The superficial dorsal horn  of the spinal cord receives and processes multimodal sensory information from skin, muscle, joints, and viscera then relay it to the brain. Neurons within the SDH fall into two broad categories, projection neurons and interneurons. The later can be further subdivided into excitatory and inhibitory types. Traditionally, interneurons within the SDH have been divided into overlapping groups according to their neurochemical, morphological and electrophysiological properties. Recent clustering analyses, based on molecular transcript profiles of cells and nuclei, have predicted many more functional groups of interneurons than expected using traditional approaches. In this study, we used electrophysiological and morphological data obtained from genetically-identified excitatory (vGLUT2) and inhibitory (vGAT) interneurons in transgenic mice to cluster cells into groups sharing common characteristics and subsequently determined how many clusters can be assigned by combinations of these properties. Consistent with previous reports, we show differences exist between excitatory and inhibitory interneurons in terms of their excitability, nature of the ongoing excitatory drive, action potential (AP) properties, sub-threshold current kinetics, and morphology. The resulting clusters based on statistical and unbiased assortment of these data fell well short of the numbers of molecularly predicted clusters. There was no clear characteristic that in isolation defined a population, rather multiple variables were needed to predict cluster membership. Importantly though, our analysis highlighted the appropriateness of using transgenic lines as tools to functionally subdivide both excitatory and inhibitory interneuron populations. The superficial dorsal horn (SDH) of the spinal cord receives and processes primary afferent input conveying noxious, thermal, tactile, and pruritic sensory signals from the body destined for the brain. A key feature of the SDH, which includes laminae I and II, is the considerable heterogeneity within the local interneurons that make up >95% of the neurons in this region Todd, . In contRecent models of spinal sensory processing have provided a framework for understanding how interneurons contribute to spinal processing mechanisms. Specifically, they have highlighted the important interplay between inhibitory gating mechanisms  receptor have shown this population to be integral for spinal itch transmission  or the vesicular glutamate transporter-2 (vGLUT2). This enabled us to study inhibitory and excitatory dorsal horn neurons, respectively. Several studies have also used reporter proteins (eg GFP/TdTomato) expressed in specific cell types to study excitatory and inhibitory populations Lowl/MwarJ, Jackson Laboratories, Bar Harbor, ME, USA, #028862] or, a VGLUT2−Cre line  with a mouse reporter line to express a Cre-dependent~Td tomato . This generated mice where TdTomato is expressed in either vGAT (inhibitory) or vGLUT2 (excitatory) positive neurons. Both Cre-lines have previously been used to manipulate these populations in the dorsal horn using chemogenetics -Slc32a1n = 3, vGAT:TdTom and vGLUT2:TdTom) were overdosed with sodium pentobarbitone (30 mg/kg i.p) and perfused transcardially with 0.9% sodium chloride solution followed by 4% fresh depolymerized formaldehyde in 0.1 M phosphate buffer (pH 7.4). Spinal cords were dissected out and post-fixed in the same fixative for 2 h. Transverse sections (30 μm thick) were cut from the lumbar enlargement (L3–L5) on a cryostat  at −20°C. Sections were then incubated in primary antibody, anti-Pax2 , reacted with a secondary antibody conjugated to Cy5 , before mounting on slides in glycerol. Image stacks  were captured using laser-scanning confocal microscopy on a Leica TCS SP8 microscope  using a 10×/0.4 apochromat air interface objective , or 40×/1.3 apochromat oil immersion objectives  to capture “whole of dorsal horn” and higher resolution images, respectively.To assess the validity of TdTom expression in vGAT expressing neurons as a marker for inhibitory interneurons in the dorsal horn, we assessed co-expression of an accepted developmental marker for all inhibitory interneurons, Pax2, in this tissue , prepared using a vibrating microtome . Slices were transferred to an air interface incubation chamber containing oxygenated ACSF (118 mM NaCl substituted for sucrose) and allowed to equilibrate at room temperature (~23°) for at least 1 h prior to recording.Spinal cord slices for patch-clamp electrophysiology were prepared using previously reported methods  to achieve a final pH of 7.3–7.4. All recordings were made at room temperature. Neurons were visualized using a 40× objective and near-IR differential interference contrast optics. To identify vGAT and vGLUT2 positive neurons that expressed red fluorescent protein , slices were viewed under fluorescence using a TRITC filter set. vGAT and vGLUT2 neurons within substantia gelatinosa (LI-II) were targeted for recordings and were easily identified by their clear somatic fluorescence , to record excitatory synaptic input and AP discharge (series resistance <40 MΩ). Neurobiotin (0.2%) was included in the internal solution for studying post hoc cell morphology. All data were acquired using a Multiclamp 700B amplifier , digitized online  using an ITC-18 computer interface  and stored on the computer. This data capture and subsequent analysis used Axograph X software .Following incubation, slices were transferred to a recording chamber and continuously superfused with Carbanox-bubbled ACSF , with rheobase defined as the first current step that induced AP discharge. AP discharge patterns were classified using previously described criteria  and the inward currents; T-type calcium and the non-specific cationic current, Ih. Input resistance and series resistance were monitored throughout all recordings and data were excluded if either value changed by more than 10%. No correction was made for liquid junction potentials.The subthreshold currents underlying AP discharge were identified in a voltage-clamp protocol. This protocol consisted of an initial hyperpolarization from a holding potential of −70 mV to −100 mV (1 s duration) followed by a depolarizing step to −40 mV (200 ms duration) and applied P/N leak subtraction for analysis (within Axograph software). Four major ionic currents previously described in dorsal horn neurons could be identified in responses, including the outward potassium currents (IA was identified by an outward positive peak in the depolarizing step of the subthreshold current protocol (−100 mV to −40 mV) whereas ICa was identified as an inward current during this step. Peak current was measured (positive or negative) and used to designate cells as exhibiting dominant IA or ICa (or no current) responses. This approach cannot exclude the possibility that some cells expressed both IA and ICa currents, though even if occurring in combination our protocol yields the current likely to have the greatest functional relevance for spiking by predominating. Ih was characterized by a sag in the hyperpolarizing step (−70 mV to −100 mV). Peak Ih current was quantified as the difference between the immediate current response to this step subtracted from the maximum steady-state current at the end of this step.All electrophysiology data were analyzed offline using Axograph X software. Input resistance was calculated from the averaged current response  generated in each cell during a 5 mV hyperpolarizing step. Spontaneous excitatory postsynaptic currents (sEPSCs) were detected using a sliding template method (a semi-automated procedure in the Axograph package). sEPSCs frequency was determined from at least 30 s of continuous recording at a membrane potential of −70 mV. Peak amplitude, rise time (10–90% of peak), and decay time constant (fitted over 10–90% of the decay phase) were measured from the averaged sEPSC obtained for each neuron. The excitatory drive was calculated as the area under the curve of the averaged sEPSC multiplied by sEPSC frequency. Resting membrane potential (RMP) was taken as the average of 30 s of current clamp recording immediately after entering the whole-cell mode (input current = 0 pA). Individual AP properties for each neuron were assessed from the first spike generated at rheobase current. APs were captured using a derivative threshold method with AP threshold defined as the inflection point during spike initiation (dV/dt ≥15 V/s). The difference between the AP threshold and its maximum positive peak was defined as the AP peak. AP half-width was measured at 50% of AP peak, and AP afterhyperpolarization (AHP) amplitude was taken as the difference between the AP threshold and the maximum negative peak following the AP. AP discharge pattern properties were all assessed in the response to multiple steps with the response exhibited at two-step injections above rheobase (Rh40) used to measure key characteristics. AP delay was the time between step onset and the first AP discharged during the response. The first interspike interval (1st AP ISI) was the time between the onset of the first and second APs in the response. AP duration was the time between the first and last AP in the step response. AP number (AP n) was the number of spikes discharged in the entire response, and spiking AP adaptation was the ratio of the first interspike interval divided by the last interspike interval. For subthreshold currents, Iz = 1 μm, the field of view 445 μm × 445 μm). The dendritic architecture was analyzed for all cell recoveries deemed adequate to assess the rostrocaudal and dorsoventral extent occupied by processes. Specifically, as all recordings were undertaken in sagittal slices, the dorsoventral and rostrocaudal axes of each image were first oriented. Only cells with a clearly differentiated filled soma and processes, not overlapping with other filled cells in the same slice were analyzed. Three measurements were taken for each cell of these images: (1) the rostrocaudal extent, which was the distance between the most rostral and caudal labeled processes in the rostrocaudal plane; (2) the dorsal extent, which was the distance between the center of the cells soma and the dorsal-most labeled process in the dorsoventral plane; and (3) the ventral extent, which was the distance between the center of the cells soma and the ventral-most labeled process in the dorsoventral plane. A series of measures were also derived from this data, with dorsal and ventral measures added to yield dorsoventral extent, rostrocaudal extent was divided by the dorsoventral extent to provide a rostrocaudal/dorsoventral (RC:DV) ratio, and dorsal extent was subtracted from the ventral extent to yield a measure of dorsoventral bias. These measures have previously been shown to efficiently distinguish neuronal morphology in the dorsal horn  was included in the recording pipette for morphological analysis of recorded neurons. Slices containing filled cells were incubated in Streptavidin-Alexafluor 488 secondary antibody . They were mounted on slides with glycerol and imaged on a Leica TCS SP8 scanning confocal microscope using a 25 × 0.95 fluotar-water immersion objective  by determining where additional clusters no longer yielded an appreciable improvement to the SSE.Clustering analyses were completed using Orange (v3.2) data analysis software . Chi-squared tests compared the distribution of AP discharge patterns and subthreshold current expression between neuron classes.All data are presented as mean ± standard deviation (SD) unless otherwise stated. One-way ANOVAs were performed with a student Newman–Keuls n = 6) and VGLUT2:TdTomato mice . In each transgenic mouse line, recordings were targeted to labeled TdTomato positive neurons  and unlabeled TdTomato negative neurons . Thus, four-cell groups are differentiated and reported on: (1) targeted inhibitory interneurons ; (2) putative excitatory interneurons ; (3) targeted excitatory interneurons ; and (4) putative inhibitory interneurons . It follows that VGAT+ and VGLUT2− samples should come from the same overall (inhibitory) population, and likewise, the VGLUT2+ and VGAT− (excitatory) samples should overlap substantially.The following results include data from 198 dorsal horn neurons recorded in the SDH (laminae I-II) of spinal cord slices prepared from VGAT:TdTomato . In contrast, TdTom+ cell counts were lower in the medial dorsal horn compared to the middle region counts in VGLUT2:Td sections . Despite this minor regional difference for the VGLUT2:Td tissue, the number of TdTom+ cells per section was similar between VGAT:Td and VGLUT2:Td animals . Likewise, the number of Pax2+ cells was similar between VGAT:Td and VGLUT:Td animals . This analysis rules out inconsistent labeling as a cause for any subsequent difference in electrophysiological or morphological comparisons. In contrast, differences in the number of TdTom+ cells that exhibited overlapping Pax2+ immunolabeling in VGAT:Td vs. VGLUT2:Td animals were dramatic. For the VGAT:Td animal: 93.9% of TdTom+ cells were Pax2+, 87.3% of Pax2+ profiles were TdTom+, and 6.1% of TdTom+ did not express Pax2. This shows cells labeled in the vGAT:Td line are mostly inhibitory, some inhibitory interneurons are undetected, and importantly the chance of falsely identifying inhibitory interneurons is low.To assess our capacity to reliably identify inhibitory and excitatory interneurons in VGAT:Td and VGLUT2:Td mice, we first undertook immunolabeling experiments. TdTomato-expression (TdTom) in the lumbar spinal cord sections from each genotype was compared to Pax2 immunolabeling , which provides a reliable marker of inhibitory neuron phenotype in the dorsal horn . Cell coAs an excitatory cell marker was not readily available for this study a similar analysis to that above was not possible for the VGLUT2:Td animals. We could, however, use an exclusion approach to assess the capacity of TdTom+ expression to reliably identify excitatory interneurons by showing 91.6% of TdTom+ cells were Pax2−, and only 8.3% of TdTom+ cells were Pax2+. These data indicate that almost all vGLUT2 profiles were not inhibitory and thus the chance of falsely identifying an excitatory interneuron was low. The fraction of excitatory interneurons captured by the VGLUT2:Td animal could not be assessed without an excitatory cell marker. This value can, however, be estimated by comparison with the VGAT:Td animal using the well-established ratio of excitatory to inhibitory interneurons in the dorsal horn . This difference was also conserved between inhibitory VGAT+ cells and putative excitatory VGAT− cells (p < 0.001). Likewise, input resistance was lower in putative inhibitory (VGLUT2−) cells vs. excitatory VGLUT2+ cells (p = 0.05), but not in the putative excitatory VGAT− population. Differences in RMP were less prominent, with only targeted inhibitory VGAT+ cells exhibiting more depolarized membrane potentials than excitatory VGLUT2+ cells (p = 0.04). Finally, the rheobase current required to initiate AP discharge did not differ across the four neuron groups.A comparison of the electrical properties across excitatory and inhibitory interneurons revealed some differences between these populations . First, p < 0.001, Student Newman–Keuls post hoc test). The amplitude of APs was larger for the inhibitory cell groups compared to the excitatory populations , and the time course  was faster in the inhibitory vs. excitatory subsets . Finally, AHP amplitude was larger in VGLUT2− vs. VGLUT2+ neurons (p = 0.02). Thus, inhibitory dorsal horn populations exhibited AP spikes that had larger amplitudes and faster time courses than their excitatory counterparts.Despite a similar overall rheobase current, the properties of rheobase APs did differ . Specifip < 0.001). The TF was the dominant response among inhibitory interneurons . In contrast, IB and DF responses were more frequent in excitatory interneurons . Finally, although rare, rapid burst discharge (We next assessed the features of AP discharge initiated by depolarizing current step injection (1 s), because the literature suggests certain discharge responses are features of inhibitory and excitatory interneurons . Consistischarge  was obsep < 0.001). Conversely, inhibitory interneurons were more likely to express T-type currents than excitatory populations . In addition, voltage steps to −90 mV often evoked slowly activating hyperpolarization-activated cation currents in the inhibitory populations, reflecting Ih that was less common in excitatory populations . Measurement of peak currents during steps to −40 mV (regardless of direction) and −90 mV provides an indication of the relative magnitude of these currents in each population. This comparison for the −40 mV response confirmed A-type currents dominate in excitatory populations . This is consistent with the greater incidence of A-type potassium currents and DF AP discharge in these cells. In contrast, the lower peak current values for the inhibitory populations (VGAT+ and VGLUT2−) reflected a subset of these cells expressing pronounced inward T-type current responses (−100 to −250 pA), which is consistent with rapid burst and initial burst responses. Consistent with reports of a high incidence of TF in inhibitory populations, peak current responses during the −90 mV step  were also larger in these cells . Thus, inhibitory dorsal horn populations tend to express voltage-gated currents that support repetitive AP discharge and TF responses dominate. In contrast, excitatory populations are more likely to express voltage-gated currents that can suppress AP discharge, with DF being more prominent in these cells.Consistent with the above distribution of AP discharge patterns, the subthreshold voltage-activated currents that shape AP discharge was also differentially expressed across the cell groupings . Specifip < 0.001), whereas the sEPSC decay time constant was slower in inhibitory interneurons . In contrast, sEPSC amplitude and rise time were similar across all groups . Together, this is consistent with excitatory populations in LI-II receiving greater ongoing excitatory drive than inhibitory populations and, based on faster sEPSC decay times, this drive may occur at synapses located closer to their soma.The excitatory synaptic drive that recruits inhibitory and excitatory dorsal horn cell populations was also assessed , Table 4post hoc recovery and characterization of cell morphology. Interestingly, recovery was more successful in inhibitory rather than excitatory interneuron populations with yields of 55% (42/76) and 44% (11/25) for VGAT+ and VGLUT− cells, respectively (p < 0001). In contrast, dorsoventral dimensions were similar across the four neuron types .Neurobiotin was included in recording pipettes for ectively . In contectively . The extectively . Inhibitp < 0.01), compared to identified and putative excitatory populations (VGLUT2+ and VGAT−). Finally, the relative bias for processes in the dorsoventral plane was assessed by calculating the difference between a cell’s ventral and dorsal extent . No difference was detected in dorsoventral bias across cell groups (p = 0.22). However, the identified excitatory interneuron group exhibited a distribution of dorsoventral measures that was heavily skewed in the ventral plane (p = 0.04). Together, these results support the view that inhibitory dorsal horn cells exhibit extensive rostrocaudal territories, whereas excitatory interneuron processes are often biased to extend in the ventral plane.A ratio of the rostrocaudal to dorsoventral cell dimensions RC/DV in  was alson = 72). Hierarchical clustering based on electrophysiological data alone yielded six distinct clusters  reliably marked excitatory and inhibitory cells (>90% of excitatory and inhibitory cells correctly labeled). Thus, we can be confident that most targeted recordings and subsequent morphological analysis assessed the intended excitatory or inhibitory cell types. We also collect data from unlabeled cells in each transgenic line under the premise that these cells should represent the opposing population . This assumption appears to largely hold with the VGAT:Td line as TdTom labeled 87% of inhibitory cells (assessed by co-labeling for Pax2) leaving a fraction of the VGAT−Td negative cells (~13%) were also inhibitory. Thus, recordings from VGAT− cells, assumed to be excitatory neurons, may include some inhibitory cells.Although the use of transgenic animals to prospectively identify excitatory and inhibitory populations has obvious advantages, it also has limitations. First, our immunolabeling indicated that transgenically labeled populations  were made in this study and showed that all recorded cells exhibited the TF discharge pattern that is typical of inhibitory interneurons. While the majority of vGAT+ cells (~75%) in our study exhibited TF, there was more diversity including; initial/rapid bursting (~18%), delayed (5%) and single spiking (1%) patterns. This suggests we captured a more heterogeneous population, which can only be uncovered in a larger sample size . Nevertheless, these data show a VGAT−Tdtom mouse is a useful tool for teasing apart, from and inhibitory standpoint, the complex circuitry of the dorsal horn.Another study has sought to clarify the inhibitory nature of labeled VGAT-positive cells, before going on to manipulate these cells and monitor behavioral outcomes , and slower sEPSC decay kinetics than the excitatory population. Many of these properties are consistent with the more excitable electrophysiological phenotype of TF inhibitory interneurons. In addition, some of these differences likely relate to the distinct morphological features of inhibitory and excitatory neurons in the dorsal horn. Specifically, our inhibitory sample showed much longer rostrocaudal dendritic profiles, whereas dorsoventral arborizations were similar to the excitatory sample. The larger dendritic trees are consistent with the lower input resistance of our inhibitory neurons (Barrett and Crill, Our morphological observations are also consistent with the well-established morphological categories for classifying dorsal horn neurons in lamina II (Grudt and Perl, Despite the above associations, we did not classify the morphology of recorded cells in this study for two reasons. First, the morphological classification described above was developed specifically for neurons in lamina II, however, our recordings sampled a region that would include lamina I cells and potentially some in lamina III. Precisely locating cells to laminae require additional immunolabeling to confidently identify laminae borders. The second reason relates to cells that do not fit the criteria for this classification. In most studies that classify dorsal horn neurons in this way, an additional group of unclassified neurons is typically reported (Grudt and Perl, The two transgenic lines utilized throughout this study, vGAT:Td and vGLUT2:TdTom, positively identify inhibitory and excitatory cells and are thus useful for studying these broad cell populations. Furthermore, in cross-referencing the electrophysiological and morphological properties of these excitatory and inhibitory neurons in the SDH, our experiments support the conclusions of several previous studies characterizing various dorsal horn subpopulations. Inhibitory interneurons in this region are generally more excitable and easily recruited with TF discharge responses dominating, while the ongoing excitatory synaptic drive to this population is weak. This population also exhibits extensive rostrocaudal dendritic arborizations and features synaptic inputs on their distal dendrites that undergo substantial filtering. In contrast, excitatory interneurons are generally less excitable with a greater degree of DF and IB discharge profiles, and excitatory synaptic drive to this population is strong. This population exhibits a general bias in their dendritic arbors in the ventral plane, consistent with dorsal horn models that assign excitatory interneurons a role in transmitting signals from ventral to dorsal aspects of this region (Torsney and MacDermott, All datasets generated for this study are included in the article.The animal study was reviewed and approved by The University of Newcastle Animal Care and Ethics Committee under Section 25 of the NSW Animal Research Act, 1985.TB, CD, RC, DH, and BG conceived and designed the research study. TB, MG, and JI conducted experiments and acquired data. TB, MG, JI, JM, DH, and BG analyzed data. BG, TB, and RC wrote the manuscript. All authors edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The unpleasant smell released from dead bodies, may serve as an alarm for avoiding certain behaviour or as feeding or oviposition attractants for animals. However, little is known about their effect on the structure and function of proteins. Previously, we reported that using the aroma form of TEMED (a diamine), representative of the "smell of death", could completely inhibit the fibril formation of HEWL, as an antibacterial enzyme, and a model protein for fibrillation studies. To take this further, in this study we investigated the kinetics of TEMED using a number of techniques and in particular X-ray crystallography to identify the binding site(s) of TEMED and search for hotspot(s) necessary to inhibit fibril formation of HEWL. Structural data, coupled with other experimental data reported in this study, revealed that TEMED completely inhibited fibril formation and stabilized the structure of HEWL through enhancement of the CH-Π interaction and binding to an inhibitor hotspot comprised of residues Lys33, Phe34, Glu35 and Asn37 of HEWL. Additionally, results from this study showed that the binding of TEMED increased the activity and thermal stability of HEWL, helping to improve the function of this antibacterial enzyme. In conclusion, the role of the "smell of death”, as an important signal molecule affecting the activity and stability of HEWL was greatly highlighted, suggesting that aroma producing small molecules can be signals for structural and functional changes in proteins. Sense of smell in animals and human beings help them to find food, detect dangerous situations and provide cognitive influences, especially in human beings. These smells or aromas, arise from the properties of some compounds, which are found in their gaseous phase due to their small size and low vapour pressure.et al., in 2016, demonstrated that flies appear to use their sense of smell as a signal to find polyamine-rich foods and that polyamine-rich diets significantly increased the number of progenies of flies  to publish the content specifically under the CC BY 4.0 license.http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:We recommend that you contact the original copyright holder with the Content Permission Form (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. 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This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at plosone@plos.org if you have any questions.In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at [Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: NoReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Comments:It is interesting to reveal the effect of unpleasant smell such as TEMED on the structure and function of proteins. The work by Seraj et al. describes TEMED could inhibit the fibril formation of Hen egg white lysozyme (HEWL). Further, they identified the binding site(s) of TEMED and HEWL, and the hotspot(s) required to inhibit fibril formation. In addition, the binding of TEMED and HEWL increased the activity and thermal stability of HEWL. In conclusion, the role of the "smell of death" could serve as an important signal molecule to affect the activity and stability of HEWL. I think the work may be interesting for the potential readers of the journal. However, the following issues should be clearly addressed before it is accepted for publication.1. The researchers identified the binding site(s) of TEMED and HEWL, and the hotspot(s) required to inhibit fibril formation through the Ligplot analysis. Could you please kindly provide more methods/techniques such as site-directed mutagenesis to confirm your findings?2. Is it specific for TEMED to inhibit fibril formation and increase the activity and thermal stability of HEWL? Does it affect the structure and activity of other proteins such as BSA?3. Is there other diamine affecting the activity and structure of HEWL?4. Could you explain why the residues Lys33, Phe34, Glu35 and Asn37 are important for the binding of TEMED and HEWL?5. The abstract should be revised carefully to describe the goal of this study, the main findings and the conclusion to help readers better understand the novelty of this study.6. The figures and tables in this manuscript could not meet the science criterion and should be thoroughly reorganized and polished to present to the readers.7. The conclusion is too long. It should be concise and highlight the most important findings and scientific significance. Please revise it carefully.8. The authors should have their work reviewed by a proper translation/reviewing service or native-English speaker before submission. The manuscript needs careful editing by someone with particular attentions to English grammar, spelling, and sentence structure so that the goal and results of the study are clear to the readers.In my opinion, the article in the present form is not suitable for publication in PLoS One, and should undergo a round of major revision before publication at least.Reviewer #2: This study tried to study how "unpleasant smell" molecules (using TEMED as a model molecule) can be signal molecules and provide a structural mechanism. However, the writing of the paper is not clear even English is good. There are two major issues for this manuscript:1. HEWL is more like a random model protein the authors chose to study how TEMED affects protein stability and fiber formation. There is no clear explanation why interaction of TEMED with HEWL will have a biological effect. Using model proteins to study impact of TEMED is acceptable, but making farfetched biological functions is not, and can be misleading.2. Although several crystal structures were determined in complex with TEMED, the omit maps  and 2Fo-Fc are not provided. The authors have to provide these two maps to show that the TEMED molecules are really in the complex, instead of artifacts. For a small molecule like TEMED, arbitrarily placing atoms is not likely to impact Rfree or R in a noticeable way. By checking the B factors of some pdb structures published in this study, some TEMED molecules have high B factors, indicating low occupancy or improper model fitting.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  2 Apr 2020Please see the file uploadedReviewer #1: Comments:It is interesting to reveal the effect of unpleasant smell such as TEMED on the structure and function of proteins. The work by Seraj et al. describes TEMED could inhibit the fibril formation of Hen egg white lysozyme (HEWL). Further, they identified the binding site(s) of TEMED and HEWL, and the hotspot(s) required to inhibit fibril formation. In addition, the binding of TEMED and HEWL increased the activity and thermal stability of HEWL. In conclusion, the role of the "smell of death" could serve as an important signal molecule to affect the activity and stability of HEWL. I think the work may be interesting for the potential readers of the journal. However, the following issues should be clearly addressed before it is accepted for publication.1. The researchers identified the binding site(s) of TEMED and HEWL, and the hotspot(s) required to inhibit fibril formation through the Ligplot analysis. Could you please kindly provide more methods/techniques such as site-directed mutagenesis to confirm your findings?Answer: Thank you for your suggestion. However, in this study, we used X-ray crystallography with high resolution crystal structures to identify the binding site(s) and in particular the hotspot for inhibition of fibril formation in HEWL. The Ligplot images are a means to present the data obtained from our crystal structures showing residues in proximity to TEMED, their distances and types of interactions. Additionally, as mentioned in our manuscript, the binding of TEMED increased the activity of HEWL further emphasising the position and residues involved in binding:Additionally, according to the results obtained in this study, position A could be strongly suggested as the "hotspot" for inhibition of fibril formation in HEWL. This position is near the active site of HEWL and binding of TEMED to this position could explain why treatment of HEWL with TEMED resulted in an increased enzymatic activity. Harada et al. 2007, claimed that existence of lysyl residues with a positive charge at the C-terminal of α-helix A, α-helix B and α-helix C  contributed to the stability of helices, resulting in conformational stability of HEWL [1]. Kawamura et al. 2008, stated that Arg114 and Phe34 are two residues which are responsible for the productive binding at right-sided binding site of HEWL and that the CH-π interaction between the guanidyl side chain (C(NH2)2) of Arg114 and the phenyl ring of Phe34 on the molecular surface could stabilize the Arg114 side chain and fix it to accept the substrate molecule via the right-sided mode [2]. Accordingly, the interaction between Arg114 and Phe34 is suggested to be the important structural factor in maintaining the native conformation of HEWL. Based on their results, Arg114 in HEWL is thought to play an important role in lysozyme catalysis, especially in transglycosylation. Now looking at our results of the enzymatic activity test and the structure of HEWL treated with TEMED, the presence of TEMED near Phe34 (with hydrophobic interactions) could have an effect on the stabilization of Arg114. Additionally, it is plausible to say that the CH-π interaction between the methylamine (N(CH₃)₂) groups in TEMED (of which there are two) and phenyl ring in Phe34 (providing the π system), in addition to the CH-π interaction between the guanidyl side chain of Arg114 and again that of the phenyl ring of Phe34, are the main contributors in enhancing the ability of HEWL to accept its substrate, such that we see greater enzymatic activity in HEWL in the presence of TEMED and greater intrinsic fluorescence emission.2. Is it specific for TEMED to inhibit fibril formation and increase the activity and thermal stability of HEWL? Does it affect the structure and activity of other proteins such as BSA?Answer: Since the aim of this research was to use HEWL as a model protein for fibrillation studies, we were not concerned with the effect of TEMED on other proteins, but rather the effect of different small molecules on inhibition of HEWL fibril formation. However, we have preliminary DSF results showing that the presence of different concentrations of TEMED for two different purified proteins, Thiosulfate transferase (hTst) and 1-deoxy-D-xylulose 5-phosphate synthase from Mycobacterium tuberculosis (mtDXS), either showed no thermal stability changes or resulted in decreased Tm, respectively .Therefore, it appears so far that TEMED is specific for increasing the thermal stability of HEWL. The DSF results are provided to answer the respectable reviewer’s question, but is preliminary and preferably not to be included in this manuscript.Additionally, Anthony et al., reported from their study that in contrast to the effects of TEMED, which we observed on HEWL, all the three similar polyamines listed in their study including putrescine, spermidine and spermine resulted in an increased alpha synuclein aggregation [3]. Another report from Luo et al. showed that cellular polyamines promote amyloid-Beta (Aβ) peptide fibrillation [4]. Furthermore, it is reported that the acceleration of aggregation by certain polyamines suggest that they may also facilitate neurodegeneration [3].DSF results of 1-deoxy-D-xylulose 5-phosphate synthase from Mycobacterium tuberculosis (mtDXS) and Thiosulfate transferase (hTst) in either the absence or presence of different concentrations of TEMED in solution. (a) 1-deoxy-D-xylulose 5-phosphate synthase from Mycobacterium tuberculosis (mtDXS). (b) Thiosulfate transferase (hTst).3. Is there other diamine affecting the activity and structure of HEWL?Answer: Yes, there is a report from Okanojo, et al., at 2005 which showed that diamines prevent thermal aggregation and inactivation of lysozyme [5].4. Could you explain why the residues Lys33, Phe34, Glu35 and Asn37 are important for the binding of TEMED and HEWL?Answer: As we mentioned in our manuscript, Kawamura et al. 2008 [6], stated that Arg114 and Phe34 are two residues which are responsible for the productive binding at right-sided binding site of HEWL. Furthermore, the CH-Π interaction between the guanidyl side chain (C(NH2)2) of Arg114 and the phenyl ring of Phe34 on the molecular surface could stabilize the Arg114 side chain and fix it to accept the substrate molecule via the right-sided mode. Accordingly, the interaction between Arg114 and Phe34 is suggested to be the important structural factor in maintaining the proper conformation of HEWL. Based on their results, Arg114 in HEWL is thought to play an important role in lysozyme catalysis, especially in transglycosylation. Now looking at our results of the enzymatic activity test and the structure of HEWL treated with TEMED, the presence of TEMED near Phe34 (with hydrophobic interactions) could have an effect on the stabilization of Arg114. Additionally, it is plausible to say that the CH-Π interaction between the methylamine (N(CH₃)₂) groups in TEMED (of which there are two) and phenyl ring in Phe34 (providing the Π system) in addition to the CH-Π interaction between the guanidyl side chain of Arg114 and again that of the phenyl ring of Phe34, is the main contributor in enhancing the ability of HEWL to accept its substrate such that we see greater enzymatic activity in HEWL in the presence of TEMED and greater intrinsic fluorescence emission.Additionally, binding of TEMED close to Glu35 as one of the residues in the active site including Glu35 and Asp52, which resulted in increasing in the activity of HEWL, was supportive data about the importance of this position.5. The abstract should be revised carefully to describe the goal of this study, the main findings and the conclusion to help readers better understand the novelty of this study.Answer: Thank you. The abstract has been improved.6. The figures and tables in this manuscript could not meet the science criterion and should be thoroughly reorganized and polished to present to the readers.Answer: Changes were applied as much as possible.7. The conclusion is too long. It should be concise and highlight the most important findings and scientific significance. Please revise it carefully.Answer: Changes were applied to the conclusion section of the manuscript.8. The authors should have their work reviewed by a proper translation/reviewing service or native-English speaker before submission. The manuscript needs careful editing by someone with particular attentions to English grammar, spelling, and sentence structure so that the goal and results of the study are clear to the readers.Answer: Thank you for your suggestion. This was done.In my opinion, the article in the present form is not suitable for publication in PLoS One, and should undergo a round of major revision before publication at least.Reviewer #2:This study tried to study how "unpleasant smell" molecules (using TEMED as a model molecule) can be signal molecules and provide a structural mechanism. However, the writing of the paper is not clear even English is good. There are two major issues for this manuscript:1. HEWL is more like a random model protein the authors chose to study how TEMED affects protein stability and fiber formation. There is no clear explanation why interaction of TEMED with HEWL will have a biological effect. Using model proteins to study impact of TEMED is acceptable, but making farfetched biological functions is not, and can be misleading.Answer: As we explained in this study, TEMED was chosen because of its pungent fishlike odour and its similarity to putrescence and cadaverine as polyamines, also known as the "smell of death”, which are produced by decarboxylation of precursor amino acids by enzymes of bacterial origin. On the other hand, based on our previous study, it was interesting for us that fibril formation in HEWL, as an antibacterial enzyme, was inhibited by TEMED with properties mentioned above. Therefore, we decided to extend our study and tried to find whether or not these results were related to any potential biological effects or roles. Interesting we found, using results from a number of techniques, that TEMED was a specific ligand for HEWL.2. Although several crystal structures were determined in complex with TEMED, the omit maps  and 2Fo-Fc are not provided. The authors have to provide these two maps to show that the TEMED molecules are really in the complex, instead of artifacts. For a small molecule like TEMED, arbitrarily placing atoms is not likely to impact Rfree or R in a noticeable way. By checking the B factors of some pdb structures published in this study, some TEMED molecules have high B factors, indicating low occupancy or improper model fitting.Answer: Omit maps are now provided in the supplementary information Figures S2-S5.Bibliography1. Harada, A., et al., Relationship between the stability of Hen Egg-White lysozymes mutated at sites designed to interact with α-helix dipoles and their secretion amounts in yeast. Bioscience, biotechnology, and biochemistry, 2007. 71(12): p. 2952-2961.2. Kawamura, S., et al., The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme. Bioscience, biotechnology, and biochemistry, 2008. 72(3): p. 823-832.3. Antony, T., et al., Cellular polyamines promote the aggregation of alpha-synuclein. J Biol Chem, 2003. 278(5): p. 3235-40.4. Luo, J., et al., Cellular polyamines promote amyloid-beta (Abeta) peptide fibrillation and modulate the aggregation pathways. ACS Chem Neurosci, 2013. 4(3): p. 454-62.5. Okanojo, M., et al., Diamines prevent thermal aggregation and inactivation of lysozyme. J Biosci Bioeng, 2005. 100(5): p. 556-61.6. Kawamura, S., et al., The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme. Biosci Biotechnol Biochem, 2008. 72(3): p. 823-32.AttachmentResponse to Reviewers_AS_ZS 13.docxSubmitted filename: Click here for additional data file. 27 Apr 2020The aroma of TEMED as an activation and stabilizing signal for the antibacterial enzyme HEWLPONE-D-19-32635R1Dear Dr. Seyedarabi,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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N-acetylphenylalanylmethionine, propanoic 3-[ carbamoyl]-2-(piperazin-1-yl) acid, 3-(propylaminosulfonyl)-4-chlorobenzoic acid, ascorbic acid (vitamin C), and 4-(dipropylsulfamoyl) benzoic acid (probenecid). Their half maximal inhibitory concentration (IC50) was determined through fluorometry. An acidic reagent 2′-O-(4-methylumbelliferyl)-α-dN-acetylneuraminic acid (MUNANA) was used as substrate for viruses of human influenza H1N1 or avian influenza H5N2. Inhibition was observed in millimolar ranges in a concentration-dependent manner. The IC50 values of the five proposed scaffolds ranged from 6.4 to 73 mM. The values reflect a significant affinity difference with respect to the reference drug zanamivir (p < 0.001). Two compounds (N-acetyl dipeptide and 4-substituted benzoic acid) clearly showed competitive mechanisms, whereas ascorbic acid reflected non-competitive kinetics. The five small organic molecules constitute five different scaffolds with moderate NA affinities. They are proposed as lead compounds for developing new NA inhibitors which are not analogous to sialic acid.Neuraminidase (NA) of influenza viruses enables the virus to access the cell membrane. It degrades the sialic acid contained in extracellular mucin. Later, it is responsible for releasing newly formed virions from the membrane of infected cells. Both processes become key functions within the viral cycle. Therefore, it is a therapeutic target for research of the new antiviral agents. Structure–activity relationships studies have revealed which are the important functional groups for the receptor–ligand interaction. Influenza virus type A NA activity was inhibited by five scaffolds without structural resemblance to sialic acid. Intending small organic compound repositioning along with drug repurposing, this study combined in silico simulations of ligand docking into the known binding site of NA, along with in vitro bioassays. The five proposed scaffolds are  Influenza is a severe and infectious respiratory disease with high mortality and morbidity rates. Two subtypes of influenza A viruses (H1N1 and H3N2) along with two lineages of influenza B viruses (Victoria and Yamagata) are cocirculating in the human population. The World Health Organization (WHO) estimates that there are approximately 290,000 to 600,000 annual deaths worldwide associated with the infection . Both glNA mediates a pivotal step in the life cycle of influenza A viruses and thereby contributes to their transmissibility and pathogenicity—all of which characterizes it as a potential viral protein target. Commercial drugs against this infectious disease in oral dosage forms have been derived from the aforementioned terminal sialic acid which constitutes the natural substrate of NA ,8. PreciChanges in the primary sequence have been detected and may reflect drug resistance phenomena, in particular, residue mutation patterns at the binding sites of both neuraminidase groups . In a moNevertheless, oral drug administration seemingly shortens the duration of disease or convalescence in addition to reducing the risk of complications or the patient’s death if the drug treatment is applied on time. Very early during the initial stages of infection, neuraminidase inhibitors have a dual effect not only as a causal cure attacking the infection cycle but also as a prophylactic tool against influenza virus infections. In recent years, emerging single or multidrug-resistant strains have threatened the therapeutic achievements. Several mutational changes have been associated with drug resistance . In 2018To this regard the present study aims at proposing non-sialic acid-like scaffolds with proven binding site affinity to address the drug resistance problem suggesting the development of scaffold derivatives to enhance binding site affinity to mutated neuraminidases.The molecules under scrutiny do not represent analogues of neuraminidase inhibitors oseltamivir and zanamivir—albeit to some extent they are structurally related to natural substrate sialic acid by a set of common features . Four drN-acetylphenylalanylmethionine was named AAMOL because it constitutes a dipeptide; hence “AA” symbolizes two amino acids. FMOL is the short name of propanoic 3-[ carbamoyl]-2-(piperazin-1-yl) acid, and alludes to the first introduction of an aromatic ring. The phenyl group was written with an “F” like the one-letter code “F” for phenylalanine or “fenil” in Spanish and therefore the scaffold was called FMOL. The “S” in STL reflects the sulfonamide class of substance: 3-(propylaminosulfonyl)-4-chlorobenzoic acid. The two repositioned or repurposed molecules were ascorbic acid (common name: vitamin C) and 4-(dipropylsulfamoyl) benzoic acid (INN drug name: probenecid). CIV and ORP are their respective inverted code names. Thanks to their coded labels, all substances could be tested in the laboratory without user bias.It is noteworthy to explain that the internal acronyms reflect certain features of the five studied molecules as potential NA inhibitors: As input data for our studies we included two influenza strains: (i) the human A/PR/8/34 (H1N1) and (ii) the avian A/Chicken/Mexico/31381-7/1994 (H5N2). Multiple sequence alignments (MSA) were performed with different sequences of influenza A neuraminidases (from N1 to N9) to identify segments of conserved amino acids which correspond to residues at the active site. Residues R118, D151, R152, R224, E276, R292, R371 and Y406 constitute the catalytically active residues within the binding site. They interact directly with the substrate, sialic acid (SA). Residues E119, R156, W178, S179, D198, I222, E227, H274, E277, N294 and E425 assist the recognition and binding of ligands ,17,18,19Molecular docking was carried out under Autodock Vina ,21 usingIn the next step, we retrieved the neuraminidase sequences of human A/PR/8/34 (H1N1) strain (NCBI GenBank: ABD77678.1) and avian A/Chicken/Mexico/31381-7/1994 (H5N2) strain (NCBI GenBank: ACZ36772.1). To obtain full-length three-dimensional structures of our target proteins, we used I-TASSER for homology modeling ,31.50).Interaction analyses allowed the observation of the residues that form hydrogen bonds with the ligands. These interaction analyses were based on the outcome of docking studies to determine how the ligand occupies the active site. These computed binding modes were found to be on an equal footing with our experimental results reflecting how efficient our molecules were with respect to the zanamivir control. Precisely, the active site of NA is extremely polar. To facilitate the inspection of the inhibitor binding modes, the active site was divided into five subsites (S1-S5) ,32,33 F. S1 is cThe experiments with the four neuraminidases were carried out in presence of the reference drug zanamivir. The catalytic residues within the active sites were identified as R118, R292 and R371 at subsite S1 and D151 at subsite S2, R152 at subsite S3, and E276 ,36,37. TThe literature reports about different structural modifications of reference drug zanamivir. Starkey et al. (1995)  synthesiThe five scaffolds were proposed since they are not analogous to sialic acid. Nevertheless, they present similarities in their functional groups  and showSTL and CIV reveal a diversification in their interaction profiles, i.e., they interact with different residues depending on the two NA subtypes. Especially, CIV can occupy the cavity in a multiple binding mode. Thanks to its small molecular size and volume it can interact with different residues in a variety of final poses at the active site. Like CIV, STL cannot be docked in an optimal way leading to affinity losses compared to reference drug zanamivir. Its aminosulfonyl group sidechain makes an unfavorable kink and subsequently the ligand shifts into less favorable locations. This, in turn, decreases the anchoring capacity of the molecule within the active site with respect to zanamivir.FMOL is a typical scaffold since it is poorly decorated with functional groups considering its molecule weight. Few hydrogen bonds, polar and nonpolar interactions contribute to target affinity. Hence, it promises huge affinity improvements upon derivatization.2 groups. Alternatively, the sulfonyl group can be moved to the meta position to reach other residues at the catalytic site.ORP can interact with Arg118 and Arg152, following a characteristic binding pattern that was seen in almost all analyzed neuraminidases. This finding creates the opportunity to enhance affinity by introducing side chains in the scaffold’s ring meta position, for instance -OH or -NHIn the in vitro experiments, Madin-Darby canine kidney (MDCK) cell lines were infected with human influenza A H1N1 and avian H5N2 viruses. The supernatants were collected by observing an extensive cytopathic effect at 96 h post-infection (hpi). The supernatants were stored in aliquots at −70 °C for the determination of enzymatic activities of the neuraminidase and viral titration by means of lytic plaques ,48.The enzymatic activity of NA was analyzed with the fluorescent substrate MUNANA. The standardized assay allowed us to compare data between different viral isolates and assays between different laboratories. Samples of cell supernatant infected with whole virus were used as source of enzymes. The numbers of either virions in the samples or the numbers of NA molecules in each virion may vary from strain to strain. Hence, we used the criteria reported by Marathe et al. in 2013 to select the amount of enzyme or viral dilution to provide adequate fluorescence for our trials. The relative fluorescence units (RFU) used in our assays ranged from 15,000 to 45,000 RFU. In this range no saturation of the system occurred and linearity existed in the quantification method.In addition, serial 1:2 dilutions were prepared from the same supernatants and it was observed that dilutions generated a proportional increase in the concentration of 4-methylumberiferone with respect to the incubation time for MUNANA substrate. This observation attests that the dilutions generated a linear increase in the fluorescent product with respect to time .−1 were calculated for the avian virus and for the human being a Km of 142 μM and a Vmax of 2.77 μmol min−1 , as well as test substances AAMOL and ORP, showed a competitive inhibition mechanism for both NA subtypes. In this type of mechanism, the substrate and the inhibitor are exclusive, which indicates that only one of them can occupy the active site of viral neuraminidase .For the CIV molecule, the type of inhibition mechanism was characterized as being noncompetitive in nature. Henceforth, the inhibitor CIV succeeded in bind to neuraminidase, but did not modify its affinity for the substrate. It modified, however, the enzymatic velocity. For this type of mechanism, the inhibitor could bind to the enzyme forming the complex E-I (enzyme-inhibitor) or the tertiary complex E-S-I (enzyme-substrate-inhibitor). Here substrate and inhibitor are no longer mutually exclusive . AnotherAAMOL, ORP and CIV inhibit the enzymatic activity in the order of the millimolar, however, these structures can be modified, adding polar residues that allow us to increase the affinity of neuraminidase for these and decrease the value of its inhibition constant, Ki, allowing the occurrence of an inhibitory action with lower concentrations of inhibitor .50 values for the human H1N1 and avian H5N2 viruses using zanamivir as a positive control of the inhibition  remain unaccounted for in molecular mechanics calculations of docking simulations. Autodock Vina was released as a direct answer to the manifold (cryptic) parameter settings of Autodock 4.2 with their need for a “case-to-case fine tuning”  to run Autodock 4.2. Back then, hydrophobic forces like entropic and solvent effects had been integrated into the docking force field by empiric calibration of published ligand–receptor training data and averaged to fit test set examples under a linear scaling approach. A trade-off between larger training sets—to treat more of the binding site and mode variations to widen the applicability range to gain user popularity—and reproducibility (predictability) concerns had to be made by the program developers. Bearing this in mind now, it becomes quite logical to experience this prediction failure with subtype N1 where the important gain in hydrophobic forces remained unaccounted for. In more general terms, empiric parameters describe hydrophobic contributions to docking energies which is a necessary over-simplification of the physicochemical first principles unless a quantum chemistry approach is used. This type of docking pitfalls was more frequent with Autodock 3 which exaggerated hydrogen bonding contributions to the final docking energy term (ligand affinity) and triggered a new release (Autodock 4) for treatment of proteins with extended hydrophobic regions like the hydrophobic subsites S4  and S5 (S246 and E277).Scaffold ORP differs significantly in its avian and human subtype IC50 in The inhibition of viral replication was evaluated in MDCK cell cultures by means of lytic plaques for reference zanamivir as well as FMOL, STL, CIV and ORP . To obse50 is the drug concentrations necessary to reduce cytotoxicity or cell viability by 50%. The CC50 values of ORP were observed in millimolar ranges, which allows us to use higher concentrations without toxic effects, likewise, AAMOL. Its cytotoxicity is lower, since it is metabolized over time as it is a compound based on two amino acids. Both substances constitute optimal candidates to develop new antiviral drugs. The other compounds can be modified to generate products with greater antiviral activities, seeking to reduce their toxic effects at the same time.AAMOL and ORP inhibited enzymatic activity competitively, whereas CIV showed a non-competitive mechanism under millimolar assay conditions. FMOL affected the size and number of plaques at concentrations of 1 mM and 100 μM. STL completely inhibited at concentrations of 1 mM, losing this effect when diluted to 100 μM. ORP only lowered the number of plaques at a concentration of 1 mM, but when the concentration was even more lowered it finally lost its inhibitory drug action , Table 2AAMOL inhibits NA in millimolar concentrations with a competitive inhibition mechanism. It constitutes a monosubstituted dipeptide, hence its LADMET properties could be expected to be favorable, especially cellular uptake, biotransformation and toxicity. Even larger quantities (to compensate the weak affinity) should not cause toxic side effects. The challenge of derivatizations is twofold: not only potency must be improved but also the compound decay by nonenzymatic hydrolysis must be must be delayed by introducing suitable modifications.ORP imitates zanamivir’s binding mode through the carboxyl group on the ring. The coinciding binding positions of their carboxyl groups and both six-membered rings allow the introduction of functional groups in ring positions that become equivalent to the functionalities of the side chains of zanamivir. In the same manner, reducing the aliphatic chain length of the sulfonyl group should enhance target affinity. As an asset for ORP counts the observation that its receptor affinity was not affected during docking simulations with mutated neuraminidases with respect to wild type NA. The simulated viral mutant types were studied because they cause known drug resistance to oseltamivir and zanamivir ,56,57.l-gulonate or 3-oxo-l-gluonate by a twofold dehydration reaction. (ii) Its chemical bond formalism shows keto-enol tautomerism. Only the enol form—and not the keto form—became detectable in solid phase or solution by crystallography or 13C-NMR, respectively. The tautomeric preference is due to the strong stabilizing effects of two intramolecular hydrogen bonds between the oxo group of the lactone (R-O-C(R)=O) and both hydroxyl groups of the endiol form (HO-C(R)=C(R)-OH). (iii) It is a bivalent Bronsted acid and its surprising acidity strength is slightly stronger than acetic acid (pKa = 4.76) for its first proteolytic step with a measured pKa value of 4.17 thanks to its vinylogous carboxylate anion formation directly stemming from its preferred endiol form (O=C(R)-C(OH)=C(R)-O− + H+). (iv) In aqueous solution it can react as a CH acidic component  in Michael addition reactions with acrolein-like agents (R2>C=C(R)-C(R)=O). (v) Finally, its prevailing endiol form strongly reduces substances in aqueous redox reactions. During this reaction ascorbic acid is converted into dehydroascorbic acid which shows its essential role in cell physiology as antioxidant [CIV is the smallest of all five molecules. It inhibits the activity of neuraminidase in a non-competitive way at millimolar concentrations. Its kinetic behavior can be explained by the formation of stable covalent bonds instead of the reversible noncovalent bonding between the other four scaffolds to the receptor residues. Prior to describing the two possible chemical reactions of covalent bond formation by vitamin C it is paramount to understand the following aspects: (i) Its relatedness to reactivity and stereo configurations of monosaccharides, precisely their acid derivatives ioxidant .l-gulonic acid which is highly reactive (instable) to form new bonds due to its two vicinal “oxo” oxygen atoms (O=C(R)=C(R)=O). The second way (B) is more likely to occur under anaerobic conditions, but also in situations with a relative absence of molecular oxygen (O2). At the initial stage, the enol form of ascorbic acid is converted into its unusual keto form (O=C(R)-C(=O)-C(R)-OH) leading to a multi-step degradation to produce furfural but also other cyclic or ring-opened aldehydes (R-C(H)=O are conceivable. The resulting carbonyl function again is highly reactive to produce more stable adducts under covalent bond formation. Potential counterparts for reaction (A) are the basic amino acids histidine and arginine or the neutral asparagine and glutamine, as well as aromatic tyrosine and tryptophan. On the other side, almost all side chains of the amino acids are amenable to covalent bond formation via (B), except for aliphatic amino acids, proline, methionine or glycine. Of note, traces of heavy metal cations—typically copper or iron ions—catalyze the decay of vitamin C, too. Intriguingly, the latter metal constitutes the central atom at the biocatalytic site of ubiquitarian ascorbic acid oxidase.Based on the aforementioned reactivity, our experimentally determined non-competitive mechanism suggests the covalent bond formation for ascorbic acid in either of two ways: (A) after oxidation and under aerobic conditions a hydrolysis of the endocyclic ester takes place in the first step, this means a decay of the lactone ring by a water moiety as an attacking nucleophilic general base. The direct product is 2,3-diketo-Thanks to its obviously irreversible blocking mechanism in addition to its already established biochemical effects as a vitamin, it could be used as a comedication for treatment. Higher dosage would be needed to compensate for its very weak potency. To circumvent its poor target affinity, future research work could explore the chemical space of its favorable derivatives. Upon administration along with neuraminidase inhibitors they could jointly achieve a synergistic effect. Fortunately, CIV offers many possibilities for structural modification to achieve more interaction at the active site. Another promising molecular feature is that thanks to its smaller size and molecular weight , it effectively possesses multiple binding modes. During our simulations it interacted with different residues in the binding cavity. It lends the opportunity to explore new locations and pockets, too. CIV can generate a larger variety of hydrogen bond networks than all other scaffolds under study here. As a downside, in its present non-substituted form, it is not capable of efficiently occupying the binding cavity. Interestingly, derivatives of ascorbic acid with pentacyclic triterpene extensions have been reported ,60. TheyFive non-sialic acid-like compounds are proposed as unsubstituted lead compounds with scaffolds whose likelihood of inherited viral resistance to NA inhibitors is fairly reduced because all five do not constitute analogs to established drugs tightly imitating sialic acid. The novel scaffolds only loosely resemble the natural substrate sialic acid at a pharmacological cost of target affinity and subsequent antiviral potency. All docked solutions of the five molecules were analyzed to establish their binding modes and compare them to the crystal structures of reference drugs. Yet, the proposed structures constitute either undecorated or poorly decorated (“under-decorated”) scaffolds providing the opportunity to follow two opposing research and development strategies: (i) to follow the known substitution of ring positions of zanamivir or oseltamivir and other commercial drugs, which is a straight-forward method used to find stabilizing effects for successful binding by imitating the drugs’ binding modes with the downside of introducing a potentially detrimental risk of suffering inherited drug resistance from virus strains of clinical relevance. (ii) To explore new substitution patterns to improve receptor affinity and at the same time reduce susceptibility to existing influenza A resistance mechanisms against existing commercial drugs thanks to the establishment of hitherto unseen binding patterns.The present work introduces opportunities to position either new or “borrowed” functional groups on new scaffolds which will help optimize the energetically favorable concert of hydrogen bonding, salt bridges, polar and nonpolar interactions. The five unusual scaffolds will occupy new areas and pockets at the binding site to reach micro or nanomolar drug potency. For instance, in future studies, ORP derivatives could be tested with reduced number of carbon atoms on its aliphatic chains. Hence, steric hindrance will be reduced at the active site. Like CIV, ORP certainly requires the rearrangement of -OH groups to achieve an optimized receptor interaction. In contrast, polar substituents “borrowed” from commercial drugs could be introduced into its basic structure too. In more general terms, a kind of chimeric “recycling” effort could reuse the substituents seen on zanamivir as well as oseltamivir.https://www.ncbi.nlm.nih.gov/genomes/FLU) [https://www.ebi.ac.uk/Tools/msa/clustalw2/) [The PDB entries were retrieved from the RCSB Protein Data Bank (RCSB PDB) . The commes/FLU) . The pristalw2/) .Molecular docking is a suitable technique for generating and labeling protein–ligand complexes according to their binding affinities. In this study, molecular docking was performed using the program PrPx-Phyton v0.8 ,21. The Prior to docking, the two missing viral neuraminidase structures of NA Puerto Rico H1N1 A/PR/8/34) and NA Chicken México H5N2 (A/Chicken/Mexico/31381-7/1994) were generated from their sequences of amino acids in the I-TASSER platform ,31. Prec and NA CIn the next step, both neuraminidase models were prepared for docking under UCSF Chimera 1.11.2 and Vega ZZ 3.1.1.42 ,66. To tThe five proposed SOMs, sialic acid and reference drug zanamivir were prepared in the program Avogadro 1.2.0 . For theFinally, the 3D target models were inspected for completeness and the monomeric target structures were used for blind docking of all five SOMs. Sialic acid and zanamivir were included as references for re-docking studies and to compare the docked poses and computed data with their crystallographically known binding modes and measured activities . DockingInfluenza A/Puerto Rico/8/1934 (H1N1) (A/PR/8/34) virus was obtained from the American Type Culture Collection (ATCC). Influenza A/Chicken/México/31381-7/1994 (H5N2) was donated by A. Aguilar Setien. Madin-Darby canine kidney (MDCK) cells  were maintained in Dubelco Eagle’s Minimum Essential Medium supplemented with 5% fetal bovine serum and a mixture of antibiotics and antimycotics . The supernatants of the infected cultures were obtained after 96 h post-infection, clarified by centrifugation at low speed and stored at −70 °C for further use. Viral inoculum was titrated through the formation of lytic plaques in the same cell line using the conventional method in medium solidified with 0.5% agarose and stained with violet crystal . Titers N-acetylphenylalanylmethionine (AAMOL) and 3-(propyl aminosulfonyl)-4-chlorobenzoic acid  were all donated by Prof. Dr. Thierry Langer, University Vienna. Ascorbic acid (CIV) and 4-(dipropylsulfamoyl) benzoic acid (ORP) were donated by Dr. Stefan Kahlert, Institute of Anatomy, Otto von Guericke University Magdeburg, Magdeburg, Germany. The compounds were dissolved in distilled water and aliquots were stored at −20 °C for later usage.The five proposed SOMs are structurally unrelated to each other . They weN-morpholino) ethanesulfonic acid, AKA MES, 4 mM of calcium chloride, pH 6.5) and added (100 mL/well) in duplicate to a flat-bottom 96-well opaque black plate . The MUNANA substrate was added to all wells (50 mL/well) to achieve a final concentration of 100 µM. Immediately after adding the MUNANA substrate, the plate was transferred to a prewarmed Synergy 2 multimode microplate reader at 37 °C. The fluorescence signal from the enzyme buffer and MUNANA substrate alone in the absence of enzyme was treated as background noise and as a direct consequence, was subtracted from the signals obtained in the other wells. A standard curve was generated for each experiment using 4-MU diluted in enzyme buffer at final concentrations of 0.05 to 200 µM [The NA activities of both influenza A viruses (H1N1 and H5N2) were measured by a fluorescence-based assay using the fluorogenic substrate MUNANA , based on the method of Potier et al. 1979 ,71,72. So 200 µM .NA enzymatic parameters were determined in reactions in flat-bottom 96-well opaque black plates . The necessary influenza virus preparations were described above, and various MUNANA substrate concentrations were also prepared. All assay components were pre-warmed for 20–30 min at 37 °C. The reaction was conducted at 37 °C in a total volume of 150 µL. In the next step, the fluorescence of the released 4-methylumbelliferone was measured during 20 min incubation. The reaction finished by the addition of the stop solution . Fluorometric determinations were quantified with a Synergy2 multi-mode microplate reader  using excitation and emission wave lengths of 360 and 460 nm, respectively . The kin50 values from NA were determined in reactions performed as described above. The five molecules AAMOL, FMOL, STL, CIV and ORP were used in different concentrations. The reference drug zanamivir was used as inhibition control. Prior to testing, all assay components were warmed up at 37 °C for 20 min. The test reaction was conducted at 37 °C in a total volume of 150 µL, and the fluorescence of the released 4-methylumbelliferone was measured after 20 min incubation time. The reaction was stopped upon addition of 0.1 M glycine in 25% ethanol at pH 10.7. Fluorometric determinations were quantified with a Synergy 2 multi-mode microplate reader  using excitation and emission wavelengths of 360 and 460 nm, respectively. The data were fitted using nonlinear regression (GraphPad Prism 6.04) to determine the IC50 values for the molecules [IColecules ,74,75,7650) was calculated using GraphPad Prism 6.04 [The substances of the five proposed scaffolds were tested on MDCK cells cultured in 96-well plates following the MTT colorimetric method. Briefly, 20,000 cells per well were incubated with several concentrations (1 nM–75mM) for 24 and 48 h. After that, the medium was removed, cells were washed twice with PBS, 100 μL of 0.5 mg/mL MTT was added into each well and incubated for 3 h at 37 °C. After that, 100 μL of 0.1% DMSO was added and the optical density was determined at 570 nm. A standard curve was included with known amounts of untreated cells. The percentage of cytotoxicity was calculated by comparing treated versus untreated cells, the 50% cytotoxic concentration . Statistical analysis was performed using one-way ANOVA and Bonferroni test. Five small organic compounds which do not resemble either each other or any of the hitherto known commercial drugs—but show promising results of in silico docking simulation results—were tested for their in vitro inhibitory potency against viral neuraminidases of human and avian influenza A viruses (H1N2 and H5N2). All five scaffolds demonstrated weak inhibition activity. Ligand docking at the known binding sites of wild type and mutant type NAs lent insight into potential binding patterns at an atomic scale. The computed binding modes for all five ligands could be brought on an equal footing with the measured activities as well as the competitive or non-competitive inhibition mode. The advantages and disadvantages of binding of each scaffold was discussed in details. The detected disadvantages lend opportunities for improvements. Hence all five are proposed here as lead compounds for future drug development cycles to strengthen target selectivity and specificity. Precisely, the missing functional groups of zanamivir and oseltamivir offer opportunities to not only follow the example of their substitution patterns, but also attach new functional groups to contribute to the hitherto unseen binding modes to circumvent existing drug resistance at the binding site. All told, we present five unusual scaffolds with experimentally determined activity to inhibit neuraminidases in the millimolar range which can be used to develop new oral drugs against influenza A.


Background and objectives: An Italian nationwide pre-participation screening approach for prevention of sudden cardiac death in athletes (SCD-A) in competitive sportspeople showed promising results but did not achieve international consensus, due to cost-effectiveness and the shortfall of a monitoring plan. From this perspective, we tried to provide an epidemiological update of SCD-A in Italy through a year-long internet-based search. Materials and Methods: One year-long Google search was performed using mandatory and non-mandatory keywords. Data were collected according to prevalent SCD-A definition and matched with sport-related figures from Italian National Institute of Statistics (ISTAT) and Italian National Olympic Committee (CONI). Results: Ninety-eight cases of SCD-A in 2019 were identified . Male/female ratio was 13:1. The most common sports were soccer (33.7%), athletics (15.3%) and fitness (13.3%). A conclusive diagnosis was achieved only in 37 cases (33 of cardiac origin), with the leading diagnosis being coronary artery disease in 27 and a notably higher occurrence among master athletes. Combining these findings with ISTAT and CONI data, the SCD-A incidence rate in the whole Italian sport population was found to be 0.47/100,000 persons per year . The relative risk of SCD-A is 3.1  for competitive compared to non-competitive athletes; 9.9 for male  with respect to female. Conclusions: We provided an updated incidence rate of SCD-A in both competitive and non-competitive sport in Italy. A higher risk of SCD-A among competitive and male athletes was confirmed, thus corroborating the value of Italian pre-participation screening in this population. Sudden cardiac death in athletes (SCD-A) is commonly defined as a death occurring within one hour of initial acute symptoms due to cardiovascular collapse (or within 24 h in unwitnessed cases) among sport practitioners in the absence of external causal factors . This deItalian law requires pre-participation screening for competitive sport practice nationwide since 1982, aimed to certify a physical status consistent with an organized and continuative athletic activity . Since tOn the other hand, the “Italian model” of preventive sports medicine has not been uniformly accepted by the international scientific community, due to a number of studies displaying a very low incidence of SCD-A in the athletic population, even lower than sudden cardiac death in the general population , a not-nAbove all, some concern may arise from the absence of a nationwide registry of SCD-A in Italy: from this point of view, not only data from competitive sport practice but also from the non-competitive and recreational sport should be regularly and extensively collected. A huge part of the general population practicing sport as a leisure activity, indeed, has no screening at all.In order to fill this gap, we tried to evaluate the incidence of SCD-A nationwide using the most powerful and feasible instrument: the internet.Since 1 January 2019 on a weekly basis, we performed a combined search on Google’s search engine of the following keywords in Italian language: “death”, “sudden”, “sport” as mandatory words , together with “match”, “race”, “training”, “certificate”, “autopsy” as non-mandatory words . At the end of the year 2019, we strengthened the results of the search adding to mandatory keywords the year of interest and the name of each of the 107 Italian provinces . We selected the results according to the previously provided definition of SCD-A [For every event, we collected the following data: age, date, practiced sport, level of sport participation , likely diagnosis and outcome of the autopsy (where available).For this purpose, we referred to the definition of competitive sport from the 16th Bethesda conference, which defines a “competitive athlete” as an individual who participates in an organized team or in a sport that requires regular competition against others, places a high premium on excellence and achievement, and requires some form of systematic and intense training . FurtherRegarding state-of-the-art sport practice in Italy, the main and most recent sources of sport-related statistical data are represented by the Annual Report developed by the Italian National Institute of Statistics (ISTAT)  and by tIn summary, we can state that today in Italy, there are about 4.703 million competitive athletes and about 15.977 million non-competitive athletes (8.342 million men and 7.635 million women).p ≤ 0.05.Quantitative variables are presented as mean ± standard deviation, categorical variables as count and percentage. The only comparisons were made on categorical variables , and chi square test was used. The software SPSS 25.0 for Mac was used for statistical analysis. A difference was considered significant when p < 0.0001). In particular, we found 46 events among male competitive athletes  and only 1 event among female competitive athletes . Conversely, in the non-competitive population, we detected 45 male deaths  and 6 female deaths .Following our Google-based research, we identified 98 cases of SCD-A on the Italian soil in the year 2019. Among them, 47 (48.0%) involved people practicing competitive sport, whereas 51 (52.0%) involved non-competitive athletes. These events concerned far more male people (92.9%), with a male-to-female ratio of about 13:1  during sport practice, in 9 cases (9.2%) at the end of the sport activity, in 14 cases (14.3%) during the night-time sleep and in 24 cases (24.5%) at rest or during the day-to-day activities. Only in one case of SCD-A could circumstances not be established.In terms of time of the SCD-A events, the mostly involved months of the year were February (12.2%), September (10.2%) and October (17.3%); however, there were no significant differences among months.Classifying events by age groups, we found 12 episodes among under 18-year-old people (12.2%), 26 among people between 18 and 35 years old (26.5%) and 60 among people older than 35 years old (61.2%) . More spConsidering the country administrative subdivision in Regions, we delineated the number of events for every Italian Region in Concerning etiopathogenetical definition of SCD-A cases, in 61 cases (62.2%), we achieved only a probable diagnosis of the cause of death ; conversely, we attained a conclusive diagnosis in 37 cases (37.8%), with a cardiac origin in 33 (33.7%). The leading diagnosis was coronary artery disease (CAD) in 27 cases (73.0%), followed by acute myopericarditis in two (5.4%), one case (2.7%) of anomalous origin of a coronary artery (single coronary artery), one of arrhythmogenic cardiomyopathy (2.7%), one of pulmonary embolism (2.7%) and one of acute heart failure (2.7%). In the remaining four cases, the SCD-A had a non-cardiac origin .p < 0.0001 in respect to athletes younger than 35), with a mean age of 51.1 years (±11.06 years). There were 10 competitive athletes in this subset (37.0%), whereas 17 were non-competitive athletes (63.0%) (p = 0.27).The most valuable etiological subset was clearly CAD: this subgroup was composed totally (100%) of men, 26 out of 27 (96.3%) aged 35 years or older (p < 0.0001). In the competitive sport population, it was 1.00/100,000 persons per year , whereas in the non-competitive sport population, it was 0.32 .Combining our data with ISTAT and CONI reports, the SCD-A incidence rate in the whole sport population was equal to 0.47 in 100,000 persons per year, 0.78 among male and 0.08 among female athletes (p < 0.0001) compared to non-competitive athletes; it was 2.5 in males  and 1.0 in females .The relative risk (RR) of SCD-A for competitive athletes was 3.1  and in the whole sport population of 18.0  in the competitive population and of 6.9  among non-competitive practitioners.Compared to female athletes, male athletes showed a RR of SCD-A of 9.9 , with a SCD-A incidence rate of 0.28/100,000 in the whole population, 0.49/100,000 among competitive and 0.22/100,000 among non-competitive practitioners.Literature is unfortunately rich with different epidemiological studies on SCD-A, with regard to aforementioned definitions used, considering population . This risk is very close to the risk demonstrated by Corrado et al.  between competitive and non-athletic population, even if this comparison is somehow different with respect to our data. Whilst we compared competitive and non-competitive athletes, Corrado et al. compared competitive athletes and the non-athletic population, where both sedentary subjects and non-competitive athletes were included, being non-competitive athletes at higher risk of SCD-A. Finally, we cannot exclude that in our series, some of the non-competitive athletes affected by SCD-A were previously disqualified by competitive practice, according to Italian pre-participation rules. All these data do confirm the usefulness of pre-participation screening for competitive sport.Analyzing the young athletic population (aged 12 to 35 years old), estimated by ISTAT in about 14 million 874 thousand people, we found 37 occurrences in our series, with an incidence rate of SCD-A of 0.25 in 100,000 persons per year: these figures are 4 times lower than incidence rate determined by Corrado et al., but they match with data from Marjion et al.  [The most obvious dissimilarity between our series and other known data regards the difference of incidence rates between genders, specifically among competitive athletes: we found an annual incidence rate in the competitive male population of 1.36/100,000 persons and 0.08 in the competitive female population, whereas Corrado et al. reported an incidence rate of 2.6 and 1.1/100,000, respectively. However, Corrado’s population comprised young people (12–35 years), whilst our data included master athletes, with a considerable number of SCD-A to be linked to CAD, especially among males ,32. ThisData from Italian National Olympic Committee (CONI) monitoring, through the total number of competitive athletes engaged in every sport, allowed us also to estimate the incidence of SCD-A per every competitive sport: from this perspective, we found the highest incidence of SCD-A in hockey , motorsports , athletics  and handball , as shown in Lastly, a well-known but always relevant aspect outlined concerns the wide difference in CAD incidence in people younger and older than 35 years old, which confirms a strong link between age and atherosclerosis-related cardiovascular diseases ,32.The availability of a nationwide registry in Italy of all pre-participation examination data would allow for more accurate information about competitive athletes, in terms of age groups, practiced sports, identified diseases and outcome of the examination .An internet-based epidemiological search depends closely on completeness and authenticity of online information: this implies the possibility of errors in data collection wherever they were incorrectly reported by media.In this respect, it is also possible that some cases of SCD-A that occurred in Italy did not get any online news coverage and, therefore, did not get included in our work. For this reason, we purposely excluded all cases of resuscitated sudden cardiac arrest, because in our opinion they are probably more prone to be under-reported by online media. Although we consider this eventuality negligible, it cannot also be excluded that some cases of SCD-A have not been detected due to discrepancy between our search keywords and keywords used by newspapers. All of these factors could lead to an underestimation of the real total number of SCD-A events in Italy, thus eventually decreasing the epidemiological value of our data. On the other hand, the several case and series reports of SCD-A published, even if showing data in a more complete and scientific fashion, miss the real number of cases even more so than a web-based approach. Some cases not fulfilling inclusion criteria or a complete study, in fact, are not reported.Lastly, we assume irrelevant the use of the online research tool in order to identify the cause of SCD-A: in our series, a definite diagnosis was determined only in 37% of cases, reasonably due to privacy protection in compliance with current regulations. Methods that are more effective should be hereafter used in order to obtain these valuable data, possibly nationwide.Our study allows us to identify a remarkable number of SCD-A events in Italy, establishing a present-day baseline incidence rate of SCD-A in both competitive and non-competitive sport practitioners.Our series confirms available incidence rates of SCD-A in the general sport population and the higher risk of SCD-A among competitive athletes, thus corroborating the value of Italian pre-participation screening in this population.A notably hot topic regards the huge difference in incidence we found between the two genders, especially evident among competitive athletes: this aspect seems to deserve further consideration.Another key point that deserves more attention in the future is the adoption of an unambiguous definition of SCD-A, which should take into account, in our opinion, all sudden cardiac events in sporting people.Further studies based on alternative approaches are certainly required to validate our findings. In any case, we consider fundamental in the near future a wide-range collection and systematic analysis of this sort of epidemiological data, taking into account both competitive and non-competitive athletes, for evaluating properly the actual impact of a pre-participation screening on the incidence rate of SCD-A. The most important target is, in our opinion, the creation of a “nationwide registry of sport-related sudden cardiac death”, which records age, practiced sport and level of participation (competitive or non-competitive), the circumstances of the event and (wherever possible) a definite cause of death.