules and actuated by chemical potential gradients. A classical drug release in nanofibers refers to the drug being transported from its initial position in nanofibers to the outer surface of the nanofibers; then, the drug is released into its surroundings . AdditioDrug molecules could be transported from nanofibers via diffusion through pores of nanofibers. It is known that nanofibers are highly porous; when nanofibers are filled with liquid, drug molecules randomly move through liquid-filled pores due to them being driven by the chemical potential gradients . As timeThe erosion of nanofibers can be divided into surface erosion and bulk erosion. Surface erosion means that the polymeric nanofibers degrade from the surface by slowly reducing the size from the outside towards the inside . SurfaceThe models of various drug release profiles are shown in However, the most common drug release is a tri-phasic profile, and it sometimes represents a bi-phasic . In a clFunctional polymeric nanofibrous biomaterials are a critical class of artificial ECM for drug delivery in biomedical applications. Biomaterials from different polymers have been employed to load various drugs and produce multi-functional nanofibers. In order to control drug release rate while maintaining the bioactivity of agents, many approaches to adsorbing drugs have been investigated. Multiaxial nanofibers might be the best method for loading multiple drugs and delivering them with controllable release, because different drugs can be encapsulated into different layers to satisfy the multi-functionalization of nanofibers. The development of nanofiber production technology also has improved dramatically, and various methods are employed to fabricate multiaxial nanofibers with multiple functions for drug delivery. However, no technology has been used to produce multi-functional nanofibers for drug delivery at an industrial scale.In the future, the mass, stable, and efficient production of multi-functional nanofibrous biomaterials should be the main the focus of technology development to translate the advanced nanofibers from the laboratories to industry implementation and eventually to clinical application. In addition, further studies should focus on combining nanofiber production and 3D printing technology. Therefore, the produced nanofibers can mimic both the micro and macro structures of different native ECMs, so as to apply functional nanofibers into individual tissues effectively and improve the functions of nanofibers in biomedical applications.


Acitretin, indicated for generalized pustular psoriasis, was effective in concomitant classic Kaposi sarcoma. We report a 60‐year‐old woman with Kaposi sarcoma successfully treated with systemic retinoid prescribed for a generalized pustular psoriasis.Kaposi sarcoma (KS) is a rare, multicentric angioproliferative spindle cell disease of lymphatic endothelial origin.We report a 60‐year‐old woman with KS successfully treated with systemic retinoid prescribed for a generalized pustular psoriasis (GPP).23). C‐reactive protein was elevated with a value of 173 mg/L. The procalcitonin test was negative. Histology showed spongiform pustules in the stratum corneum surrounded by parakeratosis and psoriasiform hyperplasia. The diagnosis of GPP was established. The patient was put on acitretin 30 mg daily (0.4 mg/kg) with an excellent improvement of both general and skin conditions. After 15 days of hospitalization, a few, red‐brown to violaceous, infiltrated papules and plaques appeared on her both forearms . Their effects are exerted through two families of nuclear receptors, retinoic acid receptors, (RARs) and retinoid X receptors (RXRs), which belong to the superfamily of steroid‐thyroid‐vitamin D3 hormone receptors. These receptors antagonize the enhancer action of NF‐IL6, a basic zipper family transcription factor and, therefore, inhibit IL‐6 promoter action.Corbeil has demonstrated that acitretin at low concentrations was sufficient to inhibit the growth of rapidly dividing early passage KS cells. Higher concentrations induced KS cells apoptosis but they are pharmacologically unachievable with oral systemic therapy.The coexistence of psoriasis and KS is rarely reported in the literature.In summary, we would like to highlight that acitretin, prescribed for GPP in our patient, was an active antitumor drug for early classic KS with complete recovery within a few weeks. Our observation is also particular given the short latency time from GPP to KS onset. The efficacy of acitretin is interesting to assess in KS.None declared.ND and AS: managed the patient and drafted the manuscript. IC: analyzed all histopathology specimens. MBS: referred the patient. MM: managed the patient and revised the manuscript. All authors have approved the final manuscript.Appropriate consent has been obtained, prior to submission, for the publication of images and data.