Dear Editor,Burns and trauma represent two of the most severe injuries that both developing and developed countries face today. Concomitant burn and traumatic injuries are significant in terms of disease burden and cost of treatment due to associated high morbidity and mortality.Existing literature for burn and traumatic injuries encompass injuries derived from terrorism, warfare, natural disasters , and accThe Singapore General Hospital (SGH) Burns Centre is the only adult burn center in Singapore and is the regional referral center in Southeast Asia. Our center receives 93% of all burn patients in Singapore including mass casualties from the region [We conducted a retrospective case-control “trauma/burns” study of 11 patients with concomitant major burn and traumatic injuries at SGH from 1998 to 2012 obtained from the SGH Burns Registry. Inclusion criteria were trauma with multiple injuries requiring activation of the trauma team  and major burns of more than 15% total body surface area (TBSA). Exclusion criteria were patients who were dead on arrival or repatriation cases. We compared this group to a control group of 11 major burn (>15% TBSA) patients without traumatic injuries admitted during the same time period. Controls were randomly matched for TBSA and age.n = 10) in trauma/burns, 81.8% (n = 9) in control). The median TBSA is 32% for both groups  and of deep dermal depth.Both groups had similar demographics and burn characteristics for comparison. The median age is 37 and 39 years in the trauma/burns group and the control group, respectively. Majority of the patients are males (90.9% (n = 8) of patients from both groups). This highlights the importance of workplace safety such as protective equipment, regular preventive maintenance and job-specific hazard analysis implementation.Occupational burns  is the majority in both study and control groups (73% (n = 1) and the international average mortality rate is lower for burn injuries alone (3.1–18%) [n = 10) in trauma/burn patients (due to their severe injuries) and 45.5% (n = 5) in the control burns only group. Patients in the trauma/burns group mostly belonged to Clavien Dindo Classification terminal grades of 4A (n = 7) and 5 (n = 4) compared to patients in the control group who were mainly grades of 3B (n = 6) and 3Bd (n = 4).In our study, the severity and high mortality 36.4%) of patients with concomitant burn and traumatic injuries highlights the focus on managing these critically ill patients well. In contrast, the mortality for control group is 9.1% (% of pati3.1–18%) . Burn inA seasoned protocol, early resuscitation, robust burn care within the golden hour and definitive surgeries done within 24 h is important for survival. The median time of injury to admission (inclusive of time needed for transfer) was 1 h (0.2–48 h) and 2.5 h (0.25–144 h) in the trauma/burns and control group, respectively. Singapore’s small geographical area ensured that all local trauma/burn patients arrived almost within 1 h of injury. Early resuscitation and communication between paramedics and the emergency department allows for standby of the trauma and burns teams. As such, none of the trauma/burns patients passed away within 24 h of resuscitation.n = 1) and severe inhalational burns (n = 2).None of the concomitant trauma that patients in the trauma/burns group suffered was immediately life threatening such as intrathoracic and intra-abdominal injuries. Rather, it was often severe inhalational injury coupled with the systemic inflammatory response syndrome (SIRS) overdrive from the trauma and burns double hit that precipitated the “early mortalities” (days 3–8) and multi-organ failure. Three out of four mortalities were secondary to respiratory failure from acute respiratory distress syndrome  amongst burn patients with sepsis-related complications. Patients with severe burn injuries are exceptionally vulnerable to infections due to large wounds, frequent invasive procedures such as artificial ventilation, tracheotomy, and arterial lines and renal replacement therapy. Hence, early prediction of sepsis onset, timely intervention, and prevention can lower mortality and morbidity.Acinetobacter baumannii and Pseudomonas infections [Median overall length of stay was 41 days 2–68 days) in trauma/burns patients compared to 16 days (3–52 days) for the control burns only group. This prolonged hospitalization is a predictor of prolonged SIRS and also the possibility of compensatory anti inflammatory response syndrome (CAIRS) [8 days infections .All trauma/burns mortalities required dialysis for acute renal failure and varying severities of inhalational injuries and respiratory failure. The American Burn Association and National Burn Repository recognizes the presence of inhalational injury as a major predictor of mortality . Pre-renIn summary, golden hour early resuscitation with the pre-hospital team, aggressive dialysis for oliguria, early intubation with cervical clearance, sepsis prevention and surgery within 24 h with early postoperative rehabilitation, and psychosocial support are hallmarks of our hospital protocol. Nonetheless, mortality is still high.More resources and healthcare planning and policy should be carefully considered to create the best setup for expedient multidisciplinary care. We have recently set up a combined national burns and trauma database that will facilitate review and formulation of evidence-based practice in the management of patients with concomitant burns and traumatic injuries.Trauma and burn patients highlight the dire need to optimize outcomes through pre-hospital education, prevention along with a streamlined hospital resuscitation protocol and timely surgery.


Recent drug discovery and development efforts have created a large arsenal of targeted and chemotherapeutic drugs for precision medicine. However, drug resistance remains a major challenge as minor pre-existing resistant subpopulations are often found to be enriched at relapse. Current drug design has been heavily focused on initial efficacy, and we do not fully understand the effects of drug selective pressure on long-term drug resistance potential. Using a minimal two-population model, taking into account subpopulation proportions and growth/kill rates, we modeled long-term drug treatment and performed parameter sweeps to analyze the effects of each parameter on therapeutic efficacy. We found that drugs with the same overall initial kill may exert differential selective pressures, affecting long-term therapeutic outcome. We validated our conclusions experimentally using a preclinical model of Burkitt’s lymphoma. Furthermore, we highlighted an intrinsic tradeoff between drug-imposed overall selective pressure and rate of adaptation. A principled approach in understanding the effects of distinct drug selective pressures on short-term and long-term tumor response enables better design of therapeutics that ultimately minimize relapse. Recent high-throughput sequencing studies revealed extensive intratumoral heterogeneity in patients13456791011Given this evidence, it follows that tumor heterogeneity and dynamics need to be considered during the diagnosis and treatment of cancer. Both stochastic13141516171819202122232425275678293134Here, we perform a systematic computational analysis to interrogate the effects of different drug-imposed selective pressures on long-term therapeutic outcome. We are especially interested in the case where two different regimens can have the same initial efficacy on the overall tumor, but differential drug-imposed selective pressures on the individual subpopulations may lead to significantly different drug sensitivities in the long-term. Systematic sampling of the parameter space brought additional insight into the dependencies of growth rates, kill rates, and subpopulation proportions on potential predictability of therapeutic outcome, and intrinsic tradeoffs in therapeutic design.We focused our analysis in the regime of large population size (e.g. at times of diagnosis), and assumed that the population is well-mixed and contains pre-existing resistant subpopulations. While stochastic drift and background mutation rates are also important driving forces in clonal evolution and resistance467We first examined a single scenario contrasting a symmetric treatment, with equal subpopulation kill, and an asymmetric treatment, with unequal subpopulation kill. Upon successive symmetric treatments, we expectedly observed comparable tumor compositions at the initial and final time points . These rks), overall kill rate (αs), growth rate (k1) and kill rate (α1) of subpopulation 1, and the initial fraction of each subpopulation (k2) and kill rate (α2) of the second subpopulation .Using these aforementioned metrics , we next studied how varying parameters and particular parameter regions affect therapeutic outcomes. Our ODE system of equations is fully defined by the following set of parameters: overall growth rate (α2/α1) versus differential growth rate (k2/k1). Traversing along the abscissa toward the right, we increase the differential killing of the tumor subpopulations . On the other hand, values deviating up or down from the center of the ordinate axis correspond to differential growth rates of the subpopulations. This visualization also suggests the existence of a diagonal line corresponding to an ‘effective’ equal growth and kill rate as we transverse in this parameter space  on long-term therapeutic efficacy. We observed that the region of high rate of adaptation , examination of this parameter gives us insight into the consequence of proliferation, as associated with a particular tumor type, on its resistance potential in light of the other parameters. Interestingly, we observed that the region of independence in the rate of adaptation no longer stretches along the ordinate axis, but rather broadens along the differential kill axis  and overall kill rate (αs). To gain an understanding of the combinatorial effects of these two parameters in relation to differential growth and kill rates, we ran our simulations again with higher overall growth/kill rates. In comparison to Thus far, we have analyzed independently the effects of overall growth rate  used even to this day. However, although this ‘hit-hard, hit-early’ approach has led to many success stories, drug resistance remains a major obstacle towards achieving a cure. In addition, recent genome sequencing of patients both longitudinally and spatially in different regions of the primary/metastatic tumor have brought greater focus to the extent of intratumoral heterogeneity in patientsEμ-myc; p19Arf−/−), where treatment of an admixed heterogeneous tumor population with olaparib  led to much greater resistance potential than paclitaxel .Here, using ordinary differential equations modeling in a tractable model, we have systemically explored how different contexts  affect outcome . We observed that the initial tumor response is often not the best predictor for eventual prognosis. We specifically highlighted this through comparing regimens where the initial overall kill (i.e. selective pressure imposed by the drug) is the same, but due to differential selective pressure on the subpopulations, these regimens can result in very different therapeutic outcomes. A systematic interrogation through parameter sampling further enabled us to rationalize this effect in the context of different magnitudes of initial subpopulation proportions, and growth and kill rates of overall and individual subpopulations. We further illustrated this using a preclinical murine model of Burkitt’s lymphoma  and greater resistance potential. In other words, while we can modulate parameters by choosing drugs that result in increased overall tumor killing, such strategies often come at the expense of drug sensitivity after repetitive treatments. This concept has been explored in antibiotic combination studies, where highly synergistic antibiotic drug combinations (i.e. greater selective pressure imposed on the population) led to a much greater rate of adaptation9de novo mutation and genetic drift as the main driver of resistance in regimes where there exists a minor, but substantially sized, population of resistant cells. There are other clinically relevant contexts where drift and de novo mutations cannot be ignored. This includes, for example, when the resistant subpopulation is not present prior to treatment and/or in very small numbers, such that mutations and drift can vastly influence the dynamics and timing of clonal occurrence, and the overall time to relapse. Several studies of tumor clonal dynamics have used various stochastic methods to model de novo mutation and/or genetic drift in cancer initiation/progression131622Our model was based on the assumption that selection dominates over P1, k1, k2) and drug killing rates . The kinetics of the two subpopulations were modeled using ODE equations for exponential growth, shown below.A heterogeneous tumor was modeled as two subpopulations (d) was described using a Heaviside step function. Given the constraints of same initial overall tumor growth and tumor reduction (for the first treatment), the system can be completely defined by k1, α1, ks, αs. The parameters ks and αs define the overall growth and kill rate, respectively. Given a set of values for these parameters and applied constraints, we can subsequently derive the values for k2 and α2.Drug treatment , the where k2Solving for k1 value such that k2 is defined and nonnegative,The maximum Lemma 1.1:ks, and as such any k2 values equal to or less than ks are always defined.Proof of Lemma 1.1:ks can be expanded toAs defined above, the expression To prove Lemma 1.1 is to demonstrate this inequality is true. The above expression (5) can be reduced toks is always a positive value, the expression exp(kst) is always greater than 1.Since ks) and kill rate (αs), such that,Per our constraint, after the first dose, the differential killing of the subpopulations should result in equivalent overall tumor burden reduction as per overall growth rate (α2Solving for P1 and P2 under symmetric growth (ks) at time point d1.where Lemma 1.2: The bound for α1 isα2 isProof of Lemma 1.2:α1 value such that α2 is defined requiresThe minimum Therefore,α2 is maximum when the logarithm term in the α2 expression is minimized, which occurs when The value α2 is not negative. Therefore, setting α2 equal to zero and rearranging the terms in (8),An additional constraint is to ensure that α1,Solving for α1 from equation (7),Similarly, solving for α2 for which α1 is defined is,which means the minimum α1 is,And the maximum value for It is clear that the expression for nds for   and , , ks, αs, log2[α2/α1], k2/k1. Here we sampled the ratios of the killing and growth rates instead of α1 or k1 to ensure uniform sampling in this transformed space – as this is the final parameter space in which we performed our subsequent analyses. From the sampled ratios, we derived the corresponding α1 via non-linear regression fit of log2[α2/α1] versus α1 to an exponential function. Similar fitting was performed to derive k1. For each given parameter set {ks, αs, k1, α1}, we calculated the corresponding k2 and α2 values according to We used a Latin hypercube sampling method to explore the global parameter space. Specifically, we performed a parameter sweep of Arf −/− B-cell lymphomas were cultured in B-cell medium . The cell line was tested and shown to be free of Mycoplasma using both PCR-based (American Type Culture Collection) and biochemical-based (Lonza) methods. Paclitaxel was obtained from LC Laboratories and olaparib was a gift from Peter Ghoroghchian.Murine Eμ-Myc; p19All shRNAs were expressed in either MSCV–LTR–MIR30–SV40–GFP (MLS)Proliferation assay was performed to determine the net growth rate of each sorted shRNA-containing population. On day 0, 10,000 cells were seeded per well in a 96 well plate and incubated for up to 72 hrs. Cell number was counted every 12 hrs using a cell analyzer FACS LSR, FACSCanto, or LSRFortessa (BD Biosciences). For dose response experiments, the kill rates were assessed for individual populations by measuring the IC50 at 48 hours. The IC50 value was determined via fitting data to the following dose response equation,Ainf is the % live cells at the highest drug concentration, A0 is the % live cells at 0 drug concentration, n is the hill coefficient, IC50 is the drug concentration where the % live cells is reduced by half, and [drug] is the concentration of the drug.where y is the % live cells, α was calculated using the following equation.Killing rate For drug selection experiments, indicated shRNA-containing populations were mixed at a 50:50 ratio and confirmed using a cell analyzer. The admixed tumor population was treated with indicated drug at IC50 concentration – a dose needed to achieve 50% cell death at 48 hours in the individual populations. Cells were cultured in drug for six days and were split as needed. Cell death was assessed with propidium iodide incorporation. After four days, the percentages of GFP+ and Tomato+ cells were analyzed on a cell analyzer. Experiments were performed with indicated shRNAs in both MLS and MLT vectors to rule out any vector-specific effects.k1 and k2 values are the experimentally determined shChk2 and shBim growth rates in the absence of drug, averaged between the two vectors. sk was solved for based on the determined values for 1k, and 2k, following the constraint equation for symmetric initial tumor burden before treatment (2). 1α and 2α values for each drug were derived from individual dose response experiments, averaged between the two vectors, and calculated based on sα was calculated using t tests, ANOVA and two-way ANOVA with Bonferroni’s post hoc tests, and Fisher’s method were performed as indicated. Error bars shown are mean ± SEM.Statistical analyses were performed using Prism v5 (GraphPad). Two-tailed Student How to cite this article: Sun, D. et al. Differential selective pressure alters rate of drug resistance acquisition in heterogeneous tumor populations. Sci. Rep.6, 36198; doi: 10.1038/srep36198 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Kiss1r-deletion on hippocampus-related behaviors of anxiety and spatial learning in adult male mice using two mouse models. In the first, global Kiss1r-null and control mice were gonadectomized (GDX KISS1R-KO). In the second, KISS1R signalling was rescued selectively in gonadotropin-releasing hormone neurons to generate Kiss1r-null mice with normal testosterone levels (intact KISS1R-KO). Intact KISS1R-KO rescue mice were found to spend twice as much time in the open arms of the elevated plus maze (EPM) compared to controls (P < 0.01). GDX KISS1R-KO mice showed a similar but less pronounced trend. No differences were detected between intact KISS1R-KO mice and controls in the open field test (OFT), although a marked reduction in time spent in the centre quadrant was observed for all GDX mice (P < 0.001). No effects of KISS1R deletion or gonadectomy were detected in the Morris water maze. These observations demonstrate that KISS1R signalling impacts upon anxiogenic neural circuits operative in the EPM, while gonadal steroids appear important for anxiety behaviour observed in the OFT. The potential anxiogenic role of kisspeptin may need to be considered in the development of kisspeptin analogs for the clinic.The roles of kisspeptin signaling outside the hypothalamus in the brain are unknown. We examined here the impact of  In 2003, the critical importance of kisspeptin for reproduction was established by seminal studies reporting that humans with mutations in KISS1R failed to go through puberty and were infertile7. Investigations in various animal models over the last decade have now explained those clinical findings by demonstrating that kisspeptin activates KISS1Rs expressed by gonadotropin-releasing hormone (GnRH) neurons to play a central role in the control of fertility10.Kisspeptin was identified in 1996 from human melanoma cell lines12 as well as within multiple brain regions outside the hypothalamus13. Indeed, kisspeptin-immunoreactive fibers are located throughout the limbic forebrain16 and cells expressing KISS1R are dispersed across an even wider distribution of the central nervous system19. In our own mapping studies, we were interested to note that the highest density of cells transcribing Kiss1r existed in the dentate gyrus of the hippocampus18. Moreover, early electrophysiological studies by Arai and colleagues20 reported that kisspeptin selectively modulated excitatory synaptic activity in dentate granule neurons. These data suggested that kisspeptin signaling may have a role in hippocampal functioning.In addition to the GnRH neurons, kisspeptin signaling occurs at cells in several body organsKiss1r impacts upon hippocampal-related behavioral tasks such as anxiety and spatial learning22. Kiss1r-null mice fail to go through puberty and remain sexually immature with very low levels of circulating gonadal steroid hormones23. This creates an experimental confound as gonadal steroids modulate the activity of many neuronal circuits in the brain, including those within the hippocampus controlling traits such as anxiety25. One strategy for overcoming this caveat has been to undertake experiments in gonadectomized (GDX) mice so that gonadal steroids are absent in both controls and Kiss1r-null mice26. However, this results in the investigation of GDX mice rather than normal animals and is, itself, problematic when gonadal steroids may play a role in the parameters being examined. An alternative approach is to use a mouse model in which Kiss1r has been deleted from all cells in the body but rescued selectively in the GnRH neurons27. This rescue approach results in Kiss1r-deleted male mice that have normal levels of circulating testosterone and fertility28. We have examined here the learning and anxiety traits of Kiss1r-null male mice in both the GDX and rescue models to explore whether kisspeptin may have a role in hippocampal function.In the present study, we have examined whether the deletion of Kiss1r−/− and littermate wild-type Kiss1r+/+ mice7  were GDX three weeks before beginning experiments when 4–6 months old. Experimental mice were labelled as “GDX KISS1R-KO” and controls as “GDX control”. Adult male Kiss1r−/−; Tg+/− and littermate Tg+/− mice27 were used for experiments at 4–6 months of age. This was a 129P2 global Kiss1R-null mouse (Kiss1rtmGstn) crossed on to a C57BL/6 J line incorporating a transgene (Tg) in which the Gnrh1 promoter drives the expression of Kiss1R29 (available from AEH). Experimental mice were labelled “intact KISS1R-KO rescue” and controls as “intact controls”.Adult male 129S6 global P = 0.009) and gonadal status  = 6.76, P = 0.015), with an interaction between the two  = 4.32, P = 0.046) and post-hoc tests showing that KISS1R-KO rescue mice spent approximately twice as much time in the open arms compared with controls  and all GDX mice (P < 0.01) and interaction between genotype and gonadal status  = 10.01, P = 0.003) was found. Post-hoc tests showed no differences between the two GDX groups or two intact groups but intact KISS1R-KO rescue mice spent significantly less time in the centre compared with both GDX groups  was identified with GDX KISS1R-KO mice spending less time in the closed arms compared with both GDX and intact controls Fig. . With reons)Fig. . For theons)Fig. .Figure 1When compiled as percent event histograms for each group Fig. , the oveP = 0.0003) with both GDX groups travelling less than both intact groups . When examined across the three trials, intact and GDX mice were found to travel the same distance as each other on Day 1  = 4.02, P = 0.054), with intact mice travelling further than GDX animals on Days 2  = 15.43, P = 0.0005) and Day 3  = 21.78, P < 0.0001)(data not shown).The average distance travelled by mice across all three trials did not vary between control and KISS1R-KO mice in either intact or GDX groups Fig. . HoweverP = 0.444), but over two-fold more crosses in intact mice compared with GDX animals  = 23.3, P < 0.0001)  = 0.603, 001)Fig. . Both GDns) Fig. .P = 0.417), but intact mice spending over 5-times longer in the inner quadrant compared to GDX mice  = 17.38, P = 0.0003)  = 0.679, 03) Fig. . The samP > 0.05 for all comparisons, repeated-measures 2-way ANOVA). Performance in the reference memory spatial learning task was also not found to be different between any groups. Swimming velocity was the same and distance travelled . Probe tests also failed to reveal any significant differences in swimming speed  whereas KISS1R signaling is important for anxiety related to the fear of heights (EPM). The possibility that these effects reflect strain differences seems unlikely as the C57BL/6 J and 129 strains perform very similarly in the EPM and OFTKiss1r-null mice was only clearly observed in intact KISS1R-KO rescue mice. The GDX KISS1R-KO mice exhibited a similar but less robust phenomenon with less time spent in the closed arms and a trend to spend longer in the center and open arms combined. This may suggest that testosterone is involved in modulating KISS1R-dependent anxiety parameters in the EPM. More generally, this observation highlights the potential importance of normal levels of circulating gonadal steroids when interrogating kisspeptin-KISS1R signaling throughout the body.We note that the anxiolytic phenotype of Kiss1r was found to have no effects upon performance in the Morris water maze. This indicates that there is no critical role for KISS1R signaling in brain circuits responsible for spatial learning and memory in male mice44. A recent study reported that infusion of kisspeptin-13 into the third ventricle ameliorated memory loss in amyloid beta-treated mice45. As this effect was inhibited by a GnRH receptor antagonist, it is possible that the memory changes in that study were indirect resulting from increases in circulating gonadal hormone levels secondary to kisspeptin activation of GnRH neurons.The deletion of 21 and it is conceivable that deleted KISS1R signaling in any of these areas could underlie the specific anxiolytic effects reported here in KISS1R-KO mice. However, of these various regions, it is only the granule cells of the hippocampal dentate gyrus that express Kiss1r at any appreciable level18. Recent hippocampal optogenetic studies have highlighted the importance of dorsal granule cells in the modulation of exploratory behavior and ventral granule cells in the regulation of anxiety46. As the EPM tests the conflict between exploration and fear of a potentially threatening environment, KISS1R signaling may be important in either or both the dorsal and ventral dentate. Although other ligands are possible for KISS1R13, it is notable that electrophysiological studies have shown that kisspeptin increases the excitability of granule cells in the dentate20. Thus, it is plausible that a potential reduction in granule cell excitability in KISS1R-KO mice may underlie their inclination to spend more time in the open arms of the EPM.A variety of limbic brain regions contribute to anxiety-related behaviorsKISS1R has not been assessed. However, roles for kisspeptin outside the human hypothalamus have been suggested recently with the finding that the peripheral administration of kisspeptin can alter limbic brain activity, including the hippocampus, in relation to sexual and emotional stimuli47. Given our present observation of a specific anxiety deficit in Kiss1r -null male mice, it may be important to consider the possibility of effects of kisspeptin on anxiety when designing new therapies for treating reproductive disorders in the clinic.To our knowledge, the anxiety of the few humans identified with mutations in Kiss1r−/− (GDX KISS1R-KO) and littermate Kiss1r+/+ (GDX control) mice7 were GDX three weeks before beginning experiments when 4–6 months old. Adult male 129P2/C57BL/6 J Kiss1r−/−; Tg+/− (KISS1R-KO rescue) and littermate Tg+/− (control) mice27 were used for experiments at 4–6 months of age. Littermate null and control mice for each experiment were housed together three to four mice per cage under a 12 h light-dark cycle (lights on 19:00) at 20 ± 1 °C with 40 ± 5% humidity, and food and water available ad libitum. Experiments were undertaken during the animals’ dark phase under sodium lighting in the University of Otago Behavioural Phenotyping Unit. All experimentation was approved by the University of Otago Ethics Committee and undertaken in accordance with the relevant guidelines and regulations.Adult male 129S6 global Mice  were tested by an investigator blind to the experimental group and genotype. Mice were habituated to the investigator by daily handling for four days prior to behavioral testing. This consisted of one trial in the EPM (day 1), three trials in an OFT (days 1–3) and testing in a MWM (days 4–10). Mice were tracked and analysed using an overhead camera and the TopScan behavioural analysis system .The maze consisted of four arms  extending out from a central square (5 × 5 cm) with two arms having 20 cm high walls. Mice were placed in the central square facing an open arm and their activity tracked for 5 min. The number of times a mouse entered fully (whole body) into an open or closed arm and the % of time a mouse spent in the closed, centre, and open arms of the maze were measured.Each mouse was tested in the OFT after completing the EPM on Day 1 and then tested again on Days 2 and 3. The open field was rectangular open box  with the mouse placed in the centre of the open field and its activity tracked for 10 min. The total distance travelled, the number of crossings into the middle portion of the arena , and percentage of time spent in the center quadrant were measured.Cued water maze training (Day 4) was undertaken by placing a blue flag on the platform located above the water in the middle of the maze and placing mice with their nose to the wall of each of the four quadrants. Each trial lasted until the mouse had located the platform or until 60 s had elapsed at which time mouse was placed on the platform for 10 s. Two trials were undertaken in the morning and a further two trials in the afternoon. The time taken for mice to locate the platform on each trial was determined. Spatial learning and memory (Days 5–9) was tested over the next 5 days by locating the platform below the water and without a flag in the middle of the north-east (NE) quadrant. Mice were then placed in the pool in each of the quadrants as above and this was repeated on 5 consecutive days. The swimming velocity and distance required for mice to reach the platform was measured. Probe tests were undertaken on Days 7 and 9 before the spatial learning test by removing the platform from the pool and placing the mice with their nose to wall into the south-west quadrant. Mice were allowed to swim freely for 60 s and their swim velocity, number of crossing over the previous location of the platform, and % time spent in the NE quadrant measured.The day after completing the OFT, mice were tested in a circular water maze 100 cm in diameter and 35 cm deep maintained at 22 °C. A rectangular platform (6 cm diameter) was placed 1 cm above or below the surface of the water in various locations within the pool. post-hoc multiple comparison tests where required. Statistical analysis was performed with GraphPad Prism 7 software.Statistical Analysis: Comparisons across the four experimental groups were assessed with normal or repeated measures 2-way ANOVA with Tukey’s The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


The acrosome reaction (AR) is a process of membrane fusion and lytic enzyme release, which enables sperm to penetrate the egg surroundings. It is widely recognized that specific sperm proteins form an active network prior to fertilization, and their dynamic relocation is crucial for the sperm-egg fusion. The unique presence of the membrane cofactor protein CD46 in the sperm acrosomal membrane was shown, however, its behaviour and connection with other sperm proteins has not been explored further. Using super resolution microscopy, we demonstrated a dynamic CD46 reorganisation over the sperm head during the AR, and its interaction with transmembrane protein integrins, which was confirmed by proximity ligation assay. Furthermore, we propose their joint involvement in actin network rearrangement. Moreover, CD46 and β1 integrins with subunit α3, but not α6, are localized into the apical acrosome and are expected to be involved in signal transduction pathways directing the acrosome stability and essential protein network rearrangements prior to gamete fusion. A great deal is known about the mammalian fertilization, and all the physiological changes that male gamete must undergo in order to be able to fertilize the egg. However, the actual sperm protein dynamics that precedes the interaction with the egg, is still covered in a veil of mystery. So far, many proteins have been selected to be the sperm-egg binding and/or fusing candidates, some of them  were proven to be essential, some of them were discovered to play an unsuspected new role 2+-dependent event that follows capacitation, enabling sperm to be fusion-competent. During this event, the plasma membrane of the acrosomal area of the sperm head fuses with the outer acrosomal membrane, intra-acrosomal proteins are released into the extracellular space and new protein domains appear on the surface of the sperm head337810This study focuses on the final, but the most dramatic mature sperm metamorphosis, called the acrosome reaction (AR), which is characterized by the controlled exocytosis of the single giant enzyme-rich secretory vesicle the acrosome. This is a critical Ca−/− deficient males show a higher rate of spontaneous acrosome reaction compared to wild type males13141718172223262829Although a function of cytoskeletal proteins, mainly actin, is well known during the AR, it remains unclear which proteins participate in directing the dynamics of its organisation. CD46  is expressed broadly on the surface of somatic cells in humans and plays a pivotal role in the cell’s self-protection against the complement, which is also used by tumour cellsOne of these multi-molecular complexes, where integrins participate, was detected on the egg plasma membrane and it is called – the tetraspanin web. The tetraspanin web is a complicated protein network formed by the interaction of members of the tetraspanin family and others proteins31323622The interaction of CD46 and β1 integrins is well known in somatic cells, including humansin vitro defined conditions.To identify the behaviour of the studied proteins, CD46 and β1 subunit of integrins, during sperm maturation and acrosome reaction, we firstly examined immunohistochemicaly their localization in the sperm head in freshly released epididymal sperm. We were interested whether CD46 would be confined only to the acrosome region of the sperm head, as previously reportedSpecific monoclonal antibodies (mAbs), see methods, were used to label the individual proteins and follow their localization over the sperm head. Epifluorescent microscopic observation detected distinct sperm head regions that showed a profound distribution of both proteins  during tThe CD46 and β1 integrin localization during the 60 or 90 min AR progress was clearly similar. Prior to the AR, or at its very beginning, both proteins could be detected in the apical acrosome cap  line I win vitro, which is in correlation with previous findingsThere was also about 15% of acrosome reacted sperm during 60 min capacitation To analyse the distribution of individual CD46 and β1 integrin relocation patterns, among groups with different times of capacitation followed by the induced AR, the depicted protein patterns were placed beside each other . The proin vitro capacitation, the majority of sperm display the acrosome cap (AC) pattern for both CD46 and β1 integrin, which reflects the localisation of appropriate molecules in an intact acrosomal membrane. During the first 60 min of capacitation, the majority of sperm still kept the AC pattern, but the percentage of patterns related to the relocation of β1 integrins to the equatorial segment, and CD46 to the residual acrosome cap (rAC) and the apical equatorial segment (aES) slightly increased, which was expected, due to a well-known spontaneous AR in mice that represents over 10% of the sperm population. The following induction of AR by calcium ionophore caused a dramatic change in the relocation dynamics for both CD46 and β1 integrin molecules. Only less than 10% of sperm now displayed the AC pattern, and on the other hand, the pattern related to the relocation of a fluorescent signal to the equatorial segment (ES) was most prominent for the CD46 molecule. β1 integrin showed even more rapid relocation dynamics with a positive pattern detectable over the whole sperm head (WSH), including the post-acrosome region (PAR) as the prominent one. In subsequent times of analysis, the percentage of sperm expressing relocation of CD46 and β1 integrins into ES and post-acrosome region covering the whole sperm head continually increased. β1 integrins still carried on with a slightly more time dependent rapid progress of its relocation to ES and PAR compared to CD46.The graphical and statistical output of our data is presented in To further support our findings about protein relocation dynamics obtained by subjective microscopic evaluation, we also performed quantitative analysis of the fluorescent intensities  comparinBased on a similar distribution of both the proteins, their parallel time relocation during the AR and published data from the somatic cellsDual immunofluorescent staining was used for the analysis of the accurate position of CD46 and integrins by STED super-resolution microscopy. The ability of STED microscopy to distinguish individual membrane structures of the mouse sperm head was previously publishedA detailed mutual localization of CD46 and β1 integrins was visualised on freshly released epididymal sperm with an intact acrosome  and onlyIn order not to ignore α subunits of integrin proteins we decided to characterize the localization of α3 and α6 subunits, which were previously detected in sperm25Dramatic membrane reorganization starts in the acrosome cap region after the initiation of the acrosome reactionRegarding the apical hook, we also show in intact sperm the presence of β1 integrin in a “bridge” like three point structure  connectiThe important part of acrosome reaction is the relocation process of proteins involved in sperm-egg binding and fusion. The key role of actin cytoskeleton has already been described during this processData collected by 3D super–resolution SIM was used for colocalization analysis of the mutual position of the CD46 and β1 integrin subunit. 3D-SIM was chosen to collect data for colocalization analysis due to its higher resolution in z-axis (in our case approximately 350 nm). The mutual position of the CD46 and β1 integrin subunit was visualised with dual immunofluorescence staining in freshly released epididymal sperm with an intact acrosome  and micrLatrunculin A, a toxin that binds to actin monomers and prevents them from polymerizing, significantly affected the ability of sperm to relocate both the CD46 and β1 integrin subunit when the acrosome reaction was induced . The co-Protein relocation during the sperm acrosome reaction plays a crucial role for the ability of sperm to fuse with the egg as shown for the primary fusion protein IZUMO11314in vitro47CD46 is a key membrane regulator of complement activation, which protects mammalian host cells from complement-mediated damage. Beside this role, CD46 is expressed in sperm solely as an unusual lower Mr hypoglycosylated isoform localized to the acrosomal cap and it only becomes surface exposed on the inner acrosomal membrane after sperm had acrosome-reactedHere we demonstrated that one of the binding partners of CD46 on sperm is the β1 integrin subunit, which share the same acrosomal membrane localization and relocation behaviour. In intact sperm, we also identified, thanks to STED and SIM super-resolution microscopy, the localization of each protein across the individual apical head membranes and showed that the key integrin pair interacting with CD46 at the beginning of AR is α3β1 and not α6β1, which however could later play an important role in CD46’s protein organization. A previous interaction of CD46 and β1 integrins was detected in different somatic cell types2223252223To present, 18α and 6β integrin subunits and 24 of their heterodimers have been described in mammals58256364Colocalization of the α6 and β1 subunit in the plasma membrane of the acrosomal cap area and apical hook indicate the possible presence of the α6β1 heterodimer in these structures. Moreover, the presence of α6β1in mouse sperm has been previously described42To visually represent our new findings about the CD46 and β1 integrin localization and put them into the context of the known sperm membrane transformation processes occurring during AR, we have drawn 3D models and visualizations combining our own data with the data from other relevant publications. The topological localization of CD46 and β1 integrin molecules among individual sperm membranes is depicted in In In conclusion, we have shown that: 1) CD46 relocates from the acrosome into the equatorial segment and over the whole sperm head during the acrosome reaction, which is an important protein behaviour prior to sperm-egg fusion; 2) CD46 associates with the β1 integrin subunit and shares the same relocation pattern, which is disrupted when actin cytoskeleton is blocked, stressing their involvement in specific signalling pathways; 3) α3β1, but not the α6β1or α6β4 integrin pair, is confined to the intact acrosome vesicle and is consequently responsible, together with CD46, for maintaining its stability; 4) α6β1 integrins are localized to the apical hook of the intact mouse sperm head, which plays a role in the sperm train assembly.Inbred C57BL/6 mice were obtained from a breeding colony of the Laboratory of Reproduction, Faculty of Science, Charles University. Mice were housed in the animal facilities of the Faculty of Science, Charles University, and food and water were supplied ad libitum. The male mice used for all experiments were a reproductive age of 10–12 weeks. All animal procedures and all the experimental protocols were approved by Local Ethics Committee of Faculty of Science, Charles University, carried out in strict accordance with the Animal Scientific Procedure, Art 2010, and subjected to review by this Local Ethics Committee of the Faculty or Science, Charles University, Czech Republic (accreditation no. 247732008-10001).2. Released sperm were assessed for motility and viability under a light inverted microscope with a thermostatically controlled stage at 37 °C. Sperm stock was diluted to the required concentration (5 × 106/ml) in 100 μl of M2 medium under paraffin oil. Sperm were left freely to capacitate. Sperm samples were collected at both 60 and 90 min experimental times or the incubation was continued by an induction of the acrosome reaction. The freshly released epididymal sperm, which had not undergone capacitation, were used for protein detection to monitor protein status prior to capacitation.Sperm from the distal regions of cauda epididymis were released into a 200 μl droplet of M2-fertilising medium  under paraffin oil  in a Petri dish and pre-tempered at 37 °C in the presence of 5% CO2.Spermatozoa from the distal regions of the cauda epididymis were capacitated as described above. AR was induced by Calcium Ionophore (A23187 (CaI), Sigma Aldrich) at a final concentration of 5 μM. At both experimental capacitating times of 60 and 90 min CaI was left in the M2 medium for 60 or 90 min. All the sperm samples were incubated at 37 °C under 5% CO2. Sperm motility and viability were assessed at every experimental time point, when a drop of spermatozoa was placed onto a glass slide and 2.5 mM PNA lectin  was added. The status of the acrosome was examined immediately under a fluorescent microscope.All the sperm samples were incubated at 37 °C under 5% COin vitro incubation time stated above. Sperm were washed twice in PBS, smeared onto glass slides and air-dried. Sperm smears were fixed with 3.7% formaldehyde in PBS (pH 7.34) at room temperature for 10 min, followed by washing in PBS. Sperm were blocked with 10% BSA in PBS for 1 h and incubated with: primary antibody anti β1 integrin  diluted 1:10 in PBS and/or primary antibody anti-CD46 MM10  diluted 1:50 in PBS over night at 4 °C, followed by Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 568 donkey anti-rabbit IgG  and/or Alexa Fluor 488 donkey anti-rat IgG  secondary antibodies 1:300 in PBS for 1 h at room temperature. In case of dual staining, both secondary antibodies were applied together. Furthermore, PNA lectin  was added at a concentration of 2.5 mM in PBS. After washing, the slides were mounted into a Vectashield mounting medium with DAPI . The samples were examined with an Olympus IX81 fluorescent microscope and photographed with Hamamatsu ORCA C4742-80-12AG, using Olympus Soft Imaging Solutions software . Representative results are shown.Sperm smears were prepared for every For every experiment, we collected sperm data from eight mice. The positive or negative signal was evaluated from a total of 200 spermatozoa on every slide. In each group, at least two samples were analysed. Data were analysed statistically.4Cl v PBS for 15 minutes. After washing 3x in PBS, sperm were smeared onto a glass slide and air-dried. Fixed sperm were permeabilized with acetone for 7 minutes in −20 °C. Further steps were the same as those described previously. For STED and SIM visualization, the following antibodies were used: primary antibodies anti- β1 integrin  1:10 in PBS, anti-CD46 MM10  1:50, anti-actin clone Ac-40  1:100, anti-α3 integrin (N19)  1:10, anti-α3 (H-43)  1:10, anti α6 integrin (F6)  1:10, anti-estrogen receptor β H-150  1:50; secondary antibodies Alexa Fluor 568 donkey anti-rabbit IgG, Alexa Fluor 488 donkey anti-rat IgG, Alexa Fluor 568 goat anti-mouse IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor donkey anti-goat 568 IgG  1:300 in PBS. Fluorescent images were collected with a Leica TCS SP8 STED 3X microscope and DeltaVision OMX™ with the Blaze SIM Module microscope . Huygens Professional version 16.05  software was used for deconvolution of STED images and 3D visualization. An open source software Fiji was used for other image processing.Freshly released, capacitated and acrosome reacted sperm were used for STED and SIM super-resolution microscopy. Sperm were collected, as described previously, with the following differences. Sperm samples were always prepared onto high precision cover glasses . Moreover, after the application of the primary and secondary antibodies, sperm were incubated for 5 minutes with DAPI  and washed 3x in PBS. At the end, sperm were washed 1x in distilled water and air-dried. Dry samples were covered with 90% glycerol with 5% anti-fade N-propyl gallate (Sigma Aldrich). In the case of anti-actin labelling the sperm were fixed for 10 minutes in 3.7% formaldehyde at room temperature, centrifuged and immediately incubated with NHSperm were left freely to capacitate for 60 min with the subsequent induction of the acrosome reaction (described above). Latrunculin A at a final concentration of 10 μM was added into the capacitating medium at the beginning of the 60 min sperm capacitation. AR was induced by 5 μM CaI, and sperm were incubated for another 60 min. Control sperm samples were prepared in the same way but without the addition of Latrunculin A. To detect the possible influence of the ongoing acrosome reaction by Latrunculin A, control samples were prepared; sperm were incubated with Latrunculin A both during capacitation and induction of AR. The status of the acrosome was detected using PNA lectin.®In Situ Red Starter Kit Mouse/Rabbit, Olink Bioscience), β1 integrin  and α tubulin  as a negative control . The interaction of experimental proteins CD46 and β1 integrin was studied using a specially prepared starter kit, which was  comprised of one PLA probe  and one Probemarker kit  was used to prepare the PLA probe anti-rat, using an unconjugated secondary antibody  which was conjugated with a short DNA strand.To detect the interaction of proteins CD46 and β1 integrin, Proximity Ligation Assay Duolink (PLA) was used. Proteins α tubulin  and β tubulin  were selected as a positive control (DUO92101 DuolinkFreshly released sperm and sperm after CaI induced AR (see above) were washed twice in PBS, smeared onto glass slides and air-dried. Sperm smears were fixed with 3.7% formaldehyde in PBS (pH 7.34) at room temperature for 10 min, followed by washing in PBS. Sperm were blocked with 10% BSA in PBS for 1 h and incubated with primary antibodies. In each experiment, two primary antibodies were used, each directed against one of the target proteins. These antibodies were raised in different species. Species-specific secondary antibodies (PLA probes) bind to primary antibodies, and each of them has a unique short DNA strand attached to it. Both DNA strands interacted through a subsequent addition of two other circle-forming DNA oligonucleotides, forming a DNA circle, which was closed by DNA Ligation. DNA circles were amplified using a DNA polymerase. The amplified DNA was detected by hybridization with labelled oligonucleotides, which produced a visible fluorescent spot. These spots were detected with an Olympus IX81 fluorescent microscope and photographed with Hamamatsu ORCA C4742-80-12AG, using Olympus Soft Imaging Solutions software . Representative results are shown.SDS electrophoresis and immunoblotting technique were used for the β1, α3 and α6 integrin and CD46 protein detection was performed by protocols based on standard methods69http://svi.nl) was used for the colocalization analysis and its visualisation. A colocalization analyser computed a Pearson’s correlation coefficient and created a colocalization map. The Pearson’s correlation coefficient expresses the rate of correlation of colocalizing channels in a dual-colour image and gives a value between minus 1 to plus 1. In this case, 1 means an absolutely positive correlation, 0 means no correlation and -1 means a perfect anti-correlation. The value between 0.5 and 1 is interpreted as colocalization. Costes method was used for a background estimation.Experimental data were visualized and analyzed using STATISTICA 6.0.  and GraphPad Prism 5.04 . The differences in the relative fluorescent intensities between the acrosome-intact and acrosome-reacted sperm among individual relocation segments in How to cite this article: Frolikova, M. et al. Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction. Sci. Rep.6, 33714; doi: 10.1038/srep33714 (2016).


Following sleep stage scoring, we detected and characterized oscillatory events across age and examined how their features corresponded to spectral changes in the sleep EEG. Results indicated a developmental decrease in the incidence of delta and theta oscillations. Spindle oscillations, however, were almost absent at 2 years but pronounced at 5 years. All oscillatory event changes were stronger during light sleep than slow-wave sleep. Large interindividual differences in sleep oscillations and their characteristics  also existed. Changes in delta and spindle oscillations across early childhood may indicate early maturation of the thalamocortical system. Our analytic approach holds promise for revealing novel types of sleep oscillatory events that are specific to periods of rapid normal development across the lifespan and during other times of aberrant changes in neurobehavioral function.Although quantitative analysis of the sleep electroencephalogram (EEG) has uncovered important aspects of brain activity during sleep in adolescents and adults, similar findings from preschool-age children remain scarce. This study utilized our time-frequency method to examine sleep oscillations as characteristic features of human sleep EEG. Data were collected from a longitudinal sample of young children ( The electroencephalogram (EEG) is a fundamental method for identifying sleep/wakefulness states, quantifying sleep-related cortical activity and assessing sleep regulation. Slow oscillations, delta activity, and sleep spindles are prominent features of the sleep EEG during nonrapid eye movement (NREM) sleep. Visual detection of some features of the sleep EEG  can be challenging because such events may be masked by high-amplitude slow waves. This is true during early childhood, a period of rapid maturation of brain activity during sleep and a time when the amplitude of slow waves is relatively high . QuantitThe sleep EEG can be analyzed in several ways: (i) the time domain , which provides information about the incidence and amplitude of waves , 3; (ii)The benefits of sleep for brain function and learning as reflected in plastic changes across the cortex during development are increasingly recognized . OscillHere, we utilized our previously published time-frequency analysis method based on autoregressive modelling of short overlapping EEG segments –14 to chEight healthy children  were studied longitudinally at three ages . Recruitment, screening, and inclusion/exclusion criteria are detailed in our previous publications , 15.During the 5 days before an overnight sleep recording, children followed a sleep stabilization schedule. They slept in their habitual environment  with a minimal sleep opportunity of 12.5 h (2Y and 3Y) or 12 h (5Y) per 24 h day. The schedule included a nap opportunity of at least 45 min at 2Y for all children and at 3Y for seven of the children (one child had stopped napping). The stabilization phase served to minimize sleep restriction and to entrain the circadian system. Compliance with the prescribed schedule was verified with sleep diaries, daily parent reports, and actigraphic recordings. Caffeine consumption and medication affecting sleep, alertness, or the circadian system were prohibited during the entire study.Overnight in-home sleep EEG was obtained after 13 hours of prior wakefulness  using a portable 16-channel EEG recorder . Four EEG derivations , EMG, and EOG were recorded. Data were sampled at 128 Hz  and at 256 Hz . Sleep stages were visually scored for 30 s epochs (C3A2) according to standard criteria  resulting in a frequency resolution of 0.25 Hz. Epochs were excluded whenever power in the 20–40 Hz and 0.75–4.5 Hz band exceeded a threshold based on a moving average determined over twenty 30 s epochs.γ ≤ 6.6 s−1; . Average sleep duration was 10.3 ± 0.4 , 10.4 ± 0.8 (3Y), and 10.2 ± 0.4 hours (5Y). The sleep structure of a representative boy at each longitudinal assessment is illustrated in hypnograms .Oscillatory events detected in the sleep EEG are superimposed on the corresponding spectrogram , 13.7 ± 1.3 (3Y), and 14.7 ± 2.9 min (5Y) and to stage 2 (sleep onset) was 12.1 ± 2.2 , 15.7 ± 1.1 (3Y), and 16.4 ± 3.0 min (5Y). No age-dependent differences in sleep latencies were observed.Alpha events (8–10 Hz) were present throughout waking and disappeared with the onset of stage 1 . Delta/tIn this longitudinal study, we examined developmental changes in oscillatory events and power density spectra of the NREM and REM sleep EEG in children ages 2Y, 3Y, and 5Y. Overall, we observed a decrease of spectral power and event ratios in the delta/theta range and an increase in the sigma frequency range with increasing age. Moreover, we found that specific oscillations disappeared (theta bursts and “ultrafast” spindles) while other ones emerged (“regular” spindles) across the preschool-age years. Similar to adults, large interindividual differences were present thus suggesting that developmental changes in EEG sleep oscillations are not consistently pronounced in all children.In contrast to standard methods for detecting sleep oscillations, such as those used to identify slow waves e.g., , 22) or  or 22]) Event ratios and power in the delta range during LightS and SWS showed a developmental decline as measured by a reduction in slow waves and “fast” delta oscillations (2–4 Hz). “Fast” delta oscillations were also a marker for the transition into sleep (see discussion below). These findings complement our previous work showing a similar decrease in SWA during afternoon naps in the same early childhood cohort  and exte“Fast” delta oscillations (>2 Hz) likely have two different generating components, originating in the cortex and/or in the thalamus . Our finEstablishing a clear border between the delta and theta frequency bands is difficult, especially during periods of rapid developmental change in the sleep EEG. In REM sleep, “bands” of oscillatory events with frequencies up to 5 Hz were observed, and fast delta/low theta events were also present in REM sleep and decreased with increasing age. The emergence of this activity was previously reported to occur between 12 and 24 months of age . Such evμV [Theta oscillations occurred in bursts  in many μV .In adults, one of the most prominent oscillatory events are sleep spindles, which are observed in the 11–15 Hz frequency range. Many researchers distinguish between slow spindles below 12 Hz that occur more frontally in comparison to those that are observed more centrally with a frequency around 14 Hz , 32. In An interesting observation in our subjects was the occurrence of “ultrafast” spindles. Although not present in all children, these oscillatory events exhibited similar properties across subjects, which clearly distinguished them from “normal” spindles in several ways: (i) they occurred in deep sleep with the same or even higher event ratios than in LightS ; (ii) thSleep spindles are a thalamocortical phenomenon. They are generated in the thalamus (reticular nucleus), but the cortical network is involved in their onset and contributes to their termination . DiffereResults from a number of recent studies indicate that spindle characteristics and sigma activity may be markers of cognitive ability. For example, in school-age children, relative sigma power is positively associated with full scale and fluid IQ but not verbal IQ, working memory, and speed of processing . AlthougOur present findings extend those from our recently published paper examining early developmental changes in sleep spindle characteristics in the same cohort  using a Sleep onset may be operationalized in various ways. The first epoch other than wakefulness—typically stage 1—is often considered the time of sleep onset , 31, 40.μV) intermixed with delta and theta activity [Our data showed that delta/theta events (2–6 Hz) herald the transition into sleep. These events, however, were present already during waking several minutes before sleep onset, highlighting that the transition into sleep is a gradual process. Delta oscillations started to appear closer to sleep onset with increasing age but still before the onset of stage 1. Furthermore, they began to slow  approximately 2 epochs (50 s) before the first occurrence of stage 1. Alpha events disappeared with the onset of stage 1 sleep, whereas sleep spindles only appeared approximately three epochs after stage 2 was scored. Thus, the slowing of delta events and the disappearance of alpha events most clearly indicated the transition into sleep. This is in line with the observations that drowsiness in children at the age of about 3 years is accompanied by the appearance of diffuse low voltage activity  likely accumulated a higher level of sleep pressure across the waking day than those at age 5Y, a time when none of the participants still napped . The anaIn this longitudinal study, we found early developmental changes in several oscillatory events in the sleep EEG as well as large interindividual differences in delta, theta, and spindle-like oscillations. Collectively, our data suggest that delta and spindle oscillations reflect maturational changes occurring in the thalamocortical system during early childhood. Whether early changes in oscillatory events are paralleled by children's maturing cognitive capabilities is an area of rich future investigation that may shed light on the underlying neurophysiological substrates and pathways that contribute to variability in the brain and behavioral functioning in the early years of life.Supplementary Figures S1 to S8: Top: Spectrograms, oscillatory events and hypnograms of individual children (A-H) recorded longitudinally at 2Y (a), 3Y (b) and 5Y (c). For details, see Figure 1. Bottom: Mean power density spectra and event ratios of individual children (A-H) recorded longitudinally. For details, see Figure 3.


A versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation and bearing a functional handle for inhibition of protein–protein interactions is described. hCys) amino acids spaced at i and i + 4 positions by double substitution. The constraint can be readily removed by displacement of the maleimide using excess thiol. This new constraining methodology results in enhanced α-helical conformation (BID and RNase S peptide) as demonstrated by circular dichroism and molecular dynamics simulations, resistance to proteolysis (BID) as demonstrated by trypsin proteolysis experiments and retained or enhanced potency of inhibition for Bcl-2 family protein–protein interactions (BID), or greater capability to restore the hydrolytic activity of the RNAse S protein (RNase S peptide). Finally, use of a dibromomaleimide functionalized with an alkyne permits further divergent functionalization through alkyne–azide cycloaddition chemistry on the constrained peptide with fluorescein, oligoethylene glycol or biotin groups to facilitate biophysical and cellular analyses. Hence this methodology may extend the scope and accessibility of peptide stapling.The development of constrained peptides for inhibition of protein–protein interactions is an emerging strategy in chemical biology and drug discovery. This manuscript introduces a versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation using BID and RNase S peptides as models. Dibromomaleimide is used to constrain BID and RNase S peptide sequence variants bearing cysteine (Cys) or homocysteine ( Constraint of a peptide in a bioactive conformation is proposed to improve target binding affinity (by preorganization),12 increase stability (e.g. by protecting against proteolysis)15 and enhance cell-uptake,17 although a number of recent studies have provided conflicting evidence for the enhanced cell permeability and cellular potency that is conferred through stapling.20 Available strategies25 to constrain a peptide in a helical conformation  which respectively bind Bcl-xL proteins41 and RNase S protein42 ormation , includereaction 28,31 or reaction  between rotein42 , to demo14 and others61 introduced α-pentenylglycine as an alternative to the widely used α,α′-pentenylalanine for cross-linking the i and i + 4 residues within a peptide  residues with dibromomaleimide (or for cysteine (Cys), a 6-atom constraint)  .1a–d bearing Cys or hCys amino acids in positions used previously62 for i → i + 4 constraints were independently oxidised to the constrained disulfide 2a–d or cross-linked with dibromomaleimide 4 to generate 3a–d with rapid and complete conversion. Alternatively, this sequence 1a–d to 3a–d could be performed in one pot and peptides 1a–d fully regenerated by addition of an excess of thiol  to each other as a result of the different cross-link length but both were more helical than the corresponding reduced peptide 1a or 1b, (24% and 29%). Molecular dynamics calculations were conducted on 1b and 3b – these further support the conclusion that the helical conformation predominates when constrained by the maleimide linker  except for the constrained peptide 3b which was slightly more potent (IC50 ∼ 4.2 μM), thus the constraining/unconstraining reagents have either a weak or no effect on the inhibitory potency for this PPI. Although inconsistent with the concept that preorganization should promote higher binding affinity, the result is consistent with our own recent studies on hydrocarbon-constrained peptides, which illustrated enthalpy-entropy compensation operates for these peptides which bind through an induced-fit mechanism.62 For inhibition of BAK/Bcl-xL both constrained peptides 3a,b were shown to be significantly superior inhibitors compared to the reduced peptides 1a,b. The differential effects upon potency for the same peptide against Bcl-xL and Mcl-1 may arise as a consequence of favourable non-covalent contacts between the maleimide and Bcl-xL; it is anticipated that future structural analyses will shed light on these differences.Peptides ractions . Constra7a–c. Using Mitsunobu alkylation,67 alkyne–dibromomaleimide 5 was prepared and conjugated to peptide 1a to generate the constrained peptide 6. Without isolation or purification, subsequent “click” reaction with biotin, fluorescein and PEG azides38 afforded complete conversion to click-conjugated constrained peptides 7a–c  and corresponding low restoration of enzymatic activity , the constrained peptides reach a greater overall α-helical content). Here, introduction of a disulfide (2d) or a maleimide (3d) crosslink results in enhanced helicity in comparison to the WT sequence . As a result 3d is much better able to restore RNase S protein activity than is 3c.The use of dibromomaleimide-based constraints for promotion of α-helicity was then further demonstrated using the RNase S-protein/S-peptide interaction with the choice of amino acid positions at which to introduce the constraint informed by previous studies by the Verdine,c and 3d . Both pec and 3d . In this Fig. S31. Molecul Fig. S31. In cont24 but no general approach has been widely adopted; we have developed a new method to rapidly and reversibly introduce a functionalized constraint into two model peptides that enhances a range of biophysical and biochemical properties. Moreover, the constraint may be further functionalized in a divergent manner with motifs useful in chemical biology and proteomics applications. The approach can be carried out using natural readily available amino acid side chains on unprotected peptides, potentially bringing peptide stapling to a larger community than hitherto have had access and extends the applications of dibromomaleimide and related reagents. Future efforts will apply this approach to a wider range of helix mediated PPIs with the aim of establishing the generality of the method. The approach may also be useful to identify transient binding pockets proximal to a native ligand binding site using the template directed approach introduced by Sharpless.70 Similarly, the reversibility of the constraint, may permit more facile delivery of a peptide-based reagent to the cell, where it can become unconstrained and less easily transported back out of the cell. Together with the additional functional handle introduced on the maleimide constraint , this could facilitate detailed cellular and biochemical analyses e.g. identification of additional targets of the constrained peptide by pull-down. These objectives will represent the focus of our future studies.In conclusion, the attractiveness of thiol-based “stapling” approaches has been recognized for some time,


Escherichia coli (E. coli) cells after conjugation to gold nanoparticles (AuNPs). The efficacy of the bioconjugates was also tested on Salmonella typhi (S. typhi) bacteria infected into cervical cancer cells (HeLa) and further improved through specific targeting via folate receptors. Our results demonstrate significantly lower inhibitory concentration values for sushi-NP assemblies as compared to free drug, especially at optimal drug loading levels. No major cytotoxicity was observed in mammalian cells alone.Bacterial drug resistance has emerged as a serious global threat mandating the development of novel methodologies that allow facile modulation of antimicrobial action in a controlled fashion. Conjugating antibiotics to nanoparticles helps to meet this goal by increasing the drug’s overall avidity, bioavailability and easier internalisation into mammalian cells, targeting bacteria that otherwise escape antibacterial action by host cell-localisation. We used polymyxin B sulfate (PMB) and sushi peptide as model drugs against Gram-negative bacteria and established their enhanced antimicrobial activity on  Conventional antibiotics suffer from several issues such as improper biodistribution, poor water solubility, lack of target specificity and loss of efficacy over time due to the emergence of drug resistance in pathogenic bacteria2. To overcome these issues, higher doses of antimicrobials are often prescribed, which further worsen the situation as the bacteria evading the action of drug become even more resistant over time. The process is more critical in Gram-negative bacteria as their cell wall structure is quite complex containing a thick lipid layer, which when degraded, has the potential to cause tremendous pathogenicity3. Since no new major antibiotics have been developed in the last 40 years, except for the recent discovery of synthetic antibacterial agents oxadiazoles4, a new strategy is perhaps required for improving the efficacy of conventional antibiotics and dealing with the resistance crisis. The efficacy can either be improved by developing a more potent derivative of a drug molecule or by improving its delivery and interaction within the bacteria. The use of a delivery vehicle can dramatically lower the antibiotic minimum inhibitory concentration (MIC) or half maximal inhibitory concentration (IC50) value by presenting the drug molecules in such a way that it facilitates the interaction of the active groups with the target molecules on the bacteria, increasing its overall efficacy8. Typically, nanoparticles (NPs) are used for improving the delivery and specificity of therapeutics10.Antibiotics are the most conventional therapeutics used for the treatment of bacterial infections, however, 50% of these drugs prescribed to humans are either not needed or not effectively utilized as prescribed. Consequently, misuse of antibiotics is the most important factor leading to the catastrophic threat of antibiotic resistance around the world13. Additionally, since chemotherapy cannot specifically target bacteria, if the bacterial infection in cancer remains untreated, the bacteria can infect the host even after the cancer cells are killed by chemotherapy. Thus, it becomes even more crucial to ensure elimination of live bacteria from the tumor region. Substantial use of antibiotics here again builds selection pressures, which ultimately leads to the emergence of resistant microorganisms14. In the immunologically impaired cancer patients, the first dose of antibiotics administered is extremely important. A recent study showed that combination antibacterial and chemotherapy treatment lead to notable reduction of tumour activity and marked survival benefit over either therapy alone15. Also, bacteriophages (viruses that infect and lyse bacteria), matched for specific bacterial isolates, have been used in the past to treat antibiotic-resistant infections in cancer patients but the therapy does not offer a facile option in acute settings due to challenges with affordability, limited host range, phage-resistance, side effects from bacterial lysis and several other regulatory issues18. Thus, new combative strategies are desperately required to deal with bacterial infections in cancer patients.Antibiotic resistance poses an even bigger problem in diseases like cancer where patients are at a higher risk of developing serious bacterial infections due to prolonged neutropenia, lymphocyte dysfunction, mucositis, and use of invasive devices19. They are recognized for being highly selective and efficacious and, at the same time, relatively safe and well-tolerated. Consequently, commercial pharmaceutical research is fast shifting its focus toward this new class of antibiotics and close to 140 peptide therapeutics are currently under clinical trials20. Despite being an attractive alternative to conventional antibiotics, the usage of peptides also has limitations in stability and delivery, which needs to be addressed before their full potential is realized. As most peptide therapeutics are injectables, there exist challenges relating to their acidic and enzymatic degradation and inefficient internalization into cellular membranes20. In addition, peptides are prone to hydrolysis and oxidation, and have a higher tendency to aggregate and undergo faster elimination. Use of NP delivery platforms can overcome this rapid degradation, instability and aggregation issues, crossing of biological membrane barrier and increasing retention time5.Recently, peptides have emerged as a new class of antimicrobials with lesser cytotoxic effects than conventional antibiotics21, sushi uses LPS as a latch and lyses the bacteria by disrupting the membrane and releasing its cytosolic content, without LPS leakage22. This may be beneficial as free LPS in the bloodstream is known to cause medical complications such as sepsis. The response of the two drugs were tested and compared in the free and NP-conjugated forms . The structure-property relationship studies for these drugs illustrate that while PMB acts by neutralizing and shedding off the cell wall component lipopolysaccharide (LPS) present on the outer cell wall of all Gram-negative bacteria23 present in the system before and after conjugation  Fig.  and UV-Vion Figs  and S3B2E. coli and S. typhi bacteria in a microtitre broth assay at 37 °C and pH 7.4. Free drug and particles alone (without drug) were used as controls. After overnight incubation, the changes in absorbance due to condition-dependent bacterial growth were measured. The results suggest that PMB when delivered with the AuNPs killed ∼10% more E. coli in comparison to free PMB  when compared to the free peptide (∼700 nM) and reached MIC at ∼700 nM. Interestingly, the conjugates with 1000x molar excess showed significantly higher antibacterial activity in comparison with conjugates having lower molar excess of peptides.The different conjugates were applied to our model PMB Fig. . In compNPs Figs  and S4B.S. typhi. The internalized live S. typhi bacteria were analysed by optical microscopy after staining with neutral red dye and fixing of the infected HeLa cells. The results showed that the least number of bacteria survived in the 1:1000 case as compared to other ratios  molecules via the amine groups present on the peptides. This conjugation was confirmed by UV-spectroscopy as FA gives a characteristic peak at 280 nm Fig. . The effcle Fig. . This iscle Fig.  and S9. cle Fig.  which cl31. Quantification of the extent of drug binding allowed us to determine the optimum drug loading required for achieving the highest antibacterial activity amongst all the NP-bioconjugates. Sushi peptides are biologically-active derivatives of the widely popular factor C protein found in horseshoe crabs32. Factor C proteins are extensively used in endotoxin detection assays such as the popular endotoxin test such as LAL tests34. As a result, the sushi peptides have a well-established and specific interaction with the LPS, although its role in clinical applications is still not fully explored. There are two reported forms of sushi peptides, S1 and S3, both of which are 34-mer with a strong affinity for LPS35. While the S1 type forms a random coil structure and functions as a monomer transforming into alpha helix, S3 generally exists in the dimeric form and forms alpha and beta sheets on interaction with LPS36. In the literature, it is reported that the binding efficiency of S3 increases as it goes from the monomeric to dimeric to tetrameric forms, i.e., a locally high concentration of peptides has a synergistic effect on its binding properties37. Therefore, we selected the S3 type sushi for our study. The site for NP conjugation was purposely designed to be terminally opposite to the LPS-binding side in order to cause minimal interference in the peptide’s activity. We hypothesized that when the peptides attach to the NPs, they behave as multimeric units providing a much larger and suitable surface area for interaction with the LPS on the bacterial membrane. It is possible that some of the peptides also leach out from the AuNP surface but for the large part, we believe that it is the peptides that remain associated to the AuNPs surface that disintegrate the cell membrane ultimately causing bacterial death. Au-thiol linkage is known to be quite stable above pH 7 with a bond strength (50 to 100 kJ/mol) close to that of a weak covalent bond39.We began our studies by evaluating the antimicrobial susceptibility of conjugates on bacterial cells before and after their conjugation to gold NPs (AuNPs). Gold was chosen due to its low reported toxicity, ease of synthesis and functionalization with small moleculesth and 7th positions in the peptide sequence from the N-terminal side) and thus, show relatively less efficient performance. Based on geometrical considerations, we roughly estimated the peptide-peptide and peptide-LPS intermolecular distances in the intermediate case where the peptides were in 1000x molar access to NPs. Our results indicated that indeed, more peptides are available for LPS-binding in this case as the outer edges of the peptides lie at distances that are commensurate with the intermolecular LPS gap sizes  conjugation on the AuNP-sushi conjugates to enhance their internalization and further improve their antibacterial activity. The overexpressed folate receptors (FR) on cancer cells increases the internalization of NPs containing FA via receptor mediated endocytosisWe have demonstrated an efficient antibiotic targeting strategy using AuNPs that increases both the antibacterial efficacy and particle uptake for destroying intracellular Gram-negative bacterial infection. Sushi peptides are more potential antimicrobials providing the window for modulation of antibacterial activity without affecting the mammalian cells even at relatively higher concentrations. In addition, the FA conjugated NPs proved to be efficient in targeting the cancer cells infected with pathogenic bacteria and showed increased antibacterial activity as compared to non-folic acid conjugated NPs. We anticipate that this strategy can be applied in future for preparation of NPs based target specific multivalent scaffolds which might help in lowering the dosage as well reducing the chances of drug resistance development. Also conjugating NPs with ligands specific for a particular type of cell/organ in which infection is prominent, especially in organ specific cancer, can be studied further and this might help in development of more potent and target specific surface engineered drug delivery vehicles.E. coli (DH5α) and S. typhi (serovar enterica) were obtained from the School of Biological Sciences at IIT Delhi. The S3 sushi peptide sequence  was synthesized by Genemed Synthesis . Gold(III) chloride trihydrate (HAuCl4.3H2O), sodium citrate dihydrate (C6H5Na3O7.2H2O), tannic acid (C76H62O48), polymyxin B sulfate (PMB), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ortho-pthaldehyde (OPA), Luria broth (LB), folic acid (FA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and boric acid were purchased from Sigma Aldrich (India). Dithiolalkane aromatic PEG6 hydrazide (DTH) was obtained from Sensopath Technologies (USA). Neutral red and gluteraldehyde (GLA) were obtained from CDH (India). Dulbecco’s modified eagle medium , fetal bovine serum (FBS), 3--2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s phosphate-buffered saline (PBS), antibiotic-antimycotic solution and calcein AM were purchased from Invitrogen (India). All solutions were prepared using ultrapure deionised (DI) Milli-Q water (∼18.8 mΩ.cm resistivity) . HeLa cell line was obtained from the National Centre for Cell Science (NCCS) .Bacterial strains of 48. For this, 5 mL of 1% w/v HAuCl4 was mixed with 395 mL of DI water. Simultaneously, a reducing solution was prepared by mixing 20 mL of 1% w/v sodium citrate, 50 µL of 1% w/v tannic acid and 80 mL of DI water. The gold and the reducing solution were then heated up to 60 °C separately and mixed together under constant stirring at 60 °C for 4 h. Once the solution turned wine red color indicating the formation of AuNPs, the suspension was quenched in an ice-bath and stored at 4 °C. Next, the AuNPs were functionalized with sushi peptides or PMB drug. The AuNP-sushi conjugates were prepared in a 10 mL batch by mixing the peptides with 2 nM AuNP suspension in different molar ratios. The mixtures were incubated overnight at 25 °C inside a shaker incubator (Sciengenics orbitek) at 100 rpm. The conjugated particles were purified by three consecutive centrifugal washes at 8690, 11175 and 14900 rcf for 20 min each and the pellet was resuspended in 40 mM HEPES buffer at pH 7.4. All the particle conjugates were stored at 4 °C until further use and were stable for at least a month.AuNPs of approximately 16 nm size were synthesized using the Turkevich method49. DTH was added to 10 mL of AuNP suspension in a molar ratio of 1:104 (AuNP:DTH) and incubated overnight (Sciengenics orbitek) at 25 °C under constant shaking at 100 rpm. The DTH-functionalized AuNPs were purified via centrifugation at 8690, 11175 and 14900 rcf for 20 min each and finally resuspended in 40 mM HEPES buffer at pH 7.4 containing 1% v/v GLA. This mixture was kept overnight at 25 °C inside the shaker incubator at 150 rpm. The GLA-DTH-AuNP suspension was again washed three times as above and resuspended in the HEPES buffer. Finally, PMB was added in different molar ratios and the mixture was incubated overnight at 25 °C at 150 rpm. In the end, PMB-conjugated AuNPs were retrieved in 40 mM HEPES buffer at pH 7.4 by following the same washing steps as stated above. The particle conjugates were stable at 4 °C for at least 6 weeks.The AuNP-PMB conjugates were prepared using a protocol reported earlierThe size of the AuNPs was determined using the Tecnai G2 transmission electron microscope (TEM). For this, the 4x AuNP suspension was dried on a carbon-coated copper TEM grid . The samples were imaged at 200 kV and the micrographs obtained at 44000X were analyzed using the open source ImageJ software. The average size of the NPs from the particle size distribution curves was obtained to be 16 ± 4 nm. The extent of peptide/PMB conjugation on the AuNPs was measured using the OPA fluorescence detection technique. For this, 8 mg of OPA reagent was added to 100 μL of 95% v/v ethanol. This OPA solution was mixed with 200 µL of β-mercaptoethanol and 10 mL of 0.4 M boric acid titrated with KOH to pH 9.7. Equal volumes of the drug/peptide and OPA solutions were mixed and the fluorescence emission at 455 nm was measured at an excitation peak of 340 nm using a microplate reader (BioTek Synergy H1). A calibration chart was prepared by taking different concentrations of drug/peptide and the extent of sushi/PMB binding was estimated by measuring the fluorescence signal response in the supernatant before and after conjugation. For attenuated total reflection-fourier-transform infrared (ATR-FTIR) spectroscopy, the spectra of the liquid sample (50 µL) were directly recorded for sushi and AuNPs conjugated with either sushi or both sushi and folic acid.E. coli cells were grown overnight in LB broth (3 mL broth in 15 mL tube) at 37 °C and 250 rpm. The concentration of the bacteria was determined by counting colony forming units (CFU) on LB agar plates as well as absorbance at 625 nm in UV-VIS spectrophotometer (Shimadzu UV-2600). The cells were diluted to 2 × 106 CFU/mL and 50 μL of this suspension was mixed with 100 μL of two times concentrated LB and 50 μL of free PMB/sushi or conjugated NPs in different molar concentrations. The samples were incubated overnight at 37 °C and the bacterial growth was measured the next day by taking absorbance readings at 625 nm (BioTek Synergy H1 microplate reader). All the samples were prepared in triplicate and each experiment was repeated three times. The final data were reported as a mean of all the values ± 1 SD (standard deviation). Two way ANOVA was performed for calculating the statistical significance . The bacterial growth without any antibiotic was taken as positive control and all the test results were normalized with it to calculate the viability.The antimicrobial tests were performed by the microdilution broth assay in 96-well cell culture plates (Nest). 2 incubator at 37 °C with 5% CO2.The HeLa cells were cultured in high-glucose DMEM with 10% FBS . When 90% confluent, the cells were detached by adding trypsin and then centrifuged and re-dispersed in complete media. The suspended cells were seeded in a 96-well cell culture plate (Nest) at a density of 10,000 cells/well. For imaging purpose, cells were also cultured on glass coverslips kept inside 24-well cell culture plates (Nest) at a seeding density of 50,000 cells/well. The culture plates were kept overnight in a COMTT assays were carried out to assess the cytotoxicity of free and conjugated drug/peptides. After incubating the cells for 24 h with different concentrations (in complete medium) of the free PMB/sushi or NP conjugates, cells were washed with PBS and then 20 μL of 5 mg/mL MTT in PBS was added to the 200 μL of complete medium in each well. The plate was incubated for 1 h to allow the formation of purple formazan crystals inside the cells. The media were replaced by DMSO to dissolve the crystals and their concentration was determined by taking optical density (O.D.) measurements at 550 nm (BioTek Synergy H1). All the data were presented as mean value ±1 standard deviation from at least three independent experiments performed in triplicate. Cells incubated with only complete medium were used as positive controls and other values were normalized using positive control.S. typhi bacteria were grown overnight in LB broth at 37 °C and 250 rpm. The late logarithmic cultures were diluted in complete medium without serum and used to infect the HeLa cell monolayer using a multiplicity of infection of 200 bacteria per cell. The bacterial cells were left to internalize by the cells for 2 h at 37 °C after which the medium was replaced with 2x concentrated antibiotic-antimycotic solution to kill any remaining extracellular bacteria. After washing with PBS, the HeLa cells were incubated with free or conjugated sushi peptides for 3.5 h at 37 °C in order to study their effect on the intracellularized bacteria. AuNPs alone were used as negative control. The plated cells were washed three times with PBS and then lysed using 100 μL of 0.1% w/v SDS per well (in the 96-well plate format). The number of viable bacteria was determined by plating 10 μL of the lysed sample onto LB agar plates and counting the growth after overnight incubation at 37 °C. For image analysis, the mammalian cells were incubated with 30 μL of neutral red stain in 300 μL of complete medium at 37 °C for 20 min and then fixed using 4% w/v paraformaldehyde in PBS for 20 min. The cells were then imaged in bright field mode using an Olympus IX 73 microscope fitted with a Q-imaging digital color camera.The viability of the infected HeLa cells was analyzed by a live/dead staining assay. The assays were carried out by staining the cultured cells with 0.3 μL of 2 μM calcein AM per 300 μL of complete medium at 37 °C. After 30 min of incubation, the stained samples were washed thrice with PBS and visualized using fluorescence microscopy with FITC filter (Olympus IX 73). The appearance of a bright green color indicated viable cells whereas; the dead cells didn’t show any fluorescence under the microscope. Images were taken using a high speed digital camera  after fixing the exposure time.S. typhi bacteria was studied using microdilution broth assay in 96-well cell culture plates. The procedure was similar to the one as described in antimicrobial activity assays method section. The final concentrations of all the NPs used was 1000 and 1500 nm. The effect of folic acid conjugated NPs on intracellular bacteria was studied by colony count method as described previously. The final concentration of all the particles as well sushi peptides used for the study was 4000 nm. The data were normalized against a positive control - HeLa cells infected with internalized bacteria.1 mg/mL stock of FA was prepared in 50 mM NaOH. A final solution of 1000 µM FA concentration, pH- 7.4, was prepared in 1 mg/mL each of EDC and NHS in HEPES buffer and was kept for 1.5 h incubation at room temperature at 100 rpm. The FA solution with activated carboxylic groups was then added to AuNP-sushi conjugated suspension in 100 molar excess (AuNP:FA) to all the synthesized ratios (AuNP:sushi) and was kept for overnight incubation at room temperature and 100 rpm. The conjugated particles were purified by three consecutive centrifugal washes at 8690, 11175 and 14900 rcf for 20 min each and the pellet was resuspended in 40 mM HEPES buffer at pH 7.4. Absorbance spectra of the synthesized FA-conjugated NPs was studied using UV-VIS spectrophotometer (Shimadzu UV-2600). Antibacterial activity of FA-conjugated and non-conjugated AuNP:sushi of different ratios on 100 µL solution of AuNPs or conjugated NPs prepared in complete media was added onto each well of 96 well plate containing overnight grown HeLa cells (10000 cells/well). Samples were incubated at 37 °C for 4 h and then washed three times with PBS, trypsinized and the cells were counted using a haemocytometer. Finally, the cells were centrifuged and digested in 100 µL aqua regia and analysed by Agilent’s 7900 ICP-MS instrument.Supplementary information