Punica granatum L. (pomegranate) fruit juice contains large amounts of polyphenols, mainly tannins such as ellagitannin, punicalagin, and punicalin, and flavonoids such as anthocyanins, flavan-3-ols, and flavonols. These constituents have high antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, and anticancer activities. Because of these activities, many patients may consume pomegranate juice (PJ) with or without their doctor’s knowledge. This may raise any significant medication errors or benefits because of food-drug interactions that modulate the drug’s pharmacokinetics or pharmacodynamics. It has been shown that some drugs exhibited no interaction with pomegranate, such as theophylline. On the other hand, observational studies reported that PJ prolonged the pharmacodynamics of warfarin and sildenafil. Furthermore, since it has been shown that pomegranate constituents inhibit cytochrome P450 (CYP450) activities such as CYP3A4 and CYP2C9, PJ may affect intestinal and liver metabolism of CYP3A4 and CYP2C9-mediated drugs. This review summarizes the preclinical and clinical studies that investigated the impact of oral PJ administration on the pharmacokinetics of drugs that are metabolized by CYP3A4 and CYP2C9. Thus, it will serve as a future road map for researchers and policymakers in the fields of drug-herb, drug-food and drug-beverage interactions. Preclinical studies revealed that prolonged administration of PJ increased the absorption, and therefore the bioavailability, of buspirone, nitrendipine, metronidazole, saquinavir, and sildenafil via reducing the intestinal CYP3A4 and CYP2C9. On the other hand, clinical studies are limited to a single dose of PJ administration that needs to be protocoled with prolonged administration to observe a significant interaction.The  In the last two decades, green medicine, such as herbs, fruits, and dietary supplements for disease management, has increased globally due to their safety, lower price, and natural components ,2. HowevWith co-administration of the drugs with herbal or dietary supplements, there is a potential risk of causing undesired adverse drug effects or even toxicity or reduction of the desired effect of the prescribed drug, which could ultimately lead to unsuccessful treatment. Drug interactions could affect the pharmacokinetic parameters, including absorption, distribution, metabolism, and elimination, and/or the pharmacodynamics properties, including changes in the drug effect or toxicity. Moreover, food-drug interactions might have caused significant medication errors, such as the well-known grapefruit interactions. Thus, the growing wave of green medicine and replacement therapy, local beverages, and unaddressed natural substances have imposed health concerns and should be spotlighted.Punica granatum L. (pomegranate) fruit juice contains large amounts of polyphenols, mainly tannins and flavonoids. Of these tannins is an ellagitannin, which is hydrolyzed to ellagic acid, punicalagin and punicalin. As for flavonoids, pomegranate juice (PJ) contains anthocyanins, flavan-3-ols, and flavonols. All the latter constituents have high antioxidant activity and anti-inflammatory functions [The unctions . For insunctions ,9,10.It has been shown that pomegranate fruit constituents also have anti-cancer activities, such as inhibiting cancer cell growth and inducing apoptosis in human prostate, breast, lung, and skin cancer cells. In addition, fruit extracts have been shown to inhibit several signaling pathways, such as mitogen-activated protein kinases (MAPK) PI3K/Akt and NFκB. Based on in-vitro and preclinical studies, clinical studies were implemented using PJ in cancer patients such as prostate cancer .Although PJ has been suggested to have strong anti-inflammatory, antioxidant, anti-obesity, anti-diabetic, and anti-tumoral properties, many patients may be consuming PJ with or without their doctor’s knowledge; the European Food Safety Authority’s (EFSA) position has been made clear. Many health claims have been rejected due to the fact that no sufficient cause-and-effect relationship between the consumption of pomegranate-derived products and health beyond basic nutrition is well-established . Thus, tIt has been shown that pomegranate constituents inhibit cytochrome P450 (CYP450) activities such as CYP3A ,14 and COur focus was on the preclinical and clinical studies that investigated the impact of PJ on the PK of drugs that are metabolized by CYP3A and CYP2C9. Therefore, our literature research in electronic databases  included the following search phrases: “pomegranate and drugs”, “pomegranate and drugs pharmacokinetics”, “pomegranate and CYP3A”, and “pomegranate and CYP2C9”. In addition, any study that was not on PK or PK-pharmacodynamics (PD) related was excluded.The preclinical studies showing the impact of PJ on the pharmacokinetics of drugs metabolized by CYP3A4 and CYP2C9 are summarized in max) of carbamazepine and its metabolite, carbamazepine-10,11-epoxide, compared to water-fed rats [Carbamazepine is an anticonvulsant medication for treating seizures, nerve pain and bipolar disorders. Carbamazepine metabolism is mediated by CYP3A . In ratsfed rats . The chafed rats . In-vitrfed rats . These iTolbutamide is a sulfonylurea that is used in type-2 diabetes to stimulate insulin secretion. Tolbutamide is a substrate for CYP2C9. In rats, a single dose of PJ increased the tolbutamide AUC 1.2-fold but did not modulate the elimination half-life . These dmax increased five-fold, and the elimination half-life increased 1.3-fold compared to buspirone alone [Buspirone is an anxiolytic drug, and it is metabolized by CYP3A. In rabbits following repeated dosing of PJ for 7 days, the buspirone AUC and Cne alone . These dmax values but did not show an increased elimination half-life compared to control-treated rabbits [max of nitrendipine, but not the elimination half-life, by around two- and five-fold for the AUC and 1.4- and four-fold for the Cmax, respectively [Nitrendipine is a calcium channel blocker and is used primarily to treat hypertension. The effect of PJ on the PK of nitrendipine was studied in a single-pass intestinal perfusion model and preclinically in rodents. Rabbits pretreated with PJ for 7 days exhibited higher nitrendipine AUC and C rabbits . Howeverectively . Since nectively , the aboectively .max and AUC of metronidazole by 1.4 and 2.3 folds, respectively, in rats [Metronidazole is an anti-protozoal and antibiotic drug currently used in intestinal, gynecologic and dental infections and others . Multipl in rats . Metroni in rats . Thus, iet al. showed that PJ increased sildenafil bioavailability in rats in a dose-dependent fashion. The area under the curve (AUC) was increased when a higher amount of PJ was given to rats. In addition, Tmax was delayed, and the elimination rate of sildenafil was decreased when combined with pomegranate [Sildenafil is a drug prescribed for the treatment of erectile dysfunction and pulmonary hypertension. In three clinical cases, Senthikumaran et al. (2012) reported that taking PJ with sildenafil prolonged erection episodes beyond orgasm . Furtheregranate .Sildenafil is metabolized mainly by CYP3A4 (79%) and, to a lesser extent, by CYP2C9 (19%) . Since pSaquinavir is a retroviral protease inhibitor used for human immunodeficiency virus infection treatment. Saquinavir is metabolized by CYP3A4 in the gastrointestinal tract and liver, and also its absorption in the intestinal mucosa is mediated by the efflux transporter P-glycoprotein ,32.max at the three doses of pomegranate. Also, the elimination half-life was increased. In an ex vivo model, PJ also increased saquinavir transport to the mucosal compartment in a time-dependent manner similar to P-glycoprotein known inhibitors [max parameters were reduced in pomegranate-treated rats in addition to delaying in Tmax and reduction in elimination half-life elimination [max, and elimination half-life several-fold and reduced its circulatory metabolite, 1’-hydroxymidazolam [In a single-dose treatment of pomegranate and saquinavir, PJ increased saquinavir’s AUC and Chibitors . Howevermination . In the mination . In the idazolam . The latmax of warfarin [Warfarin is widely used as an anticoagulant. As a narrow therapeutic index drug, interactions with juices should be addressed. A case study reported that PJ was the reason for having a stable international normalized ratio (INR) in a patient who took warfarin for several months . When thwarfarin . In anotwarfarin . Warfariwarfarin . Since pIn this section, the preclinical studies that investigated the impact of PJ on the pharmacokinetics of drugs that are not metabolized by CYP3A4 and CYP2C9 are presented in max but not AUC [max [Metformin is mainly used in diabetes mellitus to reduce blood glucose levels. The interaction between pomegranate and metformin was studied by Awad et al. This study demonstrated that pre-administration of PJ decreased metformin C not AUC . It has AUC [max . FurtherAUC [max , then thA clinical study investigating the effect of PJ intake for 8 weeks on type-2 diabetic patients on metformin found that drinking PJ for 8 weeks significantly lowered fasting blood sugar, insulin and insulin resistance compared to patients who did not drink PJ . The latPiracetam is a nootropic drug that enhances mental performance by improving cognitive functions and memory . Eighty max, and Tmax [Theophylline, a well-known narrow therapeutic index drug, is mainly used for treating asthma and chronic obstructive pulmonary disease (COPD). PJ interaction with theophylline was studied in rats where the juice was added to drinking water 12 h before the experiment, and a dose was given 30 min before theophylline administration. No significant effect was observed on PK parameters such as AUC, Cand Tmax . Theophyand Tmax ; thus, PThe clinical studies investigating the impact of PJ on the pharmacokinetics of drugs metabolized by CYP3A4 and CYP2C9 are presented in 50, whereas the IC50 increased in the case of PJ, suggesting that PJ does not inhibit CYP3A (triazolam hydroxylation) in mechanism-based action [Midazolam is a benzodiazepine medication used for sedation and anxiety. It is metabolized by CYP450 enzymes and glucuronide conjugation. Although both PJ and grapefruit juice inhibited CYP3A in vitro, Farkas et al. (2007) could not observe an effect of PJ on the clearance of oral or intravenous midazolam . The maid action . On the d action . These cd action .Artemether is a drug used to treat malaria, and it is known to be metabolized by CYP450, mainly CYP3A4, CYP1A2, CYP2B6, and CYP2C19 ,52. Follmax and AUC in comparison to the control period [Although PJ inhibited the rate of 4-OH-flurbiprofen formation from flurbiprofen by human liver microsomes, intake of PJ in humans did not change the PK parameters of flurbiprofen nor flurbiprofen metabolite, 4′-OH-flurbiprofen, in human volunteers compared to placebo and to a potent inhibitor of CYP2C9, fluconazole . In anotl period .In an observational study, the co-administration of pomegranate dietary products increased tacrolimus concentration in a patient with heart transplantation . This anThe above preclinical studies showed that PJ interacts with the intestinal metabolism of CYP3A4 and CYP2C9 enzymes by inhibiting the activity of these enzymes, which results in increasing the bioavailability of CYP3A4 and CYP2C9 mediated drugs . It shouIn clinical studies, however, the PJ effect on CYP3A and CYP2C9-mediated metabolism was not obvious. Although most of these clinical studies were performed as a single dose of PJ, two clinical studies looked at the impact of 2-week PJ administration. Furthermore, it has been suggested that PJ constituents’ inhibition of the CYP450 enzymes declines within 1–3 days, inferring that ingestion of such medications several hours apart from PJ administration will not prevent the interaction . Other rAnother observation is that the reported drugs in the clinical studies were not the same as those preclinically tested. This opens the need for more clinical studies on PJ’s impact on, for instance, sildenafil, warfarin, carbamazepine, and others. Furthermore, clinical studies should be performed on multi-dosing of PJ to observe the PK and pharmacodynamic impacts better than a single dose, such as those observed in sildenafil and warfarin human observation studies.


Notably, the biosensor exhibited no cross-reactivity when tested against the influenza virus. The entire testing process using the biosensor takes less than a minute. In summary, our biosensor exhibits promising potential in the battle against pandemic respiratory viruses, offering a platform for the development of rapid, compact, portable, and point-of-care devices capable of multiplexing various viruses. The biosensor has the capacity to significantly bolster our readiness and response to future viral outbreaks.The COVID-19 pandemic has underscored the critical need for rapid and accurate screening and diagnostic methods for potential respiratory viruses. Existing COVID-19 diagnostic approaches face limitations either in terms of turnaround time or accuracy. In this study, we present an electrochemical biosensor that offers nearly instantaneous and precise SARS-CoV-2 detection, suitable for point-of-care and environmental monitoring applications. The biosensor employs a stapled hACE-2 N-terminal alpha helix peptide to functionalize an in situ grown polypyrrole conductive polymer on a nitrocellulose membrane backbone through a chemical process. We assessed the biosensor’s analytical performance using heat-inactivated omicron and delta variants of the SARS-CoV-2 virus in artificial saliva (AS) and nasal swab (NS) samples diluted in a strong ionic solution, as well as clinical specimens with known Ct values. Virus identification was achieved through electrochemical impedance spectroscopy (EIS) and frequency analyses. The assay demonstrated a limit of detection (LoD) of 40 TCID Recent years have witnessed an escalating global concern over respiratory virus outbreaks ,2. The eNumerous approaches have been employed to detect respiratory viruses, notably the prominent SARS-CoV-2. These methodologies primarily encompass nucleic acid detection techniques, such as reverse transcription polymerase chain reaction (RT-PCR)-based methods, and point-of-care (PoC) lateral flow assays ,8,9. Whi−1. Similarly, Ghumra et al. recently introduced a portable biosensor for SARS-CoV-2 detection in exhaled breath, utilizing spike-protein-specific nanobodies on a micro-immunoelectrode. The biosensor detects as few as 10 viral particles per sample through tyrosine amino acid oxidation [In this regard, electrochemical biosensor technology emerges as a highly promising solution due to its high sensitivity, selectivity, cost-effectiveness, and fast response ,14. Elecxidation . Detailexidation ,22. A deOne of the major challenges in the development and commercialization of electrochemical biosensors pertains to the selection of materials suitable for mass production that can maintain reproducibility and stability under diverse environmental conditions. This choice of materials directly impacts the limit of detection, susceptibility to non-specific adsorption of interfering substances, and the sensor’s overall reproducibility and stability when applied in complex real-world matrices . For insCP-based electrochemical biosensors often employ electrochemical impedance spectroscopy (EIS), ideal for point-of-care use due to simplicity, high sensitivity, and speed. EIS finds applications in diverse biorecognition tasks, such as lipid bilayer monitoring , DNA tesThis study introduces a conducting polymer-based biosensor meticulously designed for the specific detection of the SARS-CoV-2 virus. The fundamental conducting material, polypyrrole, is integrated onto a nitrocellulose membrane backbone, drawing on its established role in biosensing. Leveraging bio-receptor interaction, we achieved selective and stable biosensor functionality by coupling lactam-stapled hACE2 receptor-based peptides using a glutaraldehyde linker. The choice of the hACE2 α1-helix as the immobilizing biomolecule is informed by computational and experimental studies, showing its potential to inhibit the receptor binding domain (RBD)-hACE2 complex formation and subsequent host cell infection as a therapeutic approach ,42,43. H4H5N) 98.0%  was vacuum-distilled before use. Ammonium persulphate ((NH4)2S2O8) 98.0% (APS) and glutaraldehyde (OHC(CH2)3CHO)  were obtained from Merck, Darmstadt, Germany. Nitrocellulose membrane (pore size 0.45 µm) was purchased from Fisher Scientific. All other chemicals were of analytical grade and used without further purification. Hydrochloric acid (HCl), phosphate-buffered saline (PBS. pH 7.4), and de-ionized water were obtained from Merck , SRL , and Emplura , respectively. The hACE2 peptide sequence  of the known copy number using the Coronavirus COVID-19 Genesig real-time PCR assay kit  (The viruses utilized in this study were SARS-CoV-2 lineage B.1.617.2 (Delta Variant) culture fluid  and SARS-CoV-2 lineage B.1.1.529 (Omicron Variant) culture fluid  and . For control experiments, Influenza vaccine  containing an attenuated mix of Influenza A and B viruses (A/Victoria/2570/2019 (H1N1), A/Hong Kong/2671/2019 (H3N2), B/Washington/02/2019 and B/Phuket/3037/2013) was used. We directly employed the pre-inactivation viral titers provided by the manufacturer in TCIDigh, UK) . To evalDevelopment of the biosensors on the nitrocellulose substrate was carried out following a sequential approach as shown in To confirm the specific binding of the hACE-2-stapled peptide to the SARS-CoV-2 virus, we conducted a fluorescence imaging study. Ppy-coated glass slides (5 mm × 5 mm) were immersed in a 30 µg/mL peptide solution prepared in 1× PBS for 16 h on a rocking shaker. Following this, the glass slides were washed with 1× TBST buffer and then treated with 5% skimmed milk for 90 min to block nonspecific binding sites. After another TBST wash, the glass slides were exposed to the virus for one hour. Specifically, SARS-CoV-2 delta and omicron variants served as peptide-specific controls, while an Influenza Vaccine containing a mix of Influenza A and B virus variants (as described in the ‘Viruses’ section) was used as a peptide non-specific control. A skimmed-milk-treated glass slide without virus exposure served as the negative control. Following a 1× TBST wash, the glass substrates were treated with Alexa fluor 488 (green dye) NHS-ester-tagged peptide for 30 min. Subsequently, the substrates were rinsed with 1× TBST and left to dry overnight in darkness. Fluorescence images of the glass slides were captured using a GFP filter at 10× magnification.f denotes the final impedance after one minute of sample exposure, and Zi signifies the initial impedance of the sensor. Notably, this parameter’s value is also influenced by the probing frequency. The impedance analysis was carried out within a specific frequency range of 1 kHz to 100 kHz, chosen and fine-tuned through empirical optimization.In the experimental phase, the sensor strips were affixed to electrical connectors at both ends using silver paste. These connected sensors were then inserted into an impedance analyzer, specifically the PGSTAT204 potentiostat from Autolab , for virus detection experiments. As a two-electrode sensor configuration was employed, the potentiostat’s electrode arrangement adheres to the designated scheme , involvi10TCID50/mL in artificial saliva, was applied to the biosensor and incubated for one hour at room temperature. Following a washing step, the biosensor was exposed to an Alexa 488-tagged hACE-2 peptide to interact with the virus–biosensor complex. After additional washing with 1× TBST, images were captured via fluorescence microscopy. To ascertain specificity, a heat-inactivated influenza virus was used as a control. As anticipated, the biosensor demonstrated selective binding to the SARS-CoV-2 virus, exhibiting no binding to the Influenza viruses, even at a high concentration of 120 µg/mL of haemagglutinin (HA) protein, thereby affirming no cross-reactivity  and two lower concentrations (40 and 20 TCID50/mL), and impedance measurements were conducted (50/mL). Nevertheless, the data also exhibited fluctuations and noise, particularly within the lower frequency ranges.Subsequently, impedance alterations were gauged upon the introduction of the quantified virus. Specifically, 1 µL of SARS-CoV-2 virus was added to the biosensor in five independent replicates, with high concentration  from its initial value (Z) before the introduction of the virus or control droplet. Subsequently, segregation of control data from various virus concentration data was achieved using the 3-sigma rule principle [50/mL virus concentration, alongside a normality test. Leveraging the assumed normal distribution of other data points, we computed the separation factor (sf) between the virus and control classes using the following formulacontrol and μvirus represent the means of the control and virus data, respectively, while σcontrol and σvirus represent their respective standard deviations.To mitigate the impact of noise, we embarked on data pre-processing strategies with the goal of identifying the optimal frequency or frequency range that maximizes the discernment between the control and virus classes, particularly for lower virus concentrations such as 20 and 40 TCIDrinciple , assumin50/mL), leading to the creation of a heatmap .The separation factor between the control and varying virus concentrations was computed across individual frequencies and a range of frequency bands. Subsequently, frequencies were sorted based on descending sf values specifically for the lowest virus concentration (20 TCID heatmap c. A highcontrol) and standard deviation (σcontrol) were then computed across all control test data. Then, a decisive threshold line was established based on the following equation:As stated earlier, impedance spectroscopy is a well reported approach to characterize biosensors towards detection of viruses and bacteria, but such reports are mostly focused on relative impedance measurement ,69,70. T50/mL virus concentration data from control data, as shown in Responses below this established threshold line were identified as belonging to the virus class, whereas those exceeding it were categorized as control class. The specified threshold, set at 0.35, notably exhibited exceptional sensitivity of 95% in distinguishing the 40 TCID50/mL virus concentration data from control data. Furthermore, the established threshold from the decision tree model was rigorously validated using a completely new dataset comprising 25 virus test samples and 25 control test samples, yielding remarkable outcomes of 100% sensitivity. Detailed validation results are presented in the Following a parallel methodology, an optimal frequency range was selected for data processing, with |dZ|/Z values serving as pivotal feature variables for the model. The dataset, constituting 40 test data from both control (n = 20) and virus experiments (n = 20), was randomly divided into a 75:25 ratio to form distinct training and testing datasets. A decision tree model of depth 1 was fitted, culminating in a threshold value of approximately 0.37 . This th50/mL virus concentration, affirming this determination through 20 replicates at the specified LoD, in accordance with the requirements of the Emergency Use Authorization program of the Food and Drug Administration (FDA EUA), aiming for 95% sensitivity. In parallel, 20 replicates of artificial saliva without virus were analyzed. As depicted in 50/mL) for assessment with our biosensor. Notably, the results showcased similar sensitivity to that observed with virus in artificial saliva -, medium -, and high (Cts 30 and 31)-Ct-value groups. Prior to biosensor testing, the samples were diluted in KCl solution to a final concentration of 0.45 M. Our biosensor successfully detected the low- and medium-Ct-value samples but had a a slight margin of error in detecting the high-Ct-value specimens e. We hyp50/mL as determined by the manufacturer’s pre-inactivation analysis. To facilitate fair comparisons with other studies evaluating RAT analytical performance, we determined viral RNA copies corresponding to dilutions of TCID50/mL concentrations provided by the manufacturer using RT-PCR , or RT-PCR-based viral RNA copies, introduce bias to make comparisons [10 and 7.46 log10 RNA copies per swab [10 copies/mL and 6.39 log10 copies/mL for delta and omicron variants, respectively. Put together, our data indicate that our biosensor has comparable and in some cases better analytical performance with commercially available RATs and some reported efforts with electrochemical, electrical, and optical biosensors . This sensitivity improvement is rooted in the reduced dimensions of the active binding region . Miniatu50/mL. These results are comparable to, and in many cases superior to, commercial rapid antigen tests. Subsequent clinical evaluation is necessary to assess its practical utility. Furthermore, our biosensor showcased rapid and precise SARS-CoV-2 detection, yielding results in under a minute. A noteworthy advantage of our biosensor lies in its potential for heightened sensitivity through miniaturization. This enhancement translates into improved portability for point-of-care applications, reduced power consumption, accelerated response times, and the capability to simultaneously detect multiple analytes. These attributes render our biosensor suitable for diverse applications in medical diagnostics and environmental monitoring.In summary, our study has successfully developed a rapid biosensor tailored for precise SARS-CoV-2 detection. This was achieved by functionalizing a conductive polymer biosensor with a lactam-stapled hACE-2 N-terminal alpha helix peptide, endowing it with exceptional selectivity. This selectivity was corroborated through both fluorescence microscopy and impedance measurement, enabling accurate virus detection in minuscule volumes of artificial saliva and nasal swabs. The biosensor demonstrated robust analytical performance, boasting 95% sensitivity and 100% specificity at a LoD of 40 TCIDA provisional patent application for the invention described in this paper was filed with the United States Patent and Trademark Office on 7 November 2023. The application number is 63/513,007.


Prosopis juliflora leaves extract (PJ-CDs). The synthesized PJ-CDs were investigated by physicochemical evaluation instruments such as fluorescence spectroscopy, SEM, HR-TEM, EDX, XRD, FTIR, and UV-Vis. The UV-Vis absorption peaks obtained at 270 nm due to carbonyl functional groups shifts of n→π*. In addition, a quantum yield of 7.88 % is achieved. The synthesized PJ-CDs showing the presence of carious functional groups O–H, C–H, C=O, O–H, C–N and the obtained particles in spherical shape with an average size of 8 nm. The fluorescence PJ-CDs showed stability against various environmental factors such as a broad range of ionic strength and pH gradient. The antimicrobial activity of PJ-CDs was tested against a Staphylococcus aureus, and a Escherichia coli. The results suggest that the PJ-CDs could substantially inhibit the growth of Staphylococcus aureus. The findings also indicate that PJ-CDs are effective materials for bio-imaging in Caenorhabditis elegans and they can be also used for pharmaceutical applications.Carbon dots have stimulated the curiosity of biomedical researchers due to their unique properties, such as less toxicity and high biocompatibility. The synthesis of carbon dots for biomedical application is a core area in research. In the current research, an eco-friendly hydrothermal technique was employed to synthesize high fluorescent, plant-derived carbon dots from  Emergence of drug resistant pathogens hence the antibiotic resistance has quickly become one of the world's major life-threatening problems3. Evolution and speed of bacterial drug resistance has outpaced exploration and development of new antibiotics which are hindered due to high cost and technical difficulties4. This warrants an urgent need for production of novel and new alternative antibacterial drugs. The swift advancement of nanotechnology over the last several decades has resulted in the creation of promising antibacterial treatment alternatives5. Nanomaterials containing metal nanoparticles such as Cu-NPs, Ag-NPs, and Cu-Te NPs usually produce reactive oxygen species and followed by discharge the metal ions; thereby leading to antimicrobial activity6. However, in addition to the protein inactivation, enzymatic inhibition, oxidative stress, and DNA damage have all contributed to the presence of metal nanoparticles in non-target cells which can damage the healthy tissues and this is why the metal nanoparticles have limited clinical applicability7. Carbon is a common non-metallic element that is abundant in environment, and it appears in a variety of allotropic forms including amorphous carbon, graphite, and diamond8. Kroto et al. (1985), Water solubility and high fluorescence luminescent carbon dot were first reported in 20069. The Carbon dots (CDs) are normally pseudo nano-materials having amorphous and nano-crystalline structure and consist sp2/sp3 carbon, oxygen/nitrogen-based groups, and also post -altered groups10. There are two types of CDs syntheses methods namely bottom up and top-down methods11. In traditional top-down approaches, carbon source components are initially converted into nano materials and followed by different methods including acid discharge, laser ablation, and oxidations which are used12. CDs are also prepared by diverse bottom-up methods namely pyrolysis, carbonization, hydrothermal and microwave assisted13. CD structure mainly depends on its synthesis method and is responsible for its varied luminescence properties14. In general, the CDs have a core shells-like made of base carbon core with functional groups attached15. The CDs carry carbon atoms and other molecule elements which is accountable for the function of carbon dots16. Scientists involve in the use of CDs, due to their advantages such as water solubility, cost effective, high conductivity, tunable photoluminescence, easy surface modification, high stability and high biocompatibility across the globe18. CDs have been ideally used in wide ranging applications like dye degradation21, bioimaging24 detection of light-emitting diodes26 sensing28 and catalysis30. Till date, there are several antibacterial actions of CDs which have been verified in large number of studies, like mechanical destruction, oxidative stress and reduction of bacterial metabolism31 and photo-catalysis33. Among them the reactive oxygen molecules trigger oxidative stress is the main mechanism. Which is got activated by light to create singlet molecular oxygen (O2) or superoxide anion (O2·) or to act as a catalase to oxidise other agents34.Pathogen such as bacteria, fungi, viruses, and parasites cause several diseases every year36. There are several investigations on the preparation of carbon related natural materials, including Psidium guajava leaves37, Curcuma longa leaves38, Osmanthus leaves39, milk vetch and tea leaves and others, which have recently been reported40. Prosopis juliflora is a world worst invasive plant in Asia, Africa and Australia. This plant is capable of displacing natural plants from their habitats41.Since this plant cannot provide proper shelter the bird fauna is affected. The region’s water table in severely reduced and nutritive elements from the soil are depleted43.Natural biomaterials are resourceful, economical, and eco-friendly feasible method of carbon quantum dots since they are biocompatible, renewable, and transform to the bio-waste into valuable products44. Luminescent dyes have usually been employed to stain bacterial species, fungus, and plants cells. Anionic and Cationic dyes are similarly known as luminesce, and used their capability to bind to biological constituents of bacterial cells45. Acridine orange, ethidium bromide, and fluorescence isothiocyanate are some of the fluorochromes frequently used46. However, for a more sustainable future, it is critical to use a material which is biocompatible, minimal toxic effect, cheap, and environment friendly. Luminescent nanomaterials fulfil all the above said properties and considered possible rivals to standard fluorescent dye probes, for application in biological labelling, chemical sensing and other fields48. When compared to standard fluorescent dyes, light emitting nanomaterials, have specific nanomaterial and are quantum size effect, which several advantages such as low fluorescent intensity, limited stability, and quick photo bleaching49. In the present study we synthesised facile hydrothermal process of CDs using Prosopis juliflora leaf as carbon source. A synthesised Prosopis juliflora carbon dot was evaluated for antibacterial effect on against Gram positive Staphylococcus aureus, and a Gram negative Escherichia coli. The fluorescence property of synthesized CDs was applied for selective analysis of nematode as a bioimaging application.Use of fluorescent tags to visualize either eukaryotic or prokaryotic cells which exist becoming widespread across all disciplines50. Aerial parts of Prosopis juliflora leaves were collected in December 2021, at Periyar University campus, Salem, India . The sample was stored in plastic cover and brought to the research lab within 48 hrs. The collected plant leafs were washed carefully with tap water and then soaking in double distilled water (DDW) for five times, and finally shade drying for 15 days. The dried plant leaves leaf was grounded into a fine powder with a mechanical mixer blender. Five gram of plant leaves powder was dispersed in 50 mL of DDW and placed on Teflon autoclave and heated at 180ºC for 5 hrs. After cooling down to room temperature the natural brown colour solution was passed through Whatman No 1 filter paper and then centrifuged at 8000 rpm for 15 min to remove any unwanted bulk materials. Further, the CDs solution was kept at below 4 °C for further use51. Synthesised samples were denoted by Prosopis juliflora carbon dots [PJ-CDs].The collection of plant leaf materials was done according to the International and National guidelines for the purpose1/4 1.54 nm) was conducted on a Philips PW 3050/10 advanced spectra were recorded at a scanning speed of 0.1 min−1 with 2θ angle ranging from 10° to 80°. High-resolution transmission electron microscopy experiments (HR TEM) were performed on a JEOL/JEM 2100 plus microscope (operated at 200 kV). Fourier transform infrared spectra were collected with a FTIR spectrometer (Shimadzu IR spirit) in the wavelengths range of 4000–00 cm−1 at a room temperature. Optical properties like UV-Vis absorbance were assessed by using UV-Visible Spectroscopy  and the scanning electron microscopy (SEM) . Fluorescent images were captured by placing in; fluorescence spectra of CDs were recorded with the aid of Fluorescence spectrophotometer, Jasco, FP-8200. (Japan make).XRD analysis of the PJ-CDs employing Cu Kα radiation (λStaphylococcus aureus (S.aureus) (ATCC25923) and Escherichia coli (E.coli) (ATCC25923) bacterial strains were procured from Department of Microbiology, Periyar University, Salem, India. Cultures were maintained at 37 °C under a shaking speed of 180 rpm in a nutrient broth medium. The load of bacteria was detected by evaluating OD at 600 nm via UV-Visible Spectroscopy. The bacterial solution was diluted to till the cell concentration of 106 to107 CFU/mL.In order to guarantee the sterility, all utensils were autoclaved at 121 °C for 20 min. S.aureus and E.coli were evaluated using the well plate technique52. Petri dishes and sample were sterilized at 120 °C for 20 min before the antibacterial assay. The overnight grown bacterial culture was swabbed over the surface of the nutrient ager media using a sterile cotton swab for the even spread of the bacteria. Synthesized PJ-CDs stock solution was diluted with sterile Double Distilled Water (DDW). About 1.5 mg/mL of CDs solution was poured into the well, kept for 24 hrs at 37 °C and the inhibition zones were successively observed. The experiments were carried out in triplicate. The antibacterial activity was observed by inhibition area (mm) and compared with negative control (DDW).The antibacterial activity of the synthesized PJ-CDs against 600 values were monitored by a micro-plate reader at various time durations. This experiment was carried out three times. Besides, the bacterial inhibition of 24 hrs was calculated according to the following formula53.The minimum inhibitory concentration (MIC) is calculated by micro dilution 96-well cell culture plate route according standards guidelines. Each well contains 50 µl of bacteria and 50 µl of various concentrations  of synthesized PJ-CDs. The control well is bacterial culture in the sterile nutrient broth without any drug treatment. Sample loaded plate was kept in a shaking incubator at 180 rpm and 37 °C for 24 hrs, ODCaenorhabditis elegans incubated PJ-CDs 1 mg/ml for 8 hrs, followed by fluorescence images using a fluorescence microscopy.Since the commercial florescent dyes such as propidium iodide and propidium azide have the ability to enter the cell wall of the death bacterial cells, they have generally utilised for DNA staining techniques. Due to the cost, toxic and photo-bleaching effects researchers are in search to develop such dyes with inexpensive, low toxic and high water solubility to conduct cell viability assays. Fluorescent properties of CDs can be wide range used for so many biological applications, compared to traditional organic dyes. In vivo cellular imaging studies were performed to carry out 54. In a previous report, a water-soluble CDs produced from citric acid and Curcumin with effective antibacterial and anti - biofilm activities showed the UV-Vis spectra peak value of 280 nm which stands for n-π* transition55.The optical characterises of the synthesized PJ-CDs sample were evaluated by UV-Vis spectrophotometer Figa,b. The 2SO4 was used as a standard with Q Value of 54 % at 345 nm excitation wavelength for quinine sulphate. Equation is used for the evaluation of quantum yield of carbon dots56.Quantum yield of green synthesis PJ-CDs was determined by employing quine sulphate solution as a standard. Quine sulphate solution dissolved in 0.1 M of H2SO4 in water (Ƞ= 1.76).Q is fluorescence quantum yield, I is the combined fluorescence intensity, OD is the UV-Vis absorbance, Ƞ- is the refractive index of the solvent for PJ-CDs suspension solution as water (ƞ = 1.33) and 0.5 M H40. The absolute photoluminescence quantum yield was 7.88 %. Table 2 carbon embedded in sp3 matrix57.The fluorescence study was carried out in wavelength ranges between 395 and 480 nm. Figure 64. In another study report in fluorescent carbon dots synthesis from neera plant its detection of silver ions 65.Synthesized PJ-CDs solid sample was covered on glass substrate and submitted for crystal nature characterization. The XRD results Fig. c was doc−1 is due to O-H stretching, 2918 cm−1 is due to the C–H of alkanes  is critical in enhancing the antibacterial activity of the prepared PJ-CDs66.The broad band absorption peaks at 3391 cm67.EDX is used to identify elemental composition of synthesized PJ-CDs. The biosynthesized PJ-CDs were observed on a copper-coated carbon grid under SEM. EDX scanning performance was done with a voltage range in 40 kV and mV with cu-k radiation in the 2θ range of 10 to 80  and light irradiation. Especially the reactive oxygen species generation is the most important antibacterial activity mechanism among them. The antibacterial activity may be attributed to several functional groups which present in the green synthesized PJ-CDs that interfere with cellular enzyme roles and inhibit the cellular proliferationCaenorhabditis elegans emitted green excitation at a laser excitation of 470 nm  have led to greater water-soluble properties. The antibacterial potentially PJ-CDs can also be useful in providing sterile environment for bio-image and biomedical application.The multifunctional PJ-CDs were synthesized from natural source


Tymovirales and Picornavirales and the families Partitiviridae, Totiviridae, Tombusviridae, Iflaviridae, Nodaviridae, Permutotetraviridae, and Solemoviridae, with several attributed to four unclassified RNA viruses. Interestingly, the novel putative viruses and viral sequences were mainly associated with the mosquito Coquillettidia richardii. This study aimed to increase our understanding of the viral diversity in mosquitoes found in the natural habitats of Siberia, which is characterized by very long, snowy, and cold winters.The metagenomic analysis of mosquitoes allows for the genetic characterization of mosquito-associated viruses in different regions of the world. This study applied a metagenomic approach to identify novel viral sequences in seven species of mosquitoes collected from the Novosibirsk region of western Siberia. Using NGS sequencing, we identified 15 coding-complete viral polyproteins (genomes) and 15 viral-like partial sequences in mosquitoes. The complete sequences for novel viruses or the partial sequences of capsid proteins, hypothetical viral proteins, and RdRps were used to identify their taxonomy. The novel viral sequences were classified within the orders  Peribunyaviridae, Flaviviridae, Reoviridae, and Togaviridae, and each is a potential source of viral biodiversity for viruses, with dual tropism for invertebrate and vertebrate hosts [Mosquitoes are well known to transmit numerous arboviruses that cause viral infections in animals and humans, such as the West Nile virus (WNV) and the Zika, Japanese encephalitis, Chikungunya, and dengue viruses ,2,3,4. Ite hosts ,17,18.Comparing the viromes of mosquitoes from different geographical regions has revealed their biodiversity, providing new insights into the phylogeography of mosquito-borne viruses ,6,19,20.In this study, we sought to investigate the biodiversity of mosquito-borne viruses from different mosquito species collected in western Siberia using metagenomic approaches.https://survinat.ru/2011/09/metodika-sborov-xraneniya-i-izucheniya-komarov/, accessed on 23 June 2023). Capture was conducted after sunset. The mosquitoes were transported in a thermal bag on a damp napkin at a temperature of 4 °C and stored at minus 18–24 °C. Fragments of the 16S rRNA and the COI gene of the mitogenome were sequenced via the Sanger method to determine the mosquito species [For this study, 3910 mosquitoes were collected in the Novosibirsk region during the spring–summer periods of 2017–2018. The collection sites were situated in typical mosquito habitats in western Siberia : deciduog for 5 min at 4 °C, and the supernatants were used for the analysis. The total RNA was extracted using an Extract RNA reagent  according to the manufacturer’s protocol and purified on Cleanup Mini spin columns . The pools were then processed using benzonaze [All the mosquitoes were washed with 70% ethanol and then rinsed twice with water, followed by homogenization to remove potential surface microorganisms. The homogenization of the samples was performed mechanically by grinding them in a mortar with 300 µL of sterile saline. The homogenates were centrifuged at 8000× enzonaze . The firThe dsDNA libraries were prepared and analyzed via NGS on MiSeq using Illumina technology. Cutadapt (version 1.18) and SAMtools (version 0.1.18) were used to remove the Illumina adapters and re-read. The contigs were assembled de novo using MIRA assembly (version 4.9.6). The experimentally determined sequences were deposited in GenBank. The phylogenetic analysis was performed using RNA-dependent RNA polymerase (RdRp) sequences from GenBank with an amino acid identity > 20%. The sequences were aligned, and phylogenetic trees were built in Vector NTI Advance 11, MEGA 7/10 , and Lasergen 7 . The resulting viral sequences and sequence read archive (SRA) were deposited in GenBank. The phylogenetic trees were calculated via the maximum likelihood method, using 500 replicates for bootstrap values.Aedes caspius  and Ae. mariae  were found to be the most abundant species, comprising 41.2% of the total. Anopheles messeae  accounted for 34.6%, Culex pipiens  for 8.3%, Coquillettidia richardii  for 5.5%, C. modestus  for 4.8%, and An. maculipennis  and An. sinensis  for 2.8% each. A total of 3910 mosquitoes were collected in the Novosibirsk suburbs and the rural district of the Novosibirsk region in 2017–2018. Pools of 10–40 mosquitoes were formed to identify the mosquito species for each collection point. Fragments of the 16S rRNA and the COI gene of the mitogenome were sequenced to determine the species of mosquitoes . Aedes cPartitiviridae), Hammarskog tombus-like virus (ssRNA (+), Tombusviridae), Hammarskog picorna-like virus (ssRNA (+), Picornavirales, unclassified), Lymantria dispar iflavirus 1 (ssRNA (+) virus, Iflavirus), Wenzhou noda-like virus 6 (ssRNA (+) virus, unclassified), Mayapan virus (ssRNA (+), two segments), Sanxia permutotetra-like virus 1 (ssRNA (+) virus, unclassified), and Chaq virus-like 1 . An additional 22 viral sequences were presented as putative mosquito-borne sequences, with a level of identity less than 79% for viral prototype sequences. In total, 144 putative viral sequences were selected in the first step, and 30 of these sequences with lengths greater than 1239 bp were chosen. Eight sequences with a level of identity > 80% aa were previously described as mosquito-borne viruses . These wThe proportions for classified and unclassified viral reads are presented for 13 mosquito pools of Cq. richardii . The preThe phylogenetic analysis results for the sequences obtained from the mosquitoes are presented in Cq. richardii mosquitoes. This virus has been designated as an Inya insect-associated virus 1 . The genomic positive ssRNA of the Inya insect-associated virus was identified as comprising 6526 bp and three ORF-encoding proteins  with a previously described insect-associated tymovirus 1 in Mexico (MN203215) was detected in the proteins . The ORFCq. richardii and demonstrated an identity level of 89% with an isolate from Anopheles gambiae that was collected in Liberia (KX148575). Another novel putative partitivirus was detected in the pool of Cq. richardii mosquitoes (Anopheles darlingi in Colombia (MN661058). In addition, seven suspected partitiviruses were found in the pool of Cq. richardii mosquitoes. These are the novel insect Talaya 1 and 2 viruses (MW251327–MW251330), with aa sequence identity levels of 68–71% identity with a previously described Partitivirus-like 1 ; the Tarbrook virus (MW251325), with 79% homology with a previously described Sonnbo virus in Sweden (MK440649); and Zeyabrook partiti-like_viruses 1 and 2, with similarities of 38–42% with a Beihai partiti-like virus 2 (NC_032500) from China. All the prototype sequences were isolated earlier from invertebrates . These partitiviruses were preliminarily taxonomically identified as belonging to the family Partitiviridae, unclassified Partitiviridae.Partitivirus-like 1 was detected in squitoes  and was Culex annulirostris in Australia (MT498830)  . The ZyrCq. Richiardii in 2017 [Tombusviridae was translated using a potential alternative mechanism to suppress the stop-codon-reading mechanism with the formation of a full-size protein with an elongated ORF1 C-end [Tombusviridae  are single-stranded RNA (+) genomes between 3.7 and 4.8 kb in length, currently regarded as plant viruses with a relatively limited selection of hosts, and they usually require a subviral RNA for replication ,28,29. T in 2017 . The 416Cq. richardii mosquitoes that had five OFRs encoding 175 aa, 156 aa, 121aa, 376 aa, and 2424 aa polypeptides with 98% aa identity with a previously described Hammarskog picorna-like virus (MN513381) isolated from Cq. richiardii in Sweden . The preliminary taxonomic identification for these six viruses is Picornavirales, unclassified Picornavirales.The Miltyush picorna-like virus was found to have only 36% identity with a previously detected Halhan virus 3 from Haliotis discus hannai in Korea (NC040628). Isses picorna-like virus 1 was found to have 64% identity with a Washington bat picornavirus previously discovered in the USA (KX580885). Ichacreek insect virus 3 was identified to have a 44% level of identity with a previously detected Solenopsis invicta virus 3 (GU017972) from Picornavirales also includes some iflaviruses that were found in Cq. richardii mosquitoes in this study. Lymantria dispar iflavirus 1 was detected, with its sequence having 99% identity with already-known viral isolates from the USA and Russia . The alignment and phylogenetic analysis revealed a high level of sequence identity with the representatives of Iflavirus, the family Iflaviridae.The order Cq. richardii mosquitoes, as well as the Mayapan virus, with identity levels of 87% and 90%, respectively. Previously, the Wenzhou noda-like virus 6 sequence was identified in Channeled applesnail in China (KX883260), and the Mayapan virus (MH719096) was isolated from the Psorophora ferox mosquito in Mexico , which had a 43% identity with that isolated from the Psorophora ferox mosquito in Mexico, and the insect-associated Uzakla virus with a prototype Mosinovirus (KJ632942), which had a 36% identity with that isolated from Culicidae spp. in the Cote d’Ivoire. We assembled practically the whole genome for Wenzhou noda-like virus 6 from the n Mexico . Other nCq. richardii mosquito pools showed the presence of the virus with a 93% identity with Sanxia permutotetra-like virus 1, which was previously detected in water striders in China (KX883450). The Uzakla mosquito-associated permutotetra-like virus, with a 53% identity with Vespa velutina permutotetra-like virus 2 in France , was described early on as belonging to the group of unclassified Permutotetraviridae.The genomic RNA for permutotetraviruses is 4582 bp long and encodes three ORFs overlapping in a short region . The lonCq. richardii. The Chaq virus-like 1 had 82% identity with an earlier described unclassified sequence from Anopheles gambiae in Liberia (KX148554). The ZeyaBrook chaq-like virus 2, Kamenka insect-associated virus, and Uzakla insect virus had identity levels of 33–47% with previously unclassified putative viral sequences  isolated from invertebrates. Only the Tartas insect associate virus may be classified as unclassified Solemoviridae, having an identity level of 61% with prototype Atrato Sobemo-like virus 6 (MN661101), which was detected in Wyeomyia spp. mosquitoes in Colombia. The Solemoviridae have a relatively small (4–4.6 kb) positive-sense, single-stranded, monopartite RNA genome with 4–5 ORFs, and they are usually associated with plant viruses. Two variants of Chaq virus-like 1, Tartas insect associate virus, ZeyaBrook chaq-like virus 2, Kamenka insect-associated virus, and the Uzakla insect virus were detected in Peribunyaviridae [Rhabdoviridae [Orthomyxoviridae [Flaviviridae [Reoviridae [Chuvirus [Negevirus [The application of the metagenomic approach offers novel opportunities for virome analysis ,7,20. Thaviridae , Rhabdovoviridae , Orthomyoviridae , Flaviviiviridae , and Reooviridae , as wellegevirus . Recent egevirus ,38. Numerous genetically diverse viruses have also been detected via NGS sequencing in plants, invertebrates, and vertebrates in tropical countries ,8. PhyloCq. richardii can vary from 1.7 to 99.5%, with this species usually dominating in the main forest–steppe and steppe landscapes of the rural part of the Novosibirsk region. The south of the Western Siberian Plain is characterized by a continental climate, with short, warm summers and long winters; uniform humidity; and rather abrupt changes in all weather components over relatively short periods of time . This reTymovirales and Picornavirales; the families Partitiviridae, Totiviridae, Tombusviridae, Iflaviridae, Nodaviridae, Permutotetraviridae, and Solemoviridae; and four unclassified RNA-viruses. The previously described Partitivirus-like 1, Hammarskog tombus-like virus, Hammarskog picorna-like virus, Lymantria dispar iflavirus 1, Wenzhou noda-like virus 6, Mayapan virus, Sanxia permutotetra-like virus 1, and Chaq virus-like 1 were identified as practically complete genomes with levels of identity of 82–99% with the Cq. richardii mosquito. These viruses were found earlier in Liberia (west Africa), Sweden (north Europe), the USA (America), China (Asia), and Mexico . These findings allowed us to hypothesize that these viruses may be widely distributed on a global scale. In this study, we used a metagenomic-sequencing method to identify the viromes in seven mosquito species collected in the vicinity of Novosibirsk. The metagenomic approach was used to identify the viral diversity in randomly collected mosquitoes. We identified 30 coding-complete viral genomes and viral-like partial sequences of capsid proteins and/or RdRps from the mosquitoes . These sCq. richardii mosquito, a species that is widespread in the south of western Siberia [Some novel putative viruses and viral sequences had prototype viral sequences with identity levels from 31% to 79%, with these prototypes also being found in invertebrates from almost all continents. Some of them were associated with different species of mosquitoes. In our study, the majority of the novel viruses were associated with the  Siberia . The rolTymovirales and Picornavirales; the families Partitiviridae, Totiviridae, Tombusviridae, Iflaviridae, Nodaviridae, Permutotetraviridae, and Solemoviridae; and four unclassified RNA viruses. We believe that these virus identifications can enhance our understanding of the transmission of RNA viruses by mosquitoes in north Asia. We hope that the discovery and observation of these mosquito-borne viruses can help prevent future outbreaks of viral infections in the region under study.We identified novel and known viral genomes and viral-like partial sequences in mosquitoes collected in the Novosibirsk region of western Siberia. They were classified as novel putative viruses via a bioinformatics analysis of partial sequences of capsid proteins and RdRps or whole polyproteins (genomes) within the orders


Research has shown that many patients with type 2 diabetes (T2D) are not adherent to their medication regimen.To examine the association between adherence to insulin therapy and all-cause health care costs for patients with T2D.This study used the IQVIA PharMetrics Plus Linkable to Ambulatory Electronic Medical Record data from January 1, 2012, through September 30, 2017. Patients were included if they were identified with T2D and initiated therapy on basal insulin (BAS) or basal-bolus (BAS-BOL) combination at any time from January 1, 2013, through October 1, 2016. Patients aged < 18 years, who used an insulin pump, identified as pregnant, or did not have continuous insurance coverage from 1 year before initiation on insulin therapy through 1 year after initiation were excluded. Descriptive statistics compared patient characteristics and costs (in U.S. 2017 dollars) between patients who were adherent or nonadherent to their insulin therapy in the 1-year postperiod, where adherence was defined as having proportion of days covered (PDC) of at least 80%. In addition, generalized linear models were used to compare costs between adherent and nonadherent patients, while controlling for patient characteristics, previous general health and comorbidities, resource utilization, medication use and type of insulin.P = 0.0134) and BAS-BOL combination . Drug costs comprised 39.5%-45.4% of costs among adherent patients and 23.0%-25.9% of costs among nonadherent patients. Multivariable analyses revealed that adherent patients had significantly lower adjusted all-cause total costs than nonadherent patients in the BAS cohort  and the BAS-BOL cohort .13,296 patients were included in the BAS cohort  and 10,069 in the BAS-BOL cohort . Adherent patients had significantly lower all-cause total unadjusted costs following initiation on BAS ($29,322 vs. $31,888, In patients with T2D who initiated BAS or BAS-BOL combination therapy, adherence was associated with significantly lower all-cause total health care costs, despite significantly higher drug costs. These results illustrate the potential economic benefits associated with adherence to insulin therapy. What is already known about this subjectMost patients with type 2 diabetes will eventually require insulin therapy.Adherence to insulin therapy has been shown to be associated with better patient outcomes, including improved glycemic control, reduced resource use, and decreased mortality.Previous research, which has focused on the relationship between adherence and health care costs, has found mixed results, although most studies found that increased adherence was associated with lower total costs.What this study addsThis study expands knowledge of the association between adherence to insulin therapy and health care costs by focusing on 2 common insulin regimens: basal insulin and basal-bolus combination therapy.This study provides updated information on the costs associated with adherence to insulin therapy.1 In general, adherence to insulin therapy has been shown to be associated with better patient outcomes, including improved glycemic control,2 reduced resource use,3 and decreased mortality.4 However, in a multinational survey of individuals diagnosed with diabetes and treated with insulin, one third (33.2%) of the patients reported that they had been nonadherent to their insulin therapy for at least 1 day during the previous month.5 More recently, research that captured adherence using a Bluetooth-enabled pen cap found that 100% of patients prescribed multiple daily injections had at least 1 deviation from their insulin prescription over a 1-month time period and that rates of nonadherence ranged from 24% for patients treated with bolus insulin to 36% for patients treated with basal insulin.6 Consistent with the nonadherence found by patient self-report and technology-aided measures, nearly three quarters (72.5%) of physicians surveyed reported that their patients do not take insulin as prescribed.5Most patients with type 2 diabetes (T2D) will eventually require insulin therapy.7 The previous literature focused on basal insulin,3 the method of insulin delivery,10 or comparisons between U-500 and U-100 insulin.11 However, according to guidelines from the American Diabetes Association, many T2D patients require fast-acting (bolus) insulin at mealtimes, in addition to taking basal insulin, to achieve target hemoglobin A1c.12A review of the literature on medication adherence and health care costs among patients with T2D found that, in general, improved adherence to insulin was associated with reduced health care costs.The goal of this study was to further the literature by examining the concurrent association between all-cause health care costs and adherence to either basal insulin (BAS) or basal and bolus combination (BAS-BOL) therapy. As such, this study provides updated information on the costs associated with adherence to insulin therapy. Additionally, it compared the observed unadjusted and adjusted costs of 2 common insulin regimens (BAS and BAS-BOL).Study data came from the IQVIA PharMetrics Plus Linkable to Ambulatory Electronic Medical Record data. The combined data houses information on patient demographics, clinical encounters, laboratory test results, observational information such as body mass index and blood pressure, and insurance claims (inpatient and outpatient), including claims for prescription medications. All of the data used in this study were fully deidentified, HIPAA compliant, and collected between January 1, 2012, and September 30, 2017. As a retrospective, deidentified database, approval by an institutional review board was not required.13 over the time period from January 1, 2013, through October 1, 2016 (the identification window). In addition, all patients were required to have filled their initial prescription for BAS or BAS-BOL during the identification window, with the first such date identified as the index date, and to have continuous insurance coverage from 1 year before the index date (preperiod) through 1 year after the index date (postperiod).Patients were included in the study if they were identified as having T2D based on a previously published algorithm,Patients were excluded from the BAS and BAS-BOL cohorts if at any time from the start of the preperiod through the end of the postperiod they were identified as pregnant, diagnosed with gestational diabetes, or had a record of insulin pump use. Patients were also excluded if they were aged < 18 years as of the index date. Patients in the BAS cohort were also excluded if they used any insulin during the preperiod. The final sample consisted of 13,296 individuals in the BAS cohort and 10,069 in the BAS-BOL cohort. 15 In general, PDC was constructed with a numerator of the number of unique days during the postperiod that an individual had a supply of the intent-to-treat insulin (BAS or BAS-BOL), while only counting once if multiple prescriptions for the drug of interest is prescribed with overlapping days supply, and a denominator of the length of the postperiod (365 days).16 However, although the days supply field for insulin use is typically recorded as 30 days,17 insulin use depends on a variety of factors such as package size, carbohydrate intake, physical activity, body mass, illness, and insulin resistance.18 To account for this discrepancy, the days supply field was adjusted using a previously published algorithm. Specifically, for each insulin drug and delivery unique combination, an adjustment factor was calculated as the median time between insulin claims divided by the median pharmacy days supply (30 days).17 Consistent with a wide range of previous research, a threshold of 80% was used to classify a patient as adherent.19 Costs were defined as the contracted or accepted reimbursable amount for covered medical services or supplies that the health plan agreed to pay to service providers and were adjusted to U.S. 2017 dollars using the medical component of the Consumer Price Index.20This study focused on the association between patient adherence to insulin therapy and costs. Adherence was proxied by the proportion of days covered (PDC). PDC was chosen as the measure of adherence, since it is used by the Pharmacy Quality Alliance and the Centers for Medicare & Medicaid Services when examining treatment of patients with chronic conditions such as diabetes.21 Two-part models were used to estimate acute care costs.22This study examined the unadjusted differences in patient characteristics based on adherence status using t-statistics for continuous variables and chi-square statistics for categorical variables. The primary analyses examined the relationship between adherence and all-cause or diabetes-related costs, where diabetes-related costs were defined as all costs with an accompanying diagnosis of diabetes or receipt of a glucose-lowering agent (GLA). In addition, the analyses looked at several subcomponents of total health care costs. These subcomponents included drug costs, outpatient costs, and acute care  costs. Generalized linear models with gamma distribution and log link were used to estimate all total costs, drug costs, and outpatient costs.Separate models estimated costs for adherent and nonadherent patients. For each patient, 2 estimates of costs were computed: one assuming the patient was adherent (using the first model) and another assuming that the patient was nonadherent (using the second model). The difference in costs if adherent versus nonadherent were computed for each patient and averaged across the entire sample. Confidence intervals (CI) for costs differences were calculated using nonparametric bootstrap (random sampling with replacement) with 2,000 iterations to create a distribution where the 2.5th and 97.5th percentiles were the 95% CI lower and upper limits, respectively.23 and Diabetes Complications and Severity Index [DCSI]24); certain preperiod comorbidities not captured in the CCI or DCSI ; preperiod resource utilization ; preperiod use of GLAs ; and initial insulin type prescribed in the first month of the postperiod .All analyses controlled for patient characteristics ; preperiod general health . A In addition, there were indications from both cohorts that adherent patients were in better general health compared with nonadherent patients. Specifically, in both cohorts, adherent patients had statistically significantly lower DCSI scores and had a statistically significantly lower proportion of comorbid anxiety, visits to a cardiologist, an ER visit, or a hospitalization in the preperiod. However, compared with nonadherent individuals, in the preperiod adherent patients in both cohorts had a statistically significantly higher proportion of comorbid hyperlipidemia, use of a noninsulin injectable, and laboratory orders for A1c tests. Adherent patients also were prescribed statistically significantly more distinct classes of oral antidiabetic medicines and GLAs overall in the preperiod, while nonadherent patients were more likely to receive analog insulin delivered by vials in the first month of the postperiod.In contrast, diabetes-related total costs were significantly higher for adherent patients compared with nonadherent patients in both cohorts . All-cause drug costs were also significantly higher for adherent patients in both cohorts , as was diabetes-related drug costs .29 In addition, these study findings are in concert with research that has reported an association between adherence to BAS therapy and better patient outcomes.3In this study of patients who initiated either BAS or BAS-BOL insulin regimens, unadjusted descriptive statistics and multi-variable analyses revealed that adherence was associated with significantly lower all-cause total health care costs during the same year. As such, study findings are consistent with previous research, which has found that patients who are adherent to GLAs generally have better health outcomes, including less hospitalization and ER use, complications, and short-term disability days.7 Specifically, the majority of studies have found that adherence was associated with significantly lower all-cause total costs,33 despite the significant increase in all-cause medication costs associated with improved adherence.34 Furthermore, previous research has also found that improved adherence is associated with significantly lower inpatient and/or ER costs.32A recent meta-analysis of the association between medication adherence and outcomes among patients with T2D treated with GLAs reveal that the findings of this study regarding all-cause costs are largely consistent with previous research.8 while Perez-Nieves et al. (2018) found that greater adherence with basal insulin was associated with significant reductions in total and diabetes-related total costs, although there was no difference in diabetes-related total costs between adherent and nonadherent patients when adherence was proxied by medication possession ratio rather than PDC.3Previous research that has examined the relationship between medication adherence and total and diabetes-related costs among patients with T2D who are using insulin have found more mixed results. For example, Chandran et al. (2015) found that improved adherence to insulin pen therapy was associated with significant increases in post-index diabetes-related total costs,35 Furthermore, reductions in acute care costs have been shown to be associated with reduced hospitalization and ER resources.3 Given that fewer hospitalizations may be associated with improved long-term outcomes and increased health-related quality of life,36 the reductions in acute care costs may represent humanistic benefits to patients.For the BAS and BAS-BOL cohorts, improved adherence was associated with significantly lower all-cause and diabetes-related acute care costs. These reductions in acute care costs represent potentially large savings for payers, since previous research has found that 32.7% of all U.S. direct diabetes health care spending in 2017 consisted of hospitalization and ER costs.P < 0.0001).3 Our results are also broadly consistent with those of the Chandran et al. study of insulin pen users that used data from 2006-2010 and reported mean, annual, per patient health care costs in 2011 dollars of $26,310 for the least adherent and $23,839 for the most adherent patients (P = 0.007).8Few previous studies have examined the total health care costs of T2D patients on insulin therapy, let alone compared the costs of individuals using BAS with those using BAS-BOL. However, the adjusted total, mean, annual, per patient costs of the BAS cohort in this study  are generally consistent with, if slightly higher than, the 3-year health care costs of patients on BAS therapy reported in the Perez-Nieves et al. study  during the same period as the outcome (cost), we did not fully establish the temporal order for causal inference. It is thus possible that costs (or drivers of cost) could have influenced other variables that affected later adherence and thus the outcome influences cohort assignment.Finally, the analysis focused exclusively on statistical significance and did not consider whether such differences were clinically significant.The results from this study illustrate that patients who are adherent to either BAS or BAS-BOL therapies have significantly lower all-cause total health care costs compared with nonadherent patients, despite having higher all-cause and diabetes-related drug costs and higher diabetes-related total costs. In particular, adherent patients had substantially lower all-cause and diabetes-related acute care costs, suggesting fewer or less severe episodes of hospitalization or ER visits. These findings suggest that adherence to either a BAS or BAS-BOL regimen may be associated with lower overall costs for payers during the first year of initiation of these regimens.


Small RNAs (sRNAs) are the hallmark and main effectors of RNA silencing and therefore are involved in major biological processes in plants, such as regulation of gene expression, antiviral defense, and plant genome integrity. The mechanisms of sRNA amplification as well as their mobile nature and rapid generation suggest sRNAs as potential key modulators of intercellular and interspecies communication in plant-pathogen–pest interactions. Plant endogenous sRNAs can act in cis to regulate plant innate immunity against pathogens, or in trans to silence pathogens’ messenger RNAs (mRNAs) and impair virulence. Likewise, pathogen-derived sRNAs can act in cis to regulate expression of their own genes and increase virulence towards a plant host, or in trans to silence plant mRNAs and interfere with host defense. In plant viral diseases, virus infection alters the composition and abundance of sRNAs in plant cells, not only by triggering and interfering with the plant RNA silencing antiviral response, which accumulates virus-derived small interfering RNAs (vsiRNAs), but also by modulating plant endogenous sRNAs. Here, we review the current knowledge on the nature and activity of virus-responsive sRNAs during virus–plant interactions and discuss their role in trans-kingdom modulation of virus vectors for the benefit of virus dissemination. The gene-expression regulatory activities of non-coding small RNAs (sRNAs) are well-documented for a wide range of eukaryotic organisms, including plants ,2,3. PlaThe biological processes regulated by sRNAs depend on the nature of the target mRNAs. In plants, sRNAs are known to be involved with the regulation of developmental and physiological processes, genome reprogramming and integrity, responses to abiotic stresses, and defense against plant pathogens and pests ,5. AmongIn general, sRNAs generated in plants are divided into two major classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs). Most miRNAs are plant endogenous sRNAs derived from RNA precursors with imperfect hairpin structures, while siRNAs are processed from long dsRNAs derived from different sources (endogenous or exogenous), such as inverted repeats, sense-antisense overlapping transcripts, or synthesis by RNA-dependent RNA polymerases (RDRs) ,8. Also Plant virus diseases affect many important crops and lead to considerable economic losses in agriculture worldwide. By unraveling and understanding the virus-mediated alterations leading to plant disease development and/or plant resistance mechanisms, new strategies can be developed in order to fight plant viral diseases. In addition of modulating protein, volatiles, and phytohormones content, virus infection also significantly alters the accumulation and profile of sRNAs in infected plants, not only by generating virus-derived siRNAs (vsiRNAs) as a consequence of the activation of the antiviral RNA silencing pathway, but also for altering the profile of plant endogenous sRNAs . FurtherPlants are exposed to a variety of conditions presented by their surroundings, such as abiotic and biotic stresses, that require physiological adjustments and/or immune responses that rely on their genome plasticity . RNA silAs a result of the virus–plant co-evolution processes, plant viruses widely depend on their plant hosts for replication, protein expression, assembly, and spread throughout and between plants. This cycle of virus infection is accompanied by a variety of changes in the plant host as the result of differential gene expression and likely reflecting the general and specific interactions between the virus and the plant host . GeneralWhen viruses infect susceptible plant hosts, virus-derived dsRNAs are made available (mostly as an intermediate product of virus replication) and trigger the activation of the host’s antiviral RNA silencing pathway. The virus-derived dsRNAs are cleaved by DCL proteins, producing vsiRNAs of 21–22 nts in size that direct sequence-specific and AGO-mediated posttranscriptional gene silencing (PTGS) of viral RNA. In case of DNA virus infections, 24 nts sized vsiRNAs are additionally produced, which direct the transcriptional gene silencing (TGS) of the viral DNA genome ,26,27,28After the discovery of RNAi and its antiviral activity in 1990s, plant virologists started to focus on vsiRNA profiling, studying how viruses evade the cytoplasmic RNA degradation machinery or suppress antiviral RNAi, and to exploit RNAi by development of RNAi-mediated resistance strategies. Only a decade later, this view expanded more towards RNAi-mediated (modulating) effects on the host, caused either by the virus or their vsiRNAs, as beneficial towards the establishment and maintenance of virus replication and dissemination.By using combinatorial genome-wide-based approaches to study the virus-derived sRNA regulatory network in plants, several works have demonstrated that plant virus-derived sRNAs are able to modulate host gene expression by targeting a wide range of host endogenous mRNAs ,26. HoweNicotiana tabacum by a SatY-derived vsiRNA, leading to a loss of chlorophyl and causing yellowing of leaves. Mutation of the corresponding SatY sequence led to a mismatch of the vsiRNA on the chlorophyl synthesis gene, and disappearance of the yellowing. Although this can easily be taken as a coincidence and classified as side-effect damage, yellowing of leaves attracts insect vectors and as such will contribute to the dissemination of plant viruses from plant to plant [One of the first examples is the chlorosis observed in plants after cucumber mosaic virus (CMV) infection in the presence of Satellite Y . This turned out to result from the silencing of magnesium protoporphyrin chelatase I subunit (CHLI) mRNA in the host to plant ,33 -related (CAR) 7-like protein (Nb-CAR7). Nb-CAR7 plays a critical role in ABA signaling, an important hormone in plant responses to abiotic stresses as well as to plant pathogens, including viruses. Consistently, Nb-CAR7-overexpressing plants show a decrease in TMV RNA accumulation, while dowregulation of Nb-CAR7 resulted in a higher TMV RNA accumulation [A study on tobacco mosaic virus (TMV) showed that vsiRNAs derived from the 3′ end of the TMV genome (TMV-vsiRNA) showed high sequence complementarity to a mulation  -PPases (VPs). The VPs are regulators of active proton (H+) transport through membranes and play key roles in plant responses to abiotic stresses. However, accumulation of CWMV RNA1-derived sRNA in wheat has been shown to affect the concentration of H+ in wheat cells, thus maintaining the cytoplasm alkaline, and creating a more favorable environment for CWMV replication [Chinese wheat mosaic virus (CWMV) also generates a vsiRNA that contributes to virus infection in the wheat host by targeting and silencing the gene encoding for the vacuolar  RNA 4-derived sRNA (vsiRNA-4A), for example, targets and downregulates the eukaryotic translation initiation factor 4A (NbeIf4A) in stunting . Interes protein . Thus, sTaAAED1) which encodes for a negative regulator of reactive oxygen species (ROS) in the chloroplast. Thus, WYMV-derived vsiRNA1 activates host immunity via ROS signaling, which promotes cell death and regulates defense-related genes [Another example is a vsiRNA derived from wheat yellow mosaic virus (WYMV) RNA1. WYMV-derived vsiRNA1 has been shown to activate wheat immunity by targeting and modulating the expression of the wheat thioredoxin-like gene , African cassava mosaic virus (ACMV), and east African cassava mosaic virus-Uganda (EACMV-UG), were identified targeting host plant (Jatropha curcas) genes involved in biotic response, metabolic pathways, and transcription factors [Potyviridae. Turnip mosaic virus (TuMV)-derived miRNAs, for example, were found to downregulate a host stress-responsive gene (HVA22D) in Arabidopsis [The wide range of host target mRNAs indicates that virus-derived sRNAs are determinants in the outcome of plant–virus interactions . Interes factors . However factors . Surprisbidopsis . Sugarcabidopsis . The activation of antiviral RNAi during plant viral infections not only triggers the accumulation of vsiRNAs, but it is also accompanied by the production of plant endogenous sRNAs  with modulating effects on the host to support the establishment and maintenance of virus infections or, in some cases, to activate antiviral host defense mechanisms . Viral iPseudomonas syringae pv. tomato (Pst) DC3000 [One of the first identified pathogen-induced host endogenous siRNAs involved in the regulation of plant immunity/defense against the pathogen was reported during a study on the bacterial pathogen ) DC3000 . The repN. benthamiana LAB accession, known to lack a functional RDR1 gene. Abundant vasiRNAs were also absent from tomato variety Red Defender, containing the Sw-5 resistance gene against orthotospoviruses. Many of the endogenous vasiRNAs identified in this study were similar to those identified in earlier studies [The first studies on endogenous host sRNAs activated upon virus infection started to appear after the turn of the millennium in 2000. One of the first papers reporting on this phenomenon was from 2010, in which the induction of miRNA168 (the AGO1 regulator plant miRNA) by the tombusvirus p19 RNAi suppressor protein was shown . Althoug studies ,62, furtFrom the vasiRNA-associated transcripts identified by Fletcher et al. , many weDespite the limited number of studies, and first indications pointing towards a role for vasiRNAs in a broad-spectrum resistance against viruses, a possible pro-viral activity should not be ruled out, similar to what has been observed for cytoplasmic RNA processing bodies and stress granules that, for certain viruses, exhibit a pro-viral activity, but towards others function as anti-viral . While t2+ concentration and sensing by calmodulins (CaMs) or calmodulin-like proteins (CMLs) (Ca2+ sensors), to induce/activate downstream defense responses and a rapid increase in phytohormones such as jasmonic acid (JA), to defend and protect against herbivores. For further details on this topic, readers are referred to some recent reviews presented by Ghorbel et al. [2+-transporting-ATPase genes, known to be involved in Ca2+ transport, indicating the importance of the Ca2+ signaling cascade in defense against insect attack [Viral infections are known to cause a range of physiological and metabolic changes in the host plant, which are observed to influence the behavior and fitness of insect vectors as well ,71. A nul et al. , Mostafal et al. , Parmagnl et al. , and Strl et al. . Upon het attack ,86. 2+-ATPases act as ion transporters across cellular membranes and belong to the P-type ATPases superfamily, that either localize at the ER or at the plasma membrane [Camembrane . Intrigumembrane ,65. Howemembrane ,65 is un2+ transport as part of a signaling cascade to defend against herbivores, but also in RDR1 and RDR6 dependent amplification of vsiRNAs and RDR1-dependent endogenous vasiRNAs [2+ signaling. A recent study by Wang et al.  has showvasiRNAs ,65,85,862+-induced, and further downstream, JA-mediated defense responses via the very same VSRs or other viral proteins [Viruses do not only counterdefend against antiviral RNAi by direct interference at major steps of the RNAi pathway via viral suppressor proteins of RNAi (VSRs), topics that have been extensively reviewed in the past decade ,28,88. Tproteins ,91,92,93proteins . The tobproteins . The TSWproteins ,96,97. Oproteins ,99. A stproteins  demonstrproteins  furtherm2+ -sensors. Vice versa, insects feeding on host plants leads to direct changes in Ca2+ concentrations, and their sensing by calmodulins not only activates downstream defense responses, but also simultaneously activates RDR6 and bifunctional nuclease-2 (BN2) via transcription factors, culminating with the priming and enhancing of antiviral RNAi responses. So far, the activity of sRNAs produced in plants (either in the absence of a virus or induced upon virus infection) is considered to be primarily restricted to the host in which they arise, while viruses have also evolved an intimate relation with their vectors, such as aphids, whiteflies, and thrips, for dissemination. Since viruses utilize a plethora of strategies to manipulate the host cell machinery for the benefit of replication, it is very well possible that during the modulation of the plant sRNA profile some of these sRNAs also serve to modulate the host as well as the insect vectors regarding their attraction and feeding behavior, respectively, to enhance virus dissemination. Although there is no evidence for this concept yet, this idea is not unlikely considering the intimate tritrophic interactions between virus, plant, and vector and observed changes in vector feeding behavior.While nowadays RNAi is a commonly recognized antiviral defense mechanism involving the production of vsiRNAs, the accessibility to more affordable high-throughput sequencing technologies and metagenomics studies slowly help us to unveil a view in which viruses modulate another part of the sRNA spectrum that goes beyond vsiRNAs, i.e., the induction of host vamiRNAs and vasiRNAs. Not only are the effects of these sRNAs on viral disease and host defense responses yet to be fully uncovered, but also the absolute role of VSRs in suppression of the antiviral RNAi response, the modulation of host gene regulation and the ability to indirectly modulate other pathways, such as JA, that protect and defend against herbivores, via interaction/inhibition of factors such as calmodulin CaE. coli expressing dsRNAs [What would support the idea of trans-kingdom sRNA communication that possibly might affect virus vector behavior and support virus spread? Which indications are there pointing to this? One of the first examples of trans-kingdom RNAi came from studies in which nematodes were fed with g dsRNAs , and demMeanwhile, several examples of trans-kingdom RNAi can be found in plant–fungal pathogen interactions. For an overview on this subject, readers are referred to reviews from Hua et al.  and ChalMyzus persicae and required as a chaperone to support (persistent) potato leaf roll luteovirus transmission [Aedes aegypti mosquitoes, indicating the possibilities of ncRNA communication and modulation of virus insect vectors [Crosstalk between insect pests and other species  at the level of protein or RNA effectors that affects virus vector competency and transmission has been observed. One of the first studies of a trans-kingdom protein effector to influence virus vector transmission identified GroEL, a protein produced by the endosymbiont of the aphid smission . In relasmission  when plasmission , and alt vectors . Finding vectors . They al vectors ,109.Despite the absence of reports on trans-kingdom RNAi between virus-infected plants and their virus insect vectors, a growing number of studies support the notion that during intimate tritrophic plant–virus–vector interactions, not only the host plant but also virus insect vectors are modulated by protein and sRNA  effectors, collected during virus acquisition feeding on infected plants, which ultimately affect insect behavior and virus competence for the benefit of virus dissemination. Considering that plant viruses are known to be transmitted by insects either non-persistently (stylet-borne), persistently , or in propagative way , an additional challenge would be to find out whether insect vectors also are affected differently by trans-kingdom sRNAs, or if a similar/conserved set of trans-kingdom sRNA-mediated pathways are modulated in all these cases. Furthermore, whether vsiRNAs or miRNAs and vasiRNAs play a role during virus transmission by insects, or trans-kingdom RNAi is solely restricted to vasiRNAs, remains another interesting question waiting for scientific answer. In the occurrence of trans-kingdom RNAi, the situation becomes even more complex with persistent-propagative transmitted plant viruses, e.g., orthotopoviruses, tenuiviruses, and emaraviruses , due to Considering our current knowledge on antiviral RNAi, studies on trans-kingdom RNAi between plant host, virus, and insect vectors will be one of the next challenges for the future. It will not only provide a much more integral and holistic view and help us to further decipher their tritrophic interplay and communication, but also will contribute to our understanding on the (protein and RNA) languages spoken and in specific, the importance of specific sRNAs in these. Their identification may provide new targets for the development of strategies that may help to either prime/boost host plant defense responses and increase the resilience of crops against biotic stressors, or also interfere with virus transmission and the competence of insect vectors.


Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airway surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts.Secondary infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, but did show a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially complex solutions to better mimic physiologically relevant conditions.The impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with  Environmental stability of respiratory pathogens expelled from an infected host is a key factor impacting transmission . PreviouS. pneumoniae (Spn) secondary infection in animals have shown that influenza virus facilitates transmission of S. pneumoniae  in H1N1pdm09 stability with or without Spn   . In contS. pneumoniae result in increased morbidity and a greater risk of bacterial transmission  and a condensation air sampler (Aerosol Devices Liquid Spot). The limitations of these samplers include a short sampling period from 15 min to 1 h, which captures only a snapshot of what is in the air; a lack of detection does not mean that these pathogens are not expelled into the air. In addition to the sampling time, the amount of virus detected in the air will be limited by the flow rate of the specific sampling strategy, the amount of virus expelled by an infected ferret, and the distance of the infected source to the sampling device. These factors contribute to the observed heterogeneity in microbial detection observed in our study and necessitate the need for thoughtful use of specific air sampling devices or incorporation of multiple air sampling devices at varying flow rate and times.Co-infection with pathogens can impact the transmissibility to subsequent hosts. Concurrent infections of influenza virus and smission , 14. TheS. pneumoniae, but a trend towards increased S. pneumoniae stability in the presence of influenza virus may help explain augmented S. pneumoniae transmission in addition to the increased bacterial shedding observed during co-infection (No impact was observed for influenza virus stability in the presence of nfection , 14. InvMethods can be found in the supplemental material.