Candida spp.) and a large variety of bacterial species present. We studied the relationship between the time of proper function of Provox VP, the microorganism composition of the biofilm present on it, and the degradation level of the silicone material. Evaluation of the surface of the removed VP using an atomic force microscope (AFM) has demonstrated that biofilm growth might drastically change the silicone’s mechanical properties. Changes in silicone stiffness and thermal properties might contribute to the failure of VP function. Our data can serve in future studies for the development of methods to prevent or inhibit biofilm formation on the VP surface that would translate to an increase in their durability and safety.Voice prosthesis implantation with the creation of a tracheoesophageal fistula is the gold standard procedure for voice rehabilitation in patients after a total laryngectomy. All patients implanted with a voice prosthesis (VP) have biofilms of fungi and bacteria grow on their surface. Biofilm colonization is one of the main reasons for VP degradation that can lead to VP dysfunction, which increases the high risk of pneumonia. In a 20-month evaluation period, 129 cases of prostheses after replacement procedures were investigated. Microbiological examination of the biofilms revealed that there were four of the most common fungi species ( In 2018, worldwide laryngeal cancer was diagnosed in 177,422 patients, and there were 94,771 deaths caused by it . The pioCandida albicans, Candida glabrata, Candida krusei, and Candida tropicalis. On the VP surface, Candida spp. form a tridimensional network leading to prosthesis malfunction [Candida albicans cells in the biofilm structure express the surface adhesion molecules Eap1 and Als3, which serve as specific surface receptors responsible for bacterial binding, including the viridans  groups Streptococci and Staphylococcus aureus [Candida species hyphae is crucial to initiating biofilm formation. The penetration of the yeast filaments is facilitated by the enzymatic degradation of silicone. The process of enzymatic silicone degradation might supply nutrients for growing yeast [2−) and extracellular enzymes released by monocytes and neutrophils (nicotinamide adenine dinucleotide phosphate oxidase (NADPH)) are also involved in the process of silicone damage [Candida albicans has 30 genes in which mutations could decrease adherence of hyphae. In addition to their genetic profile, the surface topography and host immunology are important factors controlling Candida albicans adherence [The most serious disadvantage of silicone polymer-based voice prosthesis and other medical devices is their colonization by fungi and bacteria ,10,11. Tfunction ,13. Cands aureus ,15. In eng yeast ,16. Moree damage . Anothere damage . Candidadherence ,20. Immudherence . Howeverdherence . Unlike dherence ,23. Howedherence ,25,26. Tdherence . Factorsdherence . Some attempts to elongate VP lifespan by reducing biofilm development include the use of nystatin, antimicrobial agents, oral wash, and to clean the VP by special brush. All have pros and cons but are still not fully satisfactory ,30. LookCandida species and a few groups of bacteria that should be considered as typical steps of VP colonization. Additionally, using scanning electron microscope (SEM) and atomic force microscope (AFM) analyses, we have characterized VP surface material damage caused by biofilm growth. Information on the qualitative composition of “microorganism inhabitants“ on VP and characteristics of the damage of the material that can be caused by biofilm growth provide a platform for further studies to develop new means to extend the time of VP exploitation and increase the safety of VP users after cancer treatment.This study has been conducted in part as a prospective cohort study and retrospective clinical study describing biofilms on Provox VPs removed from patients. We have identified four of the most common Candida krusei (identified on 55.8% of 129 VPs), Candida albicans (46.5%), Candida glabrata (42.6%), Candida tropicalis (18.6%), and Saccharomyces cerevisiae (6.2%). We observed that there is no statistically significant difference between the occurrences of certain fungal species in each group  or general (pneumonia) infections. The most common bacterial species was Staphylococcus aureus (44.2%). The second most commonly identified species were members that belong to a group of physiological oropharyngeal microbiota (32.6%). We have observed that there is no statistically significant difference between the prevalence of certain bacteria species in each group , but, in some cases, there were only bacteria (2.3%) or only fungi (14.7%) . The occTo evaluate the effects of biofilm growth on the properties of the silicone surface, collected VPs and attached biofilms were subjected to studies using SEM and a laser confocal microscope (CLSM). Prostheses from all groups that were described based on exploration time  were invn = 10). Panels A and B show control sample surfaces with remnants from the manufacturing process. Panels C–F show the topography of the silicone surface after biofilm removal using sonification. The interaction between tissue, material, and the development of biofilm might account for all material damage. Confocal microscopy revealed the porosity (blue arrows) of the silicone surface (Panels C and D). Microcracks (yellow arrows) were also visible using AFM (Panels E and F).n = 10) after biofilm removal are shown in Changes in the mechanical and thermal properties of the representative VP from the fifth group . A physician, using sterile instruments, did the replacement procedure and collected the prostheses, which were placed into sterile containers. Directly after removal from the patients’ tracheoesophageal fistula, dysfunctional voice prostheses were subjected to microbiological evaluation. After the replacement procedure, patients answered questions about their medical history. Then, the information collected from the patients was compared with the data provided by the internal digital medical history system “CliniNet”. Correlations between the groups of collected prostheses and occurrences of certain fungi or bacteria species were calculated using the chi-square test of independence . Among all included patients, there were 116 males and 13 females. The male-to-female ratio was 9:1. The average age of patients was 66. The youngest patient was 47, and the oldest was 89. Patients underwent total laryngectomy from 18 years to 3 months (average 7.1 years) before the replacement procedure included in the study. Most of them (85%) had simultaneous implantation of voice prosthesis at the time of laryngectomy. The rest of them had undergone a secondary implantation procedure. In total, 88% of included patients had undergone radiation therapy: 73% as postoperative irradiation, and 15% as primary radical radiotherapy. The average number of voice prosthesis replacements per year in the studied patients after laryngectomy was 1.5 replacements (median 1.7), and the highest was 7 replacements. The average time between the previous replacement and the present replacement of voice prosthesis for a patient was 8.2 months (median time: 7 months).All patients and prostheses were categorized into five groups depending on the time of exploitation . The mosIn recruited patients, directly after the VP replacement procedure, failed VPs were placed in sterile specimen containers and immediately transported to a microbiological laboratory. The voice prostheses were immersed in 5 mL of thioglycolate broth  and vortexed for 2–3 min. Then, 50 µL of the eluted material was seeded onto solid media for yeast and bacteria growth. The solid culture media used for microbiological examination was as follows: Sabouraud agar with and without antibiotics, Columbia agar with sheep’s blood, MacConkey agar, and hemophilus selective agar . After a minimum of 48 h incubation, predominantly yeast and bacteria were identified using routine procedures that involved colony morphology examination and Gram staining. Then, identification of the species was performed using a Vitek 2 automated system (bioMerieux) with GNI, GPI, and Yeast ID cards.Macroscopic evaluation of the prostheses was performed within 5 h after removal. Biofilm morphology, coverage, and localization of plaques were assessed. Microscopic examination (topography) and mechanical measurements of the silicone prostheses and biofilm grown on their surfaces were recorded using a laser confocal microscope Olympus LEXT OLS4000 (CLSM), a scanning electron microscope Hitachi S-3000N (SEM), and an atomic force microscope JPK Instruments/Bruker NanoWizard 4 BioScience (AFM). SEM microscopy showed the biofilm characterization on the prosthesis surface. Images were made in BSE mode in a low vacuum. The CLSM microscope, in turn, made it possible to visualize the surface of the silicone after biofilm removal and observe their damage (3D topography maps). The topography of the biofilm on silicone surfaces was recorded using an AFM. Pyramidal-shaped cantilevers (OLYMPUS OMCL-RC800) were used and characterized by a spring constant of 0.1 N/m. Due to the lateral forces during contact mode scanning, a force-curve-based imaging mode was used (JPK QI™ mode—Quantitative Imaging). Topography maps of size 50 × 50 μm and 20 × 20 μm were carried out, with the resolution of 128 pixels per line, to show characteristics of the prosthesis surface and biofilm morphology. AFM experiments were done within 1 h of sample preparation, and silicone millimeter-scale pieces with biofilm were stored and kept in distilled water during experiments at room temperature. AFM ran in force-spectroscopy mode also served as a nanodetector for biofilm and polymer stiffness measurements. Elastic modulus  of the biofilm and polymers was calculated based on force-indentation curves. For biofilm stiffness measurements, force-indentation curves were collected on a stiff substrate and on the elastic biofilm parts. Due to the biofilm’s high deformability and complexity, force-indentation curves were collected using a silicone nitride cantilever with a spring constant of 2.5 N/m and a 20-μm diameter bead attached to the end. Elasticity maps with a scan area of 50 × 50 μm, corresponding to a grid of 2 × 2 pixels, were made in various places on the sample. By separation of the force curves recorded on the silicone surface and on the biofilm structures, we obtained the properties of the biofilm grown on prostheses. Local mechanical properties (Young’s modulus) of new and used prostheses were also calculated based on the force-indentation curves collected using OLYMPUS OMCL-RC800 cantilevers characterized by a spring constant of 0.4 N/m. Elasticity maps with a scan area of 20 × 20 μm, corresponding to a grid of 10 × 10 pixels, were made. All elasticity maps were collected from various sample areas in liquid conditions. Young’s moduli of biofilm and silicone were derived from the Hertz–Sneddon model applied to force-indentation curves  using thIf material ,49 and aDifferential scanning calorimetry (DSC) studies were carried out using a DSC Discovery apparatus . The measurements were conducted in three cycles (heat–cool–heat) in a temperature range from −90 to 350 °C, with a heating rate of 10 °C/min and a cooling rate of 5 °C/min. The results discussed in the work were taken from the second heating curves . The obtained DSC curves were used to analyze melting temperature (Tm) and fusion enthalpy (∆Hm). The degree of the silicone’s crystallinity (Xc) was determined [TDS; taken as 1% of the weight loss of the sample). The Kissinger method was used to determine the activation energy of the thermal decomposition of the tested materials. This method is based on the dependence of the temperature value Thermogravimetry (TG) tests were carried out using a Q500 thermogravimetric analyzer  in a temperature range from 30 to 1000 °C and a nitrogen atmosphere, with heating rate . p-values < 0.05 were considered to be statistically significant. Results are expressed as the average from all force curves for each group. Overall average values of Young’s modulus and thermal properties are presented as mean ± SD, where mean is the average value and SD is standard deviation. Statistical significance was determined using a two-tailed Student’s 2 independence test in the column “p-value” of p test result is higher than the chosen significance level of α = 0.05, there is no reason to reject H0. Therefore, it can be concluded that the occurrence of a given species of fungus and bacteria is independent of the group of patients. For fungi and bacteria species, p > α, so we did not observe statistical significance.The lack of relationship between fungal or bacterial species occurrence and VP lifetime is presented as a result of the χCandida krusei, Candida albicans, Candida glabrata, and Candida tropicalis. The most common bacterial species was Staphylococcus aureus. We have observed that there is no statistically significant difference between the occurrence of certain fungal or bacterial species and the time of VP function upon implantation. No patients involved in this study developed any systemic infection that could be associated with microorganisms residing in VP biofilm. New polymeric material that is developed to produce VPs should be improved to prevent deformation and cracking during exploitation.The formation of biofilms and the subsequent material deterioration by fungal and bacterial growth are the main reasons for VP failure in laryngectomized patients. Changes in the mechanical and thermal properties of silicone by the process of biofilm formation were also observed. The most common fungal species on the VPs were


Whole-genome microarray analysis in small intestinal samples of 1-d old piglets (n = 6 to 8 per treatment) showed significantly expressed genes (Padj < 0.05) which were involved in processes of tight junction formation and immunoglobulin production. Furthermore, when performing morphometry analysis, the number of goblet cells in jejunum was significantly (P < 0.001) lower in piglets from amoxicillin administered sows compared with the respective control piglets. Both significantly expressed genes (Padj < 0.05) and significant morphometry data (jejunum P < 0.05 and ileum P < 0.01) indicate that the crypts of piglets from amoxicillin administered sows deepen around weaning (day 26) as an effect of the amoxicillin administration in sows. The latter might imply that the intestinal development of piglets was delayed by maternal antibiotic administration. Taken together, these results show that maternally oral antibiotic administration changes in early life can affect intestinal development of the offspring piglets for a period of at least 5 wk after the maternal antibiotic administration was finished. These results show that modulation of the neonatal intestine is possible by maternal interventions.The objective of this study is to investigate the effect of a maternal antibiotic administration during the last week of gestation on the early life intestinal development in neonatal piglets. Colonization of the gut with bacteria starts during birth and plays a major role in the intestinal and immunological development of the intestine. We demonstrate that maternal interventions induced changes in the sows ( Maturation of the gastrointestinal tract of piglets is a multifactorial process that is important for the health of piglets in production systems . Here, wIt was shown previously that a single antibiotic treatment in piglets directly after birth led to significant changes in microbiome composition of piglets that persisted over time , 2015. AThe established principles of laboratory animal use, the European Union, and Dutch laws related to animal experiments were adhered to in this study. The Dutch Commission for Animal Studies approved the study under 2012.III.05.041. The animal experiment was approved by the Ethical Committee of Utrecht University (the Netherlands), in accordance with the Dutch law on animal experiments. Animal suffering was not expected within this particular experiment due to the nature of the intervention (amoxicillin treatment).n = 6 to 8 per group). Both treatment groups received standard gestation and lactation feed (Thirty-one Hypor gilts and sows (and their offspring) divided over three different batches were allotted to one of two treatments based on BW, backfat thickness and parity 4 wk before farrowing (day 87 of gestation) , all theion feed , from 1 n = 6 to 8) was selected, based on the average BW of the piglets and in good health and euthanized by intravenous administration of Euthasol (24 mg/kg BW) and subsequent exsanguination  to collect intestinal digesta and intestinal tissue samples. Intestinal digesta was collected from jejunum to analyze the microbiota. Intestinal scrapings were collected from ileum and jejunum for gene expression analysis. Sections of intestinal tissue from jejunum and ileum were spread on cork and fixed in formalin for histological morphometric analysis. An overview of piglet samples included in downstream analyses is shown in Piglets were weighed immediately after birth, at 24 h (20 to 25 h), 7 d, at weaning (day 26) and 4 wk after weaning (day 54). On days 1 and 7, at weaning, 4 d after weaning (day 30) and 4 wk after weaning (day 54) one apparently healthy male piglet from each sow . Mechanical shearing was carried out on the pellets containing the bacterial load in Lysing Matrix B tubes using the FastPrep-24  following manufactures protocol, i.e., three times 30 s at a speed of 30 Hz. Thereafter, DNA was extracted using the “QIAamp DNA stool minikit”  according to manufacturers’ instructions. Both, quality and quantity of DNA were checked using the NANOdrop . For bacterial amplicon library preparation, PCR was performed to amplify (20 cycles) the 16S rRNA gene hypervariable region V3 fragment using forward primer V3_F (CCTACGGGAGGCAGCAG) and reverse primer V3_R (ATTACCGCGGCTGCTGG)  . The amp package .TruncLength = 140,100 and minOverlap = 10), and reads were classified with the SILVA v.132 classifier (phyloseq (version 1.28.0) (vegan (version 2.5 to 6)  to allow diversity comparisons and the final dataset contained 5,966 taxa.The phyloseq object creation and statistical analyses were performed in R 3.5.1 . Brieflyassifier . The sta 1.28.0)  and vega.5 to 6)  packagesy = time-effect × treatment-effect + residual errors with Bonferroni post hoc testing.To determine the (semi) quantitative level of microbial load per sample (1 mL), qPCR was performed. Briefly, qPCR was performed using 1:10,000 dilution of isolated DNA from the intestinal samples using the 16S rRNA gene-derived primers as described above and Powr Sybr Green PCR Master mix (Applied Biosystems) with the following PCR conditions: 10 min at 95 °C, 40× , followed by a dissociation curve using the ABI7500 (Applied Biosystems). To allow standardization of the amount of 16S rRNA and detect potential amplification inhibiting factors, an internal control is added to each reaction, to determine the PCR efficiency of each sample. This internal control, 1 ng of an internal control of nonrelated DNA (luciferase gene), was spiked to the samples and amplified in a separate qPCR using primers JR331 CCTGAAGTTCATCTGCACCA and JR328 CTTGTAGTTGCCGTCGTCCT with the following PCR conditions: 10 m at 95 °C, 40× , followed by a dissociation curve using the ABI7500 (Applied Biosystems). The (semi) absolute level of microbial load per sample was calculated by dividing the amount of 16S-amplicon signal by the amount of internal control (Green Fluorescent Protein). Data analysis was done using Graphpad software Prism (version 8.2.1) with a two-way ANOVA following g. Three hundred and fifty microliters of supernatant were used to isolate RNA using the Direct-zol kit  according to instructions of the manufacturer. Quality control in terms of quality and quantity of the isolated RNA from each sample was performed on the BioAnalyser (Agilent Technologies) and Nanodrop (Agilent Technologies). Labeling (Cyanine-3), hybridization, scanning, and feature extraction of microarrays was performed as described before (LIMMA) and arrayQualityMetrics  was used to perform Functional Annotation Clustering (FAC) for the different contrasts. The up- and downregulated genes, here adjusted P-value < 0.05 and log fold change below −1.0 or above 1.0, were separately analyzed. The fold change threshold was more lenient here, because we wanted to start with a higher number of (annotated) genes in order to have a sufficient number of genes to investigate the affected biological processes.Total RNA was extracted from 50 to 100 mg jejunum tissue scrapings. Samples  were homogenized using a TissuePrep Homogenizer in 5 ml TRIzol reagent . The homogenate was centrifuged for 5 min at 21,000 × d before , porcineyMetrics . The datyMetrics  from BioyMetrics . QuantilIntestinal tissues samples were collected in formalin. Formalin-fixed samples were embedded in paraffin, sectioned at a 5-μm thickness, and stained with hematoxylin and eosin for histologic examination. The villus length and crypt depth were determined. These morphometric measurements were performed by using the image analysis software Image Pro Plus 7 . Each measurement of villus crypt ratio was determined in triplicate, on three representative locations per slide, the average was included for analysis.PCNA) staining, a cell proliferation marker expressed by cells throughout the S to M phases of the cell cycle . There was a significant impact of antibiotic treatment on the Shannon diversity of the fecal microbiota of sows on day 1, but not on day −7 or day 7 or the vaginal microbiota at partus  of Bray–Curtis dissimilarities indicated overlap between maternal vaginal microbiota and piglet jejunal microbiota on day 1 , starting immediately after birth (day 1) where 62 genes were significantly differentially expressed until 4 wk postweaning (day 56) where 223 genes were differentially expressed. The highest number of differentially expressed genes was found at weaning (n = 243), whereas no significant differences in gene expression were found on day 7. Principal component analysis showed that besides treatment, time was also a strong parameter determining gene expression patterns for both groups  , 4 d postweaning (day 30), and 4 wk postweaning (day 56). Large numbers of genes were affected in expression level  .There were no significant differences in villus length nor in mucosa length, indicating that the absorptive capacity was comparable between treatments. There was a significant difference in crypt depth between the treatment groups, where piglets from amoxicillin treated sows showed deeper crypts . In the To determine whether the observed differences in development are also reflected by differences in proliferation, PCNA staining was used as a proliferation marker. However, no differences in proliferation of intestinal cells were found between the treatment groups. Numbers of goblet cells present in the jejunal mucosa significantly differed on day 1 . PigletsOur study shows that maternal antibiotic treatment during the last week of gestation affects intestinal development in offspring for a period of at least 7 wk. Significant effects of the maternal antibiotic treatment on the microbial colonization and development of the gut in the offspring animals were observed although the maternal antibiotic treatment did not affect any of the measured performance parameters of the piglets. The strongest effects on intestinal development, as measured by gene expression profiles, were identified close to birth (day 1) and around weaning (day 26). Since those are important transition moments, discussion of the results was focused on these time points.Different bacterial populations were changed in the vaginal microbiota at birth (day 0) and this population was comparable with (sow) fecal microbiota at day 1. On the first day of life, jejunal microbiota is very diverse, probably reflecting the early colonizers of the naïve intestine. Although little is known about transmission routes of colonizing organisms, it is very likely that piglets are colonized from maternal microbiota either via the vagina or ingestion of sow feces or colostrum . TherefoIn summary, upregulation of genes associated with “tight junctions” and “immunoglobulins” immediately after birth will probably lead to intestinal epithelium that is more rapidly closed in piglets from amoxicillin-treated sows, whereas they have lesser numbers of goblet cells able to produce a mucin barrier. Thus antibiotic treatment may negatively affect the development of the first line of defense in neonates from amoxicillin-treated sows. A recent publication has demonstrated that transient maternal colonization during gestation also has strong impact on the intestinal development of the immune system of the offspring . This maAt the second transition moment, i.e., weaning, it is known that the microbiota adapts to the transfer from liquid milk-based diets to solid feeds with vegetable protein and energy sources . IntestiMaternal antibiotic use during (late) gestation, as proof of concept for a modulation, changes the diversity in sow fecal microbiota and also affects the intestinal development of piglets in early life, however, without significant change to the piglets microbiota. This implies that it is possible to modulate the intestinal development of piglets by maternal interventions, although the impact on health of this modulation is not known. Therefore, it may be possible to steer the intestinal development of offspring using maternal (feed) interventions.skaa181_suppl_Supplementary_Table_S1Click here for additional data file.


To evaluate the analgesic effect of vertebral cancellous bone infiltration anaesthesia during percutaneous vertebroplasty (PVP).Patients treated with vertebral cancellous bone infiltration anaesthesia (intervention group) or local anaesthesia alone (control group) during PVP at our institution during 2016–2018 were reviewed. The visual analogue scale (VAS) score before the operation, during establishment of the puncture channel, during pressure changes in the vertebral body , during bone cement injection, immediately after the operation, and at 2 h and 1 day postoperatively were compared between the groups. The patient’s satisfaction with the operation was recorded and compared between groups.P > 0.05). The VAS score in the intervention group was significantly lower than that in the control group during pressure changes in the vertebral body  and bone cement injection (P < 0.05). Immediately after the operation and at 2 h postoperatively, the pain in the intervention group was also significantly lower than that in the control group (P < 0.05), but there was no significant difference between the groups at 1 day postoperatively (P > 0.05). The patient satisfaction rate was 88% (52/59) in the intervention group and 67% (35/53) in the control group (P < 0.05).A total of 112 patients were enrolled (59 cases in the intervention group and 53 cases in the control group). There was no difference in the VAS score between the groups before the operation or during establishment of the intraoperative puncture channel (Vertebral cancellous bone infiltration anaesthesia may effectively relieve intraoperative pain and improve the surgical experience of patients without affecting the clinical effect of surgery. Percutaneous vertebroplasty (PVP) has been widely used in the treatment of osteoporotic vertebral compression fractures (OVCFs); PVP can quickly relieve pain, allow early ambulation, and improve the quality of life of patients –4. GenerA series of patients who underwent PVP for a single vertebral body between June 2016 and June 2018 in our hospital were retrospectively evaluated. The inclusion criteria were as follows: (1) a single vertebral body compression fracture; (2) vertebral cancellous bone infiltration anaesthesia or local infiltration anaesthesia; (3) bone mineral density (BMD) less than − 2.5; (4) PVP with a bipedicular approach; and (5) fracture without nerve or spinal cord compression symptoms. The exclusion criteria were as follows: (1) malignancy or angioma; (2) nonpainful OVCF; (3) burst fracture of vertebral body; and (4) multisegment vertebral fracture.The patients who were finally included were divided into two groups according to the anaesthesia method. The intervention group consisted of those who underwent vertebral cancellous bone infiltration anaesthesia, and the control group consisted of those who underwent local infiltration anaesthesia alone.All patients were placed in the prone position, and the pelvis and chest were elevated with a soft pillow to suspend the abdomen. The injured vertebral body was located using C-arm fluoroscopy, and the projection of the bilateral pedicles on the body surface were marked using a marker pen. Local infiltration anaesthesia with 1% lidocaine (approximately 8 ml on both sides) from shallow to deep was performed until the bone surface of the puncture area was reached. A sharp scalpel was used to cut the skin (approximately 5 mm) after successful administration of local anaesthesia. Then, the puncture needles  or 3.5 mm (lumbar vertebrae)) were inserted and pushed forward to enter the vertebral body (anterior pedicles) through points A, B and C . After rThe patient’s information, including age, weight, height, BMD (T-score), days of hospitalization, operative duration, intraoperative blood loss volume, and injected cement volume, were collected.The visual analogue scale (VAS) score was recorded as the observation index before the operation, during establishment of the intraoperative puncture channels, during pressure changes in the vertebral body , during bone cement injection, immediately after the operation, and at 2 h and 1 day postoperatively. The VAS score was repeatedly evaluated during pressure changes in the vertebral body and bone cement injection, and the highest score was finally retained. The patient’s satisfaction with the operation was reflected by whether they were willing to accept treatment with PVP again.t test. Count data were compared using the chi-square test. The test level was α = 0.05.Statistical analysis was performed using the SPSS 24.0 software . Continuous data are expressed as the means ± standard deviation, and comparisons between the two groups were performed using the independent samples 7 to L4 in the two groups. The age, weight, height, BMD, days of hospitalization, intraoperative blood loss volume, and injected cement volume were not significantly different between the groups (P > 0.05) . There was no significant difference in the VAS score between the groups during establishment of the intraoperative puncture channels (P = 0.762). During pressure changes in the vertebral body, especially when inserting or removing pushers or needle cores, the VAS score in the intervention group was significantly lower than that in the control group (P = 0.000). The VAS score in the intervention group was also lower than that in the control group during bone cement injection (P = 0.000), immediately after surgery (P = 0.045), and at 2 h postoperatively (P = 0.017). The pain in both groups was relieved at 1 day postoperatively compared with that before surgery, and the difference was not statistically significant between groups (P = 0.171) (Table P < 0.05).The operative duration in the intervention group was longer than that in the control group  microfracture of vertebral trabecular bone stimulates peripheral nerves, causing pain , and (2)Studies on venous angiography of the vertebral body have confirmed that the drainage vein develops slowly, and some of the vertebral body veins do not develop; therefore, after the injection of a nonionic contrast agent with high water solubility and low permeability into the vertebral body via the puncture needle, the contrast agent remains in the vertebral body for a long period of time . Thus, tAccurate puncture is essential for vertebral cancellous bone infiltration anaesthesia. An excessive puncture angle may pierce the medial wall of the pedicle such that the anaesthetic may enter the spinal canal through the damaged area, resulting in epidural anaesthesia, which brings unnecessary risk. Wang Song et al.  reportedIn summary, vertebral cancellous bone infiltration anaesthesia may effectively relieve intraoperative pain and improve the surgical experience of patients while not affecting clinical outcomes.


Pantoea ananatis, can become deleterious to crops, leading to leaf blotches, die-back, bulb rot, and fruit rot. It is unknown whether honeybees sense the bacteria in the nectar of flowers. We aimed to determine if bees can perceive these bacteria in sugar solutions and if they are deterred by them. Our results indicate that honeybees can perceive these plant bacteria only in high concentrations, which deters them from drinking the respective sugar solution. They may therefore spread P. ananatis bacteria between flowers in field-realistic densities during foraging.Honeybees are important pollinators, and may contribute to the spread of plant bacteria during their foraging trips. Some of these bacteria, such as Pantoea ananatis, an emerging pathogen in some systems. P. ananatis infections can lead to leaf blotches, die-back, bulb rot, and fruit rot. 2. We isolated P. ananatis bacteria from flowers with the aim of determining whether honeybees can sense these bacteria and if the bacteria affect behavioral responses of the bees to sugar solutions. 3. Honeybees decreased their responsiveness to different sugar solutions when these contained high concentrations of P. ananatis but were not deterred by solutions from which bacteria had been removed. This suggests that their reduced responsiveness was due to the taste of bacteria and not to the depletion of sugar in the solution or bacteria metabolites. Intriguingly, the bees appeared not to taste ecologically relevant low concentrations of bacteria. 4. Synthesis and applications. Our data suggest that honeybees may introduce P.ananatis bacteria into nectar in field-realistic densities during foraging trips and may thus affect nectar quality and plant fitness. 1. Honeybees, which are among the most important pollinators globally, do not only collect pollen and nectar during foraging but may also disperse diverse microbes. Some of these can be deleterious to agricultural crops and forest trees, such as the bacterium  Apis mellifera) are important pollinators globally. About 75% of major field crops, fruits, and vegetables produced for human consumption require insect pollination [Erwinia amylovora [Pantoea ananatis, a gram-negative facultatively anaerobic bacterium which was discovered in pineapple in 1928 [Pantoea ananatis by Trüper and De’Clari [P. ananatis has induced plant diseases in 11 countries [P. ananatis can induce several disease symptoms including leaf blotches and spots [Many insects such as honeybees  and incubated overnight at 37 °C on an orbital shaker. Afterwards, optical density at 600 nm (OD600) was determined three times. The mean of the three measurements was used for calculating the number of colony-forming units. Means of all solutions were about OD600 ~ 1.0 . After pelleting the bacteria  they were re-suspended in 0.5 Vol 60% (w/v) sugar solution  and filled with 0.5 Vol 60% (v/v) glycerol. Thus, the concentration of bacteria was 125 times higher than in the growth media with OD600 ~ 1.0 and the concentration of sugar was adjusted to 30% (w/v). The glycerol stocks were stored at −20 °C and were used to prepare the test solutions (see below).We obtained the strain 26SR6 of glycerol . To prodReturning honeybee foragers from two colonies with naturally mated queens were captured individually at the hive entrance in the morning and classified as pollen foragers (filled pollen baskets) or nectar foragers (without filled pollen baskets). The latter group may have comprised water collectors, which cannot be differentiated from nectar foragers by sight, but their number is usually very low (max. 5% ) and negP. ananatis in a sugar solution affect the proboscis extension responses (PERs) compared to pure sugar solution. We studied responses to antennal stimulation with 30% pure sucrose, fructose, and glucose solutions, and the same solutions containing different concentrations of P. ananatis. For these experiments we produced five 10-fold dilutions (D1–D5) with 30% (w/v) sugar solution  based on the glycerol stocks described above [600 ~ 1.0 of Escheria coli bacteria that contained approximately 8 × 108 colony-forming units (cfu)/µl [7 cfu/µl, dilution 2 (D2): 106 cfu/µl, dilution 3 (D3): 105 cfu/µl, dilution 4 (D4): 104 cfu/µl, dilution 5 (D5): 103 cfu/µl. To aid interpretation, we used these cfu/µl values in the figures and in E. coli and P. ananatis.In this experiment we aimed to determine if different concentrations of ed above , with D1(cfu)/µl . CorrespP. ananatis. Only honeybees showing 100% responsiveness to the pure sugar solutions with PER were selected for this experiment. The comparatively large number of individual tests could not affect satiation levels, because bees were only stimulated at their antennae and were not allowed to feed on the sugar solutions [P. ananatis. At each stimulation of the antennae with sugar solution, we recorded the occurrence of the proboscis extension response (PER). Tests were performed in the following order: 30% sugar, D5, 30% sugar, D4, 30% sugar, D3, 30% sugar, D2, 30% sugar, D1. The intertrial interval between the pure sugar solution and the sugar solution containing a certain concentration of P. ananatis bacteria was 2 min to avoid intrinsic sensitization [The solutions were made fresh on each experimental day and cooled on ice. To test if honeybee foragers are capable of perceiving the bacteria in a less attractive, lower concentration of sugar solution, we stimulated the antennae of the bees either with 3% sucrose solution or with the same solution containing different concentrations of olutions . Pollen g for 5 min. This short and mild centrifugation step reduces the bacteria in the supernatant dramatically without destroying them. Centrifugation at 10,000× g was shown to reduce bacteria from the solution up to 99% [g have been shown to cause bacterial damage [P. ananatis-supernatant in 30% sucrose.In the following experiment, we aimed to determine if the reduced responsiveness to a highly concentrated sugar solution contaminated with a high concentration of bacteria resulted from the deterrence due to the taste of the bacteria or if the presence of bacteria caused changes in the properties of the solution. For instance, the solution could include the secretion of soluble metabolites or the reduction of the sugar concentration. For this experiment, we selected a 30% sucrose solution because this normally yields a high response rate of the honeybees ,36. We dp to 99% , but forl damage , which wP. ananatis) solution to her proboscis and noted whether the bee drank this solution. When the bee drank the entire 3 µl we recorded a positive response. If the bee only touched the sugar water droplet or did not drink the entire droplet, we recorded a negative response.To quantify the responsiveness of pollen foraging bees and nectar foraging bees at the proboscis, which is usually less sensitive than the antenna , we stimTo determine the extent to which honeybees are capable of sensing a certain concentration of bacteria in increasing sucrose concentrations ranging from very low (0.1%) to high 30%) with their antennae, we produced increasing sucrose concentrations with and without bacteria. We first tested responses of all nectar and pollen foragers to stimulation of the antennae with the following sucrose concentrations applied in ascending order: 0.1%; 0.3%; 1%; 3%; 10%; 30% % with th,36,39. Tr = proportion of honeybees displaying PER at a given density of bacteria in the solution and d = density of bacteria, describing binary responses and reflecting the logit link functions used for the GLMs [a and b and set r = 0.5  to calculate the density of bacteria d that is required to prevent 50% of honeybees from consuming the solution. All of these statistical analyses were performed with the statistical computing software R (R version 3.6.2 (2019-12-12)—“Dark and Stormy Night”, R Core Team 2018). The gustatory response scores were compared between foragers tested with pure sucrose solutions and those tested with increasing sucrose concentrations containing P. ananatis using Mann–Whitney U tests because these scores did not follow normal distribution .The experiments on responsiveness resulted in binary data in which the honeybees showed a proboscis extension response PER (1) or not (0). To test whether an increasing density of bacteria is associated with a reduced probability of honeybees to show a PER, we used a generalized linear models (GLM) with binomial error distribution and logit link function. We used the response probability (0 or 1) of the bees as the response variable and the assumed density of bacteria (cfu/µl in dilutions D1–D5), the honeybees’ task (nectar or pollen forager), and the interaction between density and task (density × task) as explanatory variables. This full model was reduced in a stepwise manner and finally compared to a neutral model containing no explanatory variables using an analysis of deviance . If the the GLMs . For eacP. ananatis. All solutions were applied to the antennal tips. By a significant margin, most bees showed PER at low concentrations of P. ananatis in a 30% sucrose solution  and low (3%) sugar concentrations that were inoculated with different concentrations of solution , in a 30solution , in a 30solution , or in asolution  . Howevesolution . This efsolution  to a simP. ananatis.To determine if the deterring effect for honeybees results from the bacteria themselves, the bacteria’s secreted soluble metabolites, or the reduced sugar concentration of the solution, we removed the bacteria from the solutions and tested the responsiveness of the bees to antennal stimulation with that solution. When bacteria were removed from the sugar solution, honeybees responded similar to the case of the pure sucrose solution . NeitherF1,14 ≤ 2.76; p ≥ 0.119).As an additional measure of the effect of the bacteria on the responsiveness of the bees we calculated the density of bacteria that deterred 50% of the bees from responding. We found a broad range of bacterial densities that are required to prevent 50% of the bees from showing PER . IntriguP. ananatis bacteria , suggesting that both groups of foragers did not sense the bacteria with their proboscis . However, pollen foragers were more likely to drink sucrose solutions contaminated with high densities of P. ananatis than nectar foragers  indicate that both pollen and nectar foragers did not discriminate significantly between pure sucrose solutions and solutions containing a low concentration of bacteria .In this experiment, we analyzed responsiveness of pollen and nectar foragers to increasing concentrations of sucrose that contained Pantoea ananatis is a frequent bacterium on flowers and has become a pest for some crops [me crops ,15,16,17me crops . Honeybeme crops .Bombus terrestris), for example, was shown to perceive bacteria in floral nectar and to display a reduced feeding preference for higher concentrations of these bacteria in nectar, whereas low concentrations were generally tolerated [Arion vulgaris slugs, for example, refused lettuce leaves when they were inoculated with bacteria [In this study we aimed to determine if honeybees perceive floral bacteria with their main taste organs, i.e., their antennae and proboscis, and if they avoid sugar solutions contaminated with bacteria. The bumble bee  concentrations of these microorganisms when they were applied to their antennae, and displayed a dose-dependent reduction in proboscis extension responses.Our experiments clearly show that honeybees are capable of perceiving P. ananatis bacteria (D4), however, the bees did not discriminate between pure sugar solutions and solutions containing bacteria. These data suggest that honeybees are not deterred by low concentrations of P. ananatis bacteria in nectar and may thus spread the bacteria in low and field-realistic concentrations. Sugar solutions in higher concentrations than usually found in nectar may prevent the growth of bacteria [When we stimulated the antennae of the bees with increasing sucrose concentrations containing a comparatively low concentration of bacteria , suggestP. ananatis bacteria, bees did not avoid these solutions. This points to a lower sensitivity to bacteria at the proboscis, as has been shown for sugar [Intriguingly, when we stimulated their probosces with sugar water containing a high (D1) concentration of or sugar  and whicor sugar . This siThe reduced probability of responding to the sugar solutions was not an effect of the duration of the experiments such that honeybees reduced the responses towards the sugar solution due to fatigue. This was evident because they readily responded to nearly all of the sterile sugar solutions that were offered after each sugar solution containing bacteria until the end of the experiment .4, [Little is known about naturally occurring densities of bacteria in nectar and pollen. Some of the bacterial densities that we used in our experiments likely exceeded those reported for nectar . Howeve4, , however4, , indicatP. ananatis bacteria in sugar solutions with their antennae, which inhibit their proboscis extension response. Bees are therefore unlikely to drink from these solutions. Thus, a high infestation with bacteria, such as that occurring during biological pest control, might ultimately result in reduced pollination, whereas an ecologically relevant low concentration of bacteria is unlikely to be perceived by the bees’ antennae and will not affect their behavior. The transfer of low concentrations of bacteria by honeybees from flower to flower may ultimately change the bacterial community in the floral nectar and affect the composition of nectar by changing the sucrose–fructose balance, and the pH value of the nectar in the long run [Our data suggest that honeybees are capable of perceiving high concentrations of long run ,47. ThesHow the bees perceive the bacteria remains an open question because it is unclear which kind of receptors of the honeybee are stimulated by bacteria and how this is represented at the neuronal level. It is rather surprising that the honeybee can perceive bacteria at all, given their small repertoire of gustatory receptors. Because most of the 10 gustatory receptors of the honeybee still await their functional characterization , more ti


A recent analysis of the Mediterranean Sea surface temperature showed significant annual warming. Since small picoplankton microorganisms play an important role in all major biogeochemical cycles, fluxes and processes occurring in marine systems (the changes at the base of the food web) as a response to human-induced temperature increase, could be amplified through the trophic chains and could also significantly affect different aspects of the structure and functioning of marine ecosystems. In this study, manipulative laboratory growth/grazing experiments were performed under in situ simulated conditions to study the structural and functional changes within the microbial food web after a 3 °C increase in temperature. The results show that a rise in temperature affects the changes in: (1) the growth and grazing rates of picoplankton, (2) their growth efficiency, (3) carrying capacities, (4) sensitivity of their production and grazing mortality to temperature, (5) satisfying protistan grazer carbon demands, (6) their preference in the selection of prey, (7) predator niche breadth and their overlap, (8) apparent uptake rates of nutrients, and (9) carbon biomass flow through the microbial food web. Furthermore, temperature affects the autotrophic and heterotrophic components of picoplankton in different ways. Prochlorococcus (PROC), Synechococcus (SYN), and pico-eukaryotic algae (PE)) represent the major components of the marine picoplankton (PICO) community [Marine microorganisms are an integral part of all major biogeochemical cycles, fluxes, and processes occurring in marine systems. Heterotrophic picoplankton  and autotrophic picoplankton , nutrient availability, grazing, and viral lysis ,12,13. I−1 west of the Strait of Gibraltar to 0.51 °C decade−1 over the Black Sea) [−1 has been recorded since 2008 [Global and atmospheric climate change is altering the thermal conditions in the Adriatic Sea and, consequently, the marine ecosystem. The semi-enclosed nature of the Mediterranean Sea (including the Adriatic Sea) combined with reduced inertia, due to the relatively short residence time of its water masses, makes it highly reactive to external forces, in particular global warming . A recenack Sea) . Sea surnce 2008 . Global nce 2008 . There ince 2008  and channce 2008 ,36. SincProchlorococcus (PROC) and Synechococcus (SYN) as well as pico-eukaryotic algae (PE); and (iii) protistan grazers, namely, heterotrophic nanoflagellates (HNF) and ciliates (CIL). The experiments were performed in April 2019 when water-column mixing transports nutrients to the surface layer. Previous results obtained in the Adriatic Sea [In this study, laboratory growth/grazing experiments were performed under in situ simulated conditions in order to study the structural and functional changes within the MFW after a 3 °C rise in temperature. The study involved: (i) HPP, which include two physiological groups of heterotrophic bacteria (HB) distinguished by flow cytometry, namely, high nucleic-acid bacteria (HNA) and low nucleic-acid bacteria (LNA); (ii) autotrophic picoplankton (APP) that includes two cyanobacteria (CB) groups, namely, atic Sea  confirmeatic Sea . TherefoAccordingly, the main aim of this study was to investigate the possible direction of changes in the MFW, in the global warming scenario. Specifically, how a 3 °C rise in temperature affects changes in: (1) the growth and grazing rates of the most important groups of picoplankton and their carbon biomass flow through the MFW; (2) the relationship between HNF and CIL predators in terms of their food niche breadth and their overlap, satisfying their carbon and their preference in the selection of prey; (3) apparent uptake rates of nutrients.We hypothesized that the rise in temperature would affect the qualitative and/or quantitative changes of the majority of these parameters and that temperature would affect the autotrophic and heterotrophic components of picoplankton in different ways. The results could help to gain a better understanding of the possible changes in carbon transfer and energy flow under warming conditions, as well as to detect possible consequences for marine biogeochemical cycles.All abbreviations used in this paper are in the table of Seawater was collected in April 2019 from Kaštela Bay  in order to set up a growth/grazing experiment that was performed in the laboratory under in situ simulated conditions. A total of 100 L of ambient seawater was collected from 1 m depth, transferred to the laboratory, and filtered immediately through 200 µm mesh to remove larger zooplankton predators. The experiment started two hours after seawater collection. The values of environmental parameters and ambient abundances  of the studied microbial groups are listed in the Growth and grazing parameters were estimated using the size-fraction technique ,40. A 203) and 0.25 μM phosphate (KH2PO4). This manipulation ensured the exponential growth of all study groups over a three day period, reaching plateau stages, which enabled the determination of the maximum reached abundances, i.e., the carrying capacities (K).Each size fraction of a final volume of 4 L was then transferred to 5 L incubation bottles (run in triplicate), previously cleaned with 10% HCl and rinsed with Mili-Q water. Initially, all bottles were supplied with 5 μM nitrate  and 3 °C above ambient temperature (17 °C) for 96 h. In the experiments performed at 3 °C above in situ temperature, microorganisms had been exposed to a gradual rise in temperature during the 15 h period (temperature was increased by 0.2 °C every hour). The thermostats were equipped with six daylight lamps  that were programmed to follow the day–night cycle (constant light intensity) for the day of the year. Irradiance value during experiment was 53 μmol mplankton ,42.Additionally, at both temperatures, one set of bottles with a 2 μm fraction was simultaneously incubated in the dark. During the incubation process, the bottles were shaken gently. Subsamples for all analyses were taken at the beginning of the experiments and after 24, 48, 72, and 96 h. Triplicate subsamples for cell count were poured into sterile acid-washed glass bottles, fixed immediately, and frozen (–80 °C) until analysis (within 24 h after sampling).−1) of each microorganism group were calculated during the period of their exponential growth for each incubation bottle [BN and EN represented the number of organisms at the beginning and at the end, respectively, of the period of exponential growth; and t is the duration of exponential growth in days.Net growth rates µ=(lnGrazing rates (g) were calculated as the difference between growth rates in a predator-free fraction and growth rates in the presence of predators (for more details see −1 day−1) and losses due to grazing  were estimated using the following equations:−1) at sampling time.Production rates  and prey production (PPR) removed daily by grazing was calculated according to James and Hall  and Safig is the grazing rate; and PREYN and PREDATORN are geometric mean concentrations of prey and predator, respectively, during the experiment.Number of ingested prey cells per predator cell was calculated as:Bacterial carbon respiration (BR) was estimated from the consumption of dissolved oxygen in Biological Oxygen Demand (BOD) bottles, assuming a respiratory quotient of 1. Seawater filtered through 0.8 µm polycarbonate filters  and bacterial carbon demand (BCD), bacterial respiration (BR) was measured in parallel with bacterial production (BP), which was estimated as a difference of bacterial biomass between the beginning and the end of the incubation time. Bacterial carbon demand was estimated as BCD = BP + BR, and bacterial growth efficiency as BGE (%) = (BP/BCD) × 100.HNF and ciliate gross growth efficiencies were calculated as:i is the proportion of prey item i in the diet; ei is the proportion of prey item i in the environment; and m is the number of prey items in the environment.To evaluate prey selection, the Manly–Chesson preference or selection index  was calculated ,47,48: to 1 (complete preference). If α = 1/m, the predator is feeding randomly, and the prey is consumed in proportion to its abundance in the environment; whereas α > 1/m indicates the preference and α < 1/m indicates the avoidance of prey consumption. BRR is studied biological reaction rate (P and G in this study); A is the theoretical BRR in the absence of aE; aE is activation energy (J mol−1); R is universal gas constant ; and T is temperature in Kelvin. Regression statistics for the Arrhenius plots are included in The sensitivity of microbial production (P) and grazing on microbial prey groups (G) to temperature is displayed on an Arrhenius plot , which describes the relationship between temperature and biological reaction rates:Some predators are more specialized than others and measures of niche breadth attempt to measure this quantitatively. Since the abundance of different microbial prey types differs significantly among the many ways of expressing niche breadth, we chose Smith’s  and Hurl3−, NO2−, NH4+, and PO43−) were analyzed using a Bran+Luebbe AutoAnalyser (II and III models) and applying standard colorimetric methods [Temperature and salinity were measured using CTD multiparameter probes (Idronaut and SeaBird) with >±0.01 °C and ±0.02 accuracy, respectively. Dissolved oxygen concentration was determined by Winkler titration . Nutrien methods . Daily a−1. Samples for autotrophic cells analysis (2 mL) were preserved in 0.5% glutaraldehyde, frozen at –80 °C, and stored until analysis. Samples for analysis of bacteria were preserved in 2% formaldehyde kept at 4 °C until analysis. Autotrophic cells were divided into three groups (two cyanobacteria (Synechococcus and Prochlorococcus) and picoeukaryotes), distinguished according to light scattering, cellular chlorophyll content, and phycoerythrin-rich cell signals [For cell counting, samples were analyzed using a Beckman Coulter CytoFLEX cytometer  per cell, according to Verity and Langdon [The biomass of studied PICO groups was calculated using the following cell-to-carbon conversion factors: 20 fgC cellbacteria ,60; 36 frococcus ; 255 fgChococcus ; 2590 fgic algae ; and 0.2 for HNF . The HNF HNF [−3 . In addi Langdon .Statistical operations were performed by STATISTICA 9.0 software. Data normality was assessed by applying the Shapiro–Wilk W normality test. Grazing preference  index, niche breadth measures, and niche overlap indices were calculated using Ecological Methodology Programs (version 7) by Charles J. Krebs.A review of growth and grazing parameters based on size-fraction experimental estimations is presented in Maximal growth rates (predator-free condition) were higher than total grazing rates for all studied microbial groups, showing positive net growth rates. After a 3 °C rise in temperature , a statistically significant increase in growth rate was established for almost all microbial groups, with the exception of HNA. The highest increase in growth rates were observed for two cyanobacteria groups (SYN by 276% and PROC by 49%).plateau stage of the experiment, also increased with a rise in temperature in all groups except HNA (the greatest increases were measured for SYN by 105% and HNF by 49%). At ambient temperature, growth efficiencies of heterotrophic bacteria  and HNF (48.6%) were higher than growth efficiency of CIL (22.5%). After a 3 °C rise in temperature, the growth efficiencies of HB, HNF, and CIL decreased to 22.7%, 40.1%, and 12.3%, respectively.Carrying capacity (K), estimated as the maximum abundance reached at the Total grazing rates  reveal a statistically significant decrease with increasing temperature for heterotrophic prokaryotes (HNA and LNA groups), and a statistically significant increase for autotrophic picoplankton (APP) groups  and HNF. As with the growth rates, the highest increase in grazing rates was also observed for two cyanobacteria groups (SYN by 98% and PROC by 94%). This pattern was followed by the HNF ingestion rate (expressed as carbon biomass consumed per HNF cell), evidenced as an increase in the consumption of APP  and as a decrease in consumption of heterotrophic prokaryotes (HNA and LNA), after a 3 °C rise in temperature. Likewise, the ingestion rate of ciliates showed an increase in consumption of PROC (by 91%) and HNF (by 31%), and a decrease in consumption of LNA (by 65%), SYN (by 41%), and PE (by 19%) after the rise in temperature.In warming conditions, relative standing stock removal (SSR%) decreased for heterotrophic prokaryotes (HNA and LNA) and increased for all APP groups , particularly due to HNF grazing . Relativa) derived from the regression slope of the Arrhenius plots  and PE (from 20% to 30%), whereas the contribution of PROC to total ingested biomass was negligible , mostly at the expense of the PE which decreased from 29% to 24% in terms of contribution in total ingested biomass Even more noticeable changes after a rise in temperature occurred in ciliate grazing pressure. The contribution of HNF and SYN in satisfying CIL carbon demand increased by 10% and 6%, respectively .The results of the normalized Manly–Chesson preference or selection index  showed that HNF consumed a higher proportion of HNA and a lower proportion of LNA and PROC than their proportion in the environment at ambient temperature 14 °C; A. After Ciliates also showed a strong preference for HNA, while the affinity for PE and HNF was of slightly less intensity at ambient temperature. After the experimental rise in temperature, the preference for SYN and HNF increased markedly, mostly at the expense of HNA, although they still preferred them. On the other hand, ciliates avoided PE completely B. A statConsidering PICO biomasses overall (HNA+LNA+PROC+SYN+PE), 45% of that biomass was consumed by HNF and as much as 55% by CIL, at ambient temperature. After the rise in temperature, these values changed to 49% and 51%, consumed by HNF and CIL, respectively. These shifts are also evident in the composition of the prey. In warm conditions, the contribution of HNF to total grazing increased for LNA and PE, and decreased for SYN, whereas CIL grazing pressure showed the opposite pattern A.−1) accounted for 50% of the total number of CIL at the beginning of the log phase. During the experiment, their share to CIL biomass increased to 72% and 87% at ambient temperature and the 3 °C elevated temperature, respectively  and the end of the experiment (end of log phase before reaching the lag phase) showed that the relative contribution of CIL to total grazing, increased for groups with smaller cells  per se, but on the relative changes of these parameters after a rise in temperature, as well as between the beginning and the end of the log phase of growth (in the case of niche overlap). The values of Smith’s and Hurlbert’s measure of niche breadth increased with the rise in temperature for both HNF and CIL predators . This poFurthermore, during the experiment, food-niche overlap between HNF and CIL increased, mostly because of change in the size structure of the CIL community towards smaller sized fractions. On the other hand, the rise in temperature reduced food-niche overlap between HNF and CIL, mostly because of the marked increase of HNF contribution to total biomass ingested by CIL . In gene3− and PO43− decreased, while the uptake of NH4+ increased , followed by LNA (17%), PE (16%), SYN (3%), and PROC (<1%). Such picoplankton biomass distribution is in accordance with HNA (within heterotrophic prokaryotes) and PE (within autotrophic picoplankton) dominance in coastal regions, particularly during a spring period with high supply of nutrients. On the other hand, LNA bacteria and CB (particularly PROC) preferred oligotrophic waters %, follow,66,67,68In general, after a 3 °C rise in temperature, the growth rate increased for all studied microbial groups with, however, quantitatively different responses depending on the group. Negative relationships with temperature were found for the HNA to LNA biomass ratio , PE to CB biomass ratio (from 6.9 to 4.7), and for the HPP to APP biomass ratio (from 4.5 to 2.5). A similar relationship between the PE to CB ratio and temperature has also been reported by Otero-Ferrer et al. , howevers with temperature are supported by the study of Huete-Stauffer et al. [A reverse relation of carrying capacity K with temperature in HNA (negative relationship) compared to LNA (positive relationship), as well as correlated changes of growth rate and Kr et al. . Higher r et al. , and thir et al. ,74.Within the HPP group, at ambient temperature, LNA showed a much lower growth rate compared to HNA. However, LNA were more sensitive to temperature rises, as also confirmed by the results of Morán et al. . StudiesCompared to HB (both HNA and LNA), CB (PROC and SYN) were much more sensitive to temperature (both production and grazing), suggesting that heterotrophic as well as autotrophic picoplankton components respond differently to temperature rises. Contrary to HB, where the impact of temperature was more pronounced at temperatures below 16 °C and levelled off at higher temperatures, this impact on APP  was linear along the entire range of investigated temperatures (from 10 °C to 26 °C) . ResponsThe difference in temperature sensitivity between HPP and APP  is in accordance with the idea that planktonic autotrophs and heterotrophs are characterized by different activation energies . MoreoveSensitivity of grazing and growth rates to temperature showed similar patterns, with the exception of extremely high sensitivity of grazing on HNF to temperature. However, the data analysis showed consistent higher sensitivity of grazing mortality than production to temperature in all groups except SYN. They showed a marked increase in production at the elevated temperature that was not accompanied by an equal increase in grazing pressure. Our results for HPP (HNA and LNA) are not in accordance with the study of Sarmento et al. , who fouA positive effect of temperature on prokaryotic grazing rates and consequently an increase in their biomass transfer to higher trophic levels has been reported ,88. Afte−1 biomass, such as Lohmanniella oviformis and Strombidium sp., and small tintinnids, such as as Stenosemella nivalis and Tintinnopsis nana, became dominant in the ciliate community. The preferred prey size of CIL is positively related to oral diameter and is estimated at about 20% of lorica diameter [The relative contribution of different types of prey to total PICO biomass ingested by predators was mainly related to prey growth rate rather than their abundance/biomass. Moreover, during the experiment, the community size structure of CIL changed in order to adapt better to the consumption of faster-growing picoplankton groups with smaller cells. This emphasizes that apart from abundances, it is also important to know the community structure of ciliates, e.g., microzooplankton assemblages . The taxdiameter . A shiftdiameter ,96. TherIn warming conditions, both HNF and CIL switched their preference to SYN, a PICO component that showed the highest increase in growth rate. In such temperature conditions, considerable increase in CIL preference for HNF was also established. This is consistent with the results of Stoecker and Capuzzo  who founObviously, trophic interactions between HNF and CIL in the marine environment are complex. Since, both plankton groups are sympatric predators of picoplankton prey, they are expected to be competitors for picoplankton prey . However3- and PO43- decreased, while the uptake of NH4+ increased. This could be explained primarily by APP consumption, accompanied by an increase in APP production and their relative contribution to total picoplankton biomass. Increased concentration of NH4+ may influence the preference of phytoplankton for NH4+ vs. NO3-, mainly favoring APP production [4+ regeneration during the experiment as a possible consequence of bacterial regeneration, viral lysis, and/or excretion of predators. Bacterial regeneration of NH4+ occurred in low DOC/DON conditions, which determine net bacterial excretion rather than NH4+ uptake [After the rise in temperature, the apparent uptake of NOoduction . This im+ uptake . Such co4+/NO3− ratio showed a very strong positive relationship with APP [4+ (66%) and urea (17%). A strong preference of APP for a reduced form of nitrogen (NH4+) [4+ as their source of nitrogen. Furthermore, Casey et al. [3- as their source of N, despite their genetic capability to utilize NO3− [3- concentration. Moreover, the ratio of prokaryotic to pico-eukaryotic photoautotrophic biomasses in the north–western Mediterranean Sea decreased with increasing NO3- supply [3− supply, whereas a unimodal function was shown for PE.Changes in the NHwith APP . Wafar ewith APP  found thn (NH4+) ,105 is pn (NH4+)  reportedy et al.  found thize NO3− . The stuize NO3−  indicate- supply , suggest- supply . Further- supply  reportedIn general, after an experimental temperature rise of 3 °C, the proportion of picoplankton biomass channeled through the microbial food web towards the higher trophic levels increased. Similar results were found by several other studies ,22,86. A


Homo sapiens retinoic acid receptor) peptide was designed from retinoic acid receptor responder protein 2 of Homo sapiens, and was conjugated with synthesized CQDs (carbon quantum dots) for enhanced antibacterial activity in combination, as individually they are not highly effective. The HSER–CQDs were characterized using spectrophotometer, HPLC coupled with electrospray-ionization quadrupole time-of-flight mass spectrometer (ESI–qTOF) mass spectrometer, zeta potential, zeta size, and FTIR. Thereafter, the antibacterial activity against Vancomycin-Resistant Staphylococcus aureus (VRSA) and Escherichia coli (carbapenem resistant) was studied using growth curve analysis, further supported by microscopic images showing the presence of cell debris and dead bacterial cells. The antibacterial mechanism of HSER–CQDs was observed to be via cell wall disruption and also interaction with gDNA (genomic DNA). Finally, toxicity test against normal human epithelial cells showed no toxicity, confirmed by microscopic analysis. Thus, the HSER–CQDs conjugate, having high stability and low toxicity with prominent antibacterial activity, can be used as a potential antibacterial agent.Antibiotic-resistant bacterial infections have become global issues for public health, which increases the utter need to develop alternatives to antibiotics. Here, the HSER ( The evolution of bacteria with every generation to acquire resistance towards new antibiotics have made it a challenge to treat pathogenic bacteria, as their pattern of resistance differs . Thus, bStaphylococcus aureus (VRSA) is at the highest threat level and the carbapenem-resistant Enterobacteriaceae (CRE) is in the critical level. The last resort for the gram positive and negative bacteria is vancomycin and carbapenem, but these are also becoming resistant, creating global epidemiological and clinical burden. The VRSA is resistant due to its ability to reject the entry of vancomycin into its own system and prevent its own altered autolysis, along with gene transcription alteration [The World Health Organization in the year 2017 listed the antibiotic-resistant bacteria in three different categories depending on the threat level priorities: critical, high, and medium . The Vanteration . Whereasteration . Antimicteration ,10. Thusteration ,11. ThisAntimicrobial peptides (AMPs) can be an interesting alternative to traditional antibiotics due to its natural occurrence in almost all species, their amphipathic nature, and their assist innate immunity. They have the ability to provide highly effective and non-specific defensive activity against invading pathogens ,13,14. FHomo sapiens retinoic acid receptor) is a 22-amino acid residue peptide derived from retinoic acid receptor responder protein 2 of Homo sapiens. Retinoic acid receptor responder protein 2 was known to exhibit defensive response against gram positive and negative bacteria [E. coli was seen to be poor. Furthermore, the carbon quantum dots (CQDs) were synthesized based on our previous studies [The formation of conjugates using peptides with nanomaterials or other chemicals is possible and provides a wide range to produce new particles with medical applications . HSER . Further, the fluorescence live/dead cell imaging and phase contrast microscope imaging against VRSA and E. coli was studied. Thereafter, the mechanism of the HSER–CQDs were studied. Finally, the toxicity tests like hemolytic assay and MTT assay were performed. Thus, the HSER–CQDs showed antibacterial activity that can be used as a better treatment strategy against multidrug-resistant bacterial infections and a potential substitute for antibiotics with medicinal values.The present experiment is mainly based on the molecular dynamics (MD) simulation of the designed HSER peptide to understand the structure of the peptide, which was accompanied by the synthesis and characterization of the HSER-CQDs conjugate. Thereafter, the antimicrobial activities of HSER–CQDs were tested against VRSA, and Homo sapiens due to its defensive response against gram positive and negative bacteria. The structure was constructed directly from sequence (CCVRLEFKLQQTSCRKRDWKKP) using the build structure tool in UCSF Chimera v1.10.2, with standard Φ/Ψ angle values for alpha helix  and Dunbrack rotamer library for side-chains, then hydrogens were added. Molecular dynamics (MD) simulation was prepared and run on GPU (NVIDIA GTX1080Ti) using Gromacs v2018 with Amber99SB force field. The standard leap-frog integrator was used for calculation of trajectories in steps of 2 fs. The Verlet scheme was used for neighbor searching, while the cut-off method was used for Van der Waals interactions at 1.2 nm. Particle mesh Ewald (PME) was used with all electrostatics calculations at 1.2 nm cutoff. Briefly, the peptide was solvated in SPC water model in dodecahedron periodic box and neutralized with Cl− ions. Only 335 steps of steepest descent minimization were required to achieve convergence with maximum force <1000 kJ/mol/nm. NVT equilibration was done for duration of 1 ns using LINCS constraints for H-bonds, and modified Berendsen thermostat coupling (300 K) in two groups (protein and non-protein). NPT equilibration was done for duration of 1 ns using LINCS constraints for H-bonds, modified Berendsen thermostat coupling (300 K), and isotropic Brendsen pressure coupling (1 bar). MD Production was performed in NPT for 1000 ns using LINCS constraints for H-bonds, modified Berendsen thermostat coupling (300 K), and Parrinello-Rahman isotropic pressure coupling (1 bar). Trajectories and energies were saved every 10 ps and the total number of trajectory frames was 100,000. Post MD analysis was done by centering the protein in the box, followed by fitting of backbone and removal of all water molecules from the system. Time evolution of secondary structure was performed according to the standard DSSP method. Visualization and further analysis was done in UCSF Chimera. Clustering analysis of minimal backbone in steps of 50 frames was used to identify the most stable conformation (largest cluster). Root mean square deviations (RMSD) for the backbone (87 atoms) and for all atoms in peptide (191 atoms) were calculated with reference to the first frame (α-helix) and to the representative frame of top cluster.The peptide was derived from the retinoic acid receptor responder protein 2 of All the chemicals, reagents for peptide synthesis, CQDs synthesis, and standards were purchased from Sigma-Aldrich  in ACS purity, unless noted otherwise. All the experiments were done maintaining sterile condition to obtain the best results.According to our previous studies the preparation of the water soluble CQDs were done and briefly the synthesis of CQDs is described . To the ex) of 270 and 312 nm . A multifunctional microplate reader Tecan Infinite m200 PRO  was used to measure the absorbance and fluorescence of CQDs, HSER, and HSER -CQDs [−1; drying temperature, 300 °C. Mass range was set from 50 to 3000 m/z. Positive ion mode was used. Prior to HPLC-MS analysis, a mass spectrometer was calibrated using ESI-TOF tuning mix.The HSER-CQDs were prepared and visualized under a UV transilluminator at excitation wavelengths , which has quasielastic laser light scattering. The 1.5 mL of the CQDs solution, HSER, and HSER–CQDs were added to the polystyrene latex cell and measured at an detector angle of 173° at 633 nm wavelength, with temperature of 25 °C, the refractive index of 0.30 and real refractive index of 1.59. Fourier transform infrared spectroscopy (FTIR) analysis was done by film of each freeze dried samples using an FTIR spectrophotometer MATTSON 3000 FT-IR  with a wavenumber range of 400–4000 cmStaphylococcus aureus (VRSA) CCM 1767, was obtained from the Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Brno, Czech Republic, and Escherichia coli ATCC BAA 2340 was obtained from American Type Culture Collection (ATCC), United States. The media used for the growth of the bacteria was Mueller Hinton (MH) media of 15 mL in Erlenmeyer flasks and the bacteria was inoculated and cultures were kept in shaker at 37 °C at 130 rpm for 24 h. Further, dilution of the culture was done using the MH broth to obtain 0.1 Absorbance (0.5 MacFarland standards) at OD600 nm and used for consecutive experiments [Bacterial strain, vancomycin-resistant eriments .The standard broth micro-dilution method  was used to determine the susceptibility of bacterial cultures against HSER-CQDs and its individual component, with the detection done by the unaided eye. MIC was calculated using the solutions from high to low concentration gradually. The different concentration of HSER–CQDs  was added to the microplate wells and mixed with the bacterial cultures . Then the plate was incubated at 37 °C for 24 h. The well with lowest concentration of antibacterial agent with almost no bacterial growth was considered the MIC and the control was bacteria without any treatment .The growth curve was obtained using the Multiskan EX  by measuring the absorbance to determine the antibacterial activity. Different concentrations of the HSER–CQDs, HSER, and CQDs were used to check the antibacterial effects on gram positive and negative bacteria. The positive control was only bacteria without any treatment. To compare the activity of HSER–CQDs, the bacteria was treated with different concentration of HSER and CQDs, respectively. Further, the viability percentage was calculated from the absorbance value after 24 h, comparing it with the absorbance value of the positive control .E. coli. The number of cells observed per sample was observed from 10 randomized microscopic grid fields.Initially the samples were incubated with bacteria and HSER–CQDs conjugate (respective MIC) for 4 h at 37 °C in shaking incubator. The optical Olympus BX51 fluorescence microscope equipped with a 40× phase contrast lens was used to study the antibacterial activity of the HSER–CQDs against VRSA and The live/dead cell assay is based on two fluorescent dyes, namely, SYTO9, which permeates both damaged and intact cell membranes, and propidium iodide (PI), to stain only the cells with damaged cell membranes . A totalInitially, VRSA was treated with HSER–CQDs (25 µg/mL) and incubated at 37 °C for 4 h in shaking condition. Further, the bacterial cell membrane damage and cell cytoplasmic leakage after the treatment by HSER–CQDs was studied by centrifuging the samples, and the supernatant was used as the template for PCR and the amplification was done using 16S rRNA gene primers , and confirmation was done using agarose gel electrophoresis. The control was the supernatant of VRSA culture without treatment and the 100 bp ladder was used. The gels were visualized and documented under Azure c600 .Similarly, the genomic DNA (gDNA) from VRSA was isolated and then incubated with HSER–CQDs and kept for 1 h. Then, the samples were used as the template for PCR reaction and the amplification was done using 16S rRNA gene primers, and confirmation was done using agarose gel electrophoresis with respect to the 100 bp ladder. The control was the gDNA of VRSA culture without treatment. The gels were visualized and documented under Azure c600 .2. After 24 h treatment, 10 µL of MTT ) was added to the cells and incubated for 4 h at 37 °C. After that, MTT-containing medium was replaced by 100 µL of 99.9% dimethyl sulfoxide (DMSO) and incubated for 5 min, the absorbance of the samples was determined at 570 nm using Infinite m200 PRO  [The PNT1A , HBL-100 , MDA-MB-468 (mammary gland adenocarcinoma cells), and Du-145 (prostate adenocarcinoma cells) human cell line were cultured and used. The MTT -2,5-diphenyltetrazolium bromide) assay was used to check the viability. Briefly, in 50 µL medium, the suspension of 5000 cells was added to each well of microtiter plates, followed by incubation for 24 h at 37 °C with 5% COzerland) .h is percentage of hemolysis; cA is the absorbance of the supernatant from negative control ; tA is the absorbance of the supernatant from the samples incubated with the HSER-CQDs; and A100% is the absorbance of the supernatant of positive control (0.1% Triton X-100), which causes complete lysis of RBCs [Further, fresh human blood was collected from the volunteer, with signed informed consent, by a venipuncture from an antecubital vein. RBCs were isolated from blood and the suspensions were washed with 150 mM NaCl solution three to five times. RBCs was incubated with the HSER-CQDs for 1 h at 37 °C. The positive and the negative control was 0.1% Triton X-100 and PBS, respectively. The degree of hemolysis was determined by measuring the absorbance of the supernatant at 540 nm, after centrifugation, and calculated according to the following equation: of RBCs .2O2 showing positive ROS production [Bacterial endogenous ROS production was measured by using DCFDA Cellular Reactive Oxygen Species Detection Kit (Abcam Ab113851) according to the given protocol and instructions for bacterial cells ,30. To toduction .2 to ensure cell growth. After 24 h treatment with HSER–CQDs, the cells were observed under an inverted Olympus IX 71S8F-3 fluorescence microscope  equipped with Olympus UIS2 series objective LUCPlanFLN 40× , a mercury arc lamp X-cite 12 , and a Camera Olympus DP73 to obtain the bright field microscopic image using Stream Basic 1.7 Software. The number of cells observed per sample was observed from 10 randomized microscopic grid fields.The MDA-MB-468 (mammary gland adenocarcinoma cells), Du-145 (prostate adenocarcinoma cells), PNT1A , and HBL-100  human cell line were cultured and used. Briefly, the suspension of 5000 cells in 300 µL medium supplemented with growth factors was added to each well of microtiter plates, followed by incubation for 24 h at 37 °C with 5% COE. coli, was studied and the MIC was determined. Further the toxicity test showed it has no toxicity against eukaryotic cell lines . The mechanism of action was based on cell wall damage and leakage of cellular content. Some interaction of the HSER–CQDs with gDNA was observed, and assay showed negligible ROS generation. VRSA and E. coli was used as model organism to test the antibacterial effect of the HSER–CQDs due to its pathogenicity [The synthesis and characterization of HSER–CQDs, along with its antibacterial efficacy against VRSA and genicity .The largest protein conformation cluster was reached after ~550 ns and represented a nearly continuous duration of 21.85% of entire simulation (437 of 2000 frames) A. The reIn both the average structure of largest cluster and the representative frame, hydrogen bonds were observed rather in the side-chains than the backbone. Namely, Glu6 and Asp18 side-chains provided acceptor oxygens to the amides of arginines and glutamines . This observation is important for interpretation of FTIR vibrational spectra. Additionally, indicating that possible change in protonation states (due to pH change) will be detrimental for stable folding.The ime evolution graph of the secondary structure C shows aRMSD analysis confirmed these observations D. OveralThe HSER peptide and CQDs in 1:1 ratio helps in the formation of the HSER–CQDs conjugate, which was dialyzed and observed under UV–transilluminator and showed a bluish fluorescence. The spectrophotometric analysis was done and the absorbance was detected in the spectral range from 230 to 1000 nm and the maxima absorption peak for HSER and the HSER-CQDs conjugate was obtained at 280 nm A. Our ex−1, which are characteristic for secondary amines and carboxylates, respectively [−1 (−1) is often used as an indicator of secondary structure. This is based on the contributions of C=O stretching and C-N stretching, which are influenced by the strengths of hydrogen bonds involving them [−1, whereas the β-sheet structure is correlated with frequency range 1640–1625 cm−1. Fortunately, due to available data from MD simulations, it is clear that such interpretation of structure can be biased in this situation. Predicted HSER structure appeared to be stabilized by amide-based electrostatic interactions of side-chains (Hydrogen bonds). In a more precise description, the vibrations of the backbone do not involve many hydrogen bonds and thus they can be assigned to the disordered secondary structure with frequency range 1657–1642 cm−1 [−1 in HSER–CQDs is an indicator of short range backbone aggregation, while the decrease in 1650 cm−1 correlates with loss of random backbone and interrupted side-chain interactions in HSER–CQDs. The contribution of amide bands is dominated by the backbone peptide bond, nevertheless, several side-chain vibrations fall in the amide I and II range [−1cm−1) contributions in the amide I are in the following range: Arg (ε = 300–490), Gln (ε = 360–380), and Lys (ε = 60–130). As for the amide II region (in the 1500s cm−1) they are in the following range: Gln (ε = 220–240), Asp (ε = 290–380), Glu (ε = 450–470) and Lys (ε = 60–130). Furthermore, the Cys thiol (–SH) stretching peak at 2551 cm−1 [−1) between peptide backbone and secondary amines, carboxylates, and hydroxyl groups of the CQDs surface.The FTIR analysis A of the ectively . The brovely [−1 B indicating them . AccordiII range . Their e551 cm−1  was abseHowever, the particle size distribution showing average particle size and the zeta potential of the prepared HSER–CQDs were analyzed using DLS after purification, using dialysis in D-Tube maxi dialyzer tubes against Mili-Q C,D. TherE. coli was initiated by determining the MIC using broth micro-dilution method. The MIC value of HSER-CQD against VRSA and E. coli was seen to be 25 and 50 µg/mL, respectively, and the concentrations below showed turbid solutions proving the presence of the bacteria, whereas the HSER and CQDs were found to have no prominent antibacterial effects or a higher concentration was needed to be applied (>125 µg/mL) , but at 25 µg/mL the inhibition was more than 73%. Whereas, the individual components of HSER showed no prominent activity against VRSA, with an inhibition of 32.32% at 125 µg/mL, and in case of E. coli the inhibition was around 22.23% at 125 µg/mL, but CQDs too showed no prominent antibacterial activity against VRSA (32.67%) and E. coli at 125 µg/mL ), as shown in 25 µg/mL . Thus, tE. coli. Decrease in the viable bacterial cell numbers and disruption of the cell with loss of cell integrity due to rupture of the cell wall cause a huge change in bacterial morphology, cytoplasmic leakage, and the presence of cell debris, which were seen under the microscope for both VRSA and E. coli after the treatment with HSER–CQDs, whereas the control bacterial samples with no treatments showed high number of viable cells with full cell integrity, no change in morphology, and absence of cell debris, as shown in The phase contrast condition in the optical Olympus BX51 fluorescence microscope was used for further confirmation about the antibacterial activity of the HSER–CQDs against VRSA and E. coli after treatment with HSER-CQDs was observed by the live/dead assay using an inverted fluorescence microscopy. The bacterial samples treated with the HSER-CQDs showed a considerable decrease in the viability of the bacterial cells, showing almost negligible numbers of green fluorescent cells (SYTO9 stained), but a significant increase in number of dead cells with red fluorescence (PI stained). On the contrary, in case of controls cells, the number of live green fluorescent cells were high with almost no dead cells were observed, as shown in E. coli. Furthermore, to validate the results, the viability of the VRSA and Thus, the results showed prominent reduction in the viability of bacterial cells due to the presence of HSER-CQDs causing cell death by disrupting the cell wall, causing loss of cell morphology and cell death, proving its high-antimicrobial activity against both bacterial strains . Thus, tThe previous studies under microscope showed the loss in cell integrity and disruption of the cells. To further confirm the interaction of HSER–CQDs with the bacterial cell wall causing disruption and leakage of the cellular content, the HSER–CQDs were treated with VRSA cells, and the supernatant after 4 h of treatment was used to study the presence of DNA. The treated VRSA cells supernatant on PCR amplification, using the 16S rRNA primers, confirmed the presence of the band of DNA at 106 bp, whereas the control without any treatment showed no band, as shown in Based on net charge, cell-penetrating peptides can be either hydrophobic, amphipathic, or cationic . The sec2O2. The HSER and CQDs treated cells showed higher ROS generations in comparison to HSER–CQDs-treated cells.We have showed in this work that the HSER peptide adapted a loop-shaped structure in MD simulation, and displayed random secondary structure signature by FTIR analysis (that can be interpreted as α-helix in other cases). Following the immobilization of HSER on CQDs, β-sheet structure signature was detected, which also correlates with short-range inter-chain interactions. The data clearly shows that the immobilized peptide backbone structure is different from the free one. Based on previous insights, this change in secondary structure can explain the change in peptide antimicrobial activity. The β-sheet structure realigns the peptide sequence in an alternating and amphipathic way that can break some of the electrostatic strains in the side-chains; allowing them to directly interact with the membrane. Lastly, the ROS generation against the bacterial cells was seen using DCFDA probe and spectrophotometer. The results showed in Finally, the mechanism of interaction for HSER–CQDs is based on the change of structure of the HSER after coating on CQDs surface, which causes cell wall interaction leading to cell wall disruption, with loss of cell integrity causing the leakage of intercellular component with no ROS generations, as shown in The cytotoxicity of HSER–CQDs was estimated using the MTT assay PNT1A , Du–145(prostate adenocarcinoma cells), MDA MB 468 (mammary gland adenocarcinoma cells), and HBL–100 . MTT assay is based on reduction of MTT by succinate dehydrogenase reflecting metabolic healthy conditions of cells ,51. The Further, the hemolytic effects of the HSER–CQDs at different concentrations was studied using blood with positive control as 0.1% Triton–X (100% hemolysis) and negative control as PBS (no hemolysis). The 75 and 50 μg/mL concentrations of the HSER–CQDs conjugate showed negligible hemolysis, almost below 1.5% of hemolysis, as shown in Finally, the cytotoxicity was confirmed by microscopic observation under an inverted microscopy. The results after treatment of HBL–100, PNT1A, and MDA MB 468 using HSER–CQDs showed no morphological changes in comparison to the control cells. Whereas in case of Du–145, the treated cells, when compared to control cells, showed considerable change in the morphology of few cells with loss of cell integrity and cellular leakage shown in . Thus, tHomo sapiens due to its defensive response against gram positive and gram negative bacteria. The structural analysis of HSER was computationally done using MD simulation and synthesis was performed. But the peptide showed no prominent antibacterial activity against VRSA and E. coli. Thereafter, the synthesis of the CQDs was done based on our previous work and conjugated with HSER to increase its activity. The characterization was done using spectrophotometer, HPLC coupled with ESI–qTOF mass spectrometer, UV-transilluminator, zeta potential and size, and FTIR. The prominent antibacterial activity was seen against VRSA and carbapenem-resistant E. coli using growth curve analysis and viability percentage, along with the determination of MIC against respective bacteria. The antibacterial activity was further confirmed by microscopic analysis, showing loss of cell integrity, leakage of cellular content due to cell disruption, and with cellular debris. The live/dead cells assay showed the presence of a high amount of dead bacterial cells. The mechanism of action is based on change in structure of HSER after interacting with the CQDs, which was supported by FTIR results showing that the cell wall disruption occurred along with some interaction in presence of gDNA, which stop its amplification. Finally, the HSER–CQDs were found to be compatible with RBCs and nontoxic against normal human epithelial cells, with the help of MTT and hemolytic assay and further confirmed by microscopic analysis. Thus, HSER–CQDs can be used as a substitute to antibiotics.In the present study, HSER was designed from retinoic acid receptor responder protein 2 of