This study investigates the therapeutic efficacy of combining Metformin (MET) and Celecoxib (CXB) in the treatment of breast cancer. Niosomes, which are drug carriers, were prepared using the thin-film hydration technique. These niosomes were characterized and found to have stable properties when stored at 4 °C for three months. The encapsulated drugs in the niosomes showed increased cytotoxicity compared to their free drug counterparts in both two-dimensional and three-dimensional viability assays. The combination of Metformin Niosomal Particles (MET NPs) and Celecoxib Niosomal Particles (CXB NPs) also led to decreased cell viability in both models. However, the efficacy of the niosomes’ combination was not superior to that of the free drug combination in preventing cell migration. Overall, this study provides valuable insights into the potential application of combining MET and CXB nanoparticle delivery systems for breast cancer treatment.p < 0.05). In conclusion, the results of this study provide valuable insights into the potential application of combining MET and CXB nanoparticle delivery systems to breast cancer treatment. Exploring the in vivo application of this drug delivery system could open new avenues for more effective and targeted therapeutic approaches for breast cancer patients.Breast cancer continues to be a prominent worldwide health concern and requires continued investigation into innovative therapeutic approaches. Here, we report the first investigation into the therapeutic efficacy of combining Metformin (MET) and Celecoxib (CXB), both in free and niosomal form, for the treatment of breast cancer. Our investigation encompassed the characterization of these niosomal drug carriers, their stability assessment, and their effect on breast cancer cell models. The thin-film hydration technique was employed to prepare niosomes with spherical, uniform-size distributions and high encapsulation efficiencies. The niosomes were characterized by TEM, particle size analyzer, and ATR-FTIR. The niosomes with an average size of 110.6 ± 0.6 and 96.7 ± 0.7, respectively, for MET and CXB were stable when stored at 4 °C for three months with minimal drug leakage, minor changes in encapsulation efficiency and size, and unchanged physicochemical parameters. Evaluation in two-dimensional (2D) and three-dimensional (3D) viability assays demonstrated an increased cytotoxicity of encapsulated drugs when compared to their free-drug counterparts. Additionally, the combination of Metformin Niosomal Particles (MET NPs) and Celecoxib Niosomal Particles (CXB NPs) led to decreased cell viability in both 2D and 3D models compared to each drug administered individually. When comparing the effect of the niosomal versus the free combination of the drugs on cell migration, we found that both interventions effectively prevented cell migration. However, the efficacy of the niosomes’ combination was not superior to that of the free drug combination ( Breast cancer is a highly prevalent and potentially lethal disease that significantly impacts women’s health globally . The manDespite the advances in cancer research and the wide range of therapeutic options currently available, there are still persistent hurdles in the development of effective and selective drugs for breast cancer. In addition, the use of existing chemotherapeutic agents for breast cancer is limited by many factors such as loss of efficacy due to the development of drug resistance, inadequate selectivity, and the occurrence of adverse effects . For thiThe repurposing of drugs in breast cancer has emerged as a promising strategy to increase available therapeutic options and enhance patient survival. This approach accelerates the drug development process, leveraging established agents’ existing safety profiles and providing novel and cost-effective treatment options for breast cancer patients. For instance, CXB, a nonsteroidal anti-inflammatory drug, and MET, a widely used medication for type 2 diabetes, have been investigated as potential candidates for repurposing due to their established pharmacological properties and safety profiles. Preclinical studies have demonstrated that CXB can suppress breast cancer growth through its anti-inflammatory effects . SimilarFurthermore, the utilization of the 3D spheroid model is increasingly recognized as a crucial instrument in the field of cancer research due to its ability to represent an intermediate level of complexity, bridging the gap between 2D monolayer models and in vivo solid tumors . FurtherTM 60, and cholesterol were purchased from Sigma Aldrich . Ammonium acetate ≥97.0% ACS was purchased from VWR Chemicals BDH®, glacial acetic acid from Honeywell® , Penicillin-Streptomycin Solution 100× from Euroclone® , RPMI 1640 medium with L-Glutamine from Caisson Labs , Dulbecco’s Phosphate Buffered Saline from Euroclone® , L-Glutamine 100X 200 mM from Euroclone® , Sodium Pyruvate Solution from Eurobio® Scientific , Cytiva® HyClone Fetal Bovine Serum from Global Life Sciences® , CellTiter-Glo® 3D Cell Viability Assay from Promega® , Thizolyl Blue Tetrazolium Bromide Powder BioReagent from Sigma-Aldrich Inc.® , dimethyl sulfoxide (DMSO) from Sigma-Aldrich Inc.® , and ROS-Glo™ H2O2 assay from Promega® .MET HCL and CXB were generously donated by Dar al Dawa Pharma . Chloroform, diethylamine, triethylamine, as well as HPLC-grade acetonitrile, methanol, and water were purchased from Tedia . A phosphate buffered saline (PBS) tablet, SpanThe preparation of niosomes was performed using the thin film hydration method. Niosomes loaded with MET and CXB were formulated by dissolving Span 60 and cholesterol or Span 60, cholesterol, and Tween 80, as presented in ® digital camera. The niosomes were diluted in distilled water at a volume-to-volume ratio of 1:2% and afterward applied onto a carbon-coated copper grid. The samples were then let to dry before being subjected to imaging. ImageJ® 154a software was employed to analyze the niosomes’ morphology.The morphology of the niosomes was examined using TEM, which was equipped with a Mega View IIThe particle size (PS) and polydispersity index (PDI) of the chosen formulation were assessed utilizing dynamic light scattering (DLS) through a Brookhaven 90-plus particle size analyzer . The electrophoretic light scattering (ELS) technique was employed for the measurement of the zeta potential (ZP) of particles. The sample was prepared by dispersing 50 µL of niosomal suspension in 950 µL of distilled water at 25 °C.The optimization of the purification method for niosomal drugs was achieved by investigating the kinetics of drug movement through a dialysis membrane with a molecular weight cutoff of 12–14 kDa. For MET NP purification, a drug solution was prepared by dissolving 100 mg of MET in 15 mL PBS, and the membranes were filled with 1 mL of the solution and immersed in beakers with 50 mL PBS while being stirred. HPLC-UV analysis of the samples taken at various time intervals over four hours determined the time required to remove all free drugs from the dialysis bag. The same method was applied to evaluate the purification of CXB NP, where 10 mg of CXB NP was dissolved in 15 mL of methanol, and 1 mL of the solution was transferred to the dialysis bag immersed in 200 mL of PBS. Samples were taken and analyzed to determine the optimal time for the purification step.After the purification process, the MET in its free form was removed from the dialysis membrane, leaving pure MET NP inside the dialysis bag. To determine the drug encapsulation efficiency (EE) of MET NPs, a 1 mL sample was collected from the dialysis medium at the 1 h mark and diluted with PBS to a final volume of 10 mL. Subsequently, the sample was subjected to HPLC with ultraviolet detection (HPLC-UV) to quantify the EE, as follows:Here, “Total amount of MET” denotes the initial quantity of MET used for the preparation of MET NPs, while “Free amount of MET” represents the portion of MET that remained unencapsulated within the niosomal formulation.For the purification of CXB, the process required 2 h, after which the contents of the dialysis bag were transferred to a centrifuge tube. A 100 μL sample of purified niosomes was solubilized with 10 mL of isopropyl alcohol, sonicated for 15 min, and subsequently analyzed using HPLC-UV. A 1 mL sample was withdrawn from the solution for analysis. The EE of CXB was calculated using the following equation:In this equation, “Entrapped amount of CXB” represents the quantity of the drug effectively encapsulated within the niosomes, while “Total amount of CXB” signifies the total amount of CXB utilized during the preparation process.The formulated MET NP (M2) and CXB NP (O2) were separated from the unentrapped drug and subjected to freeze drying at −50 °C for 48 h after overnight freezing at −80 °C. The resulting white powder niosomes were stored in a tightly sealed glass container at 4–8 °C.−1 with a resolution of 2 cm−1, capturing 32 scans for each sample. The spectrum data obtained were subjected to analysis using Ira® FTIR data explorer V 1.0 software. The ATR-FTIR technique was utilized to analyze the spectra of MET, CXB, and Span 60, Cholesterol and the physical mixture of the components were used for niosomal formulations and the blank niosomal formulations.The Perkin Elmer UATR-II instrument  was utilized to conduct ATR-FTIR spectroscopy in order to evaluate potential incompatibilities. The samples underwent examination in the wavenumber range of 4000–400 cmIn vitro release of the (M2) MET NP and (O2) CXB NP was assessed using a dialysis membrane with a molecular weight cutoff (MWCO) of 12–14 kDa as described . Prior tA short-term stability assessment was performed to evaluate the alterations in PS, PDI, and EE% of the optimal niosomal formulations (M2 and O2) over a duration of four months. These formulations were selected based on their characterization results. Throughout the study, the desired formulations were stored at a temperature of 4 °C.® , and the mobile phase was set to flow at a rate of 1.0 mL/min. The injection volume for HPLC analysis was 10 μL, and the drug detection was conducted at λmax 233 nm. The HPLC analysis was performed at 25 °C, and the mobile phase consisted of a mixture of Acetoni-trile and 0.02 M Ammonium Acetate Buffer at a ratio of 80:20, v/v%. In contrast, the chromatographic separation of CXB NP was performed utilizing the aforementioned column, with the mobile phase being adjusted to a flow rate of 1.5 mL/min. The injection volume utilized for HPLC analysis was 20 μL, while the detection of the drug was performed at a wavelength of maximum absorption (λmax) of 253 nm. The temperature was adjusted to 25 °C, and the mobile phase consisted of a mixture of the Acetoni-trile and the 0.02 M Ammonium Acetate Buffer at a ratio of .A Shimadzu HPLC system  equipped with a SIL-20A autosampler, SPD-20A detector, CTO-20A temperature regulator oven, and LC-20AT pump was used for the quantification of both MET NP and CXB NP. Chromatographic separation of MET NP was achieved using a Zorbax C8 column  from AgilantThe MCF-7 and MDA-MB-231 cell lines used in this study were obtained from the American Type Culture Collection (ATCC), Manassas, VA, USA.2. The medium used in this study was RPMI 1640, supplemented with 10% v/v fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% v/v Penicillin —Streptomycin (10 mg/mL).The MCF-7 and MDA-MB-231 cell lines were cultivated and maintained in a humidified atmosphere at 37 °C with 5% CO3 cells/mL and were allowed to attach and grow for 24 h. Subsequently, the cells were treated for 72 h with varying concentrations of MET, MET NP (50–2.5 mM), CXB, and CXB NP (100–5 μM) to compare the free and nanoparticle drug forms. Cell viability was assessed using the MTT assay  following the manufacturer’s instructions. Briefly, after the treatment period, 20 μL of the MTT solution (5 mg/mL) was added to each well, and the plate was incubated at 37 °C for 4 h. Following this, the aqueous solution was removed, cells were lysed using DMSO, and the optical density of each well was measured at 570 nm. The percentage of viable cells was calculated relative to the untreated control cells, enabling the assessment of the inhibitory effects of different drug concentrations on cell viability. To construct dose-response curves for each drug in both its free form and niosomal formulation, a comprehensive range of concentrations was tested to evaluate the inhibitory effects on proliferation. Non-linear regression analysis with curve fitting using GraphPad Prism Version 9.0.0 was employed to determine the IC10, IC20, and IC50 values for each compound representing the drug concentrations causing 10%, 20%, and 50% inhibition of the biological response, respectively.MCF-7 and MDA-MB-231 breast cancer cells were seeded in a 96-well plate at a concentration of 5 × 10A suspension of MCF-7 cells, consisting of 20% methylcellulose (MC), was introduced into the wells of an ultra-low attachment plate at concentrations of 2000 and 10,000 cells per well. The plate underwent centrifugation at a speed of 1000 (rcf) for a duration of 10 min. After a period of 24 h, spheroids were formed and monitored for growth by measuring the diameter for 7 days.® 3D Cell viability assay , a specialized assay built expressly for this objective.In order to assess the viability of spheroids inside the 3D cultures, we utilized the Cell Titer-GloFollowing a 72 h period of spheroid formation, we administered different treatments, namely MET (ranging from 2.5 to 100 millimolar) and MET NP (ranging from 2.5 to 50 millimolar), as well as CXB (ranging from 10 to 200 micromolar) and CXB NP (ranging from 10 to 100 micromolar). Control groups, including untreated spheroids and spheroids treated with blank niosomes at equivalent drug concentrations, were also used. The plate was incubated for 72 h to facilitate the attainment of the utmost drug effect.® reagent was added. Subsequently, the luminescence signal was measured using the Glomax® Multi detection system. Viability was calculated using the formula:Following the incubation period, the spheroids were transferred to white opaque-walled 96-well plates, and the CellTiter-Glo10, IC20, and IC50 values for each compound were established by non-linear regression analysis with curve fitting, and GraphPad Prism Version 9.0.0 was utilized to ascertain these values for each compound.The ICIn order to verify the penetration of drugs into the spheroids, drugs were introduced into the wells of 3-day-old spheroids at concentrations of 100 mM MET, 100 mM MET NP, 100 µM CXB, or 100 µM CXB NP. To accurately measure the quantity of drug that did not enter the spheroids, we collected 100 µL samples from the surrounding media at specific time intervals: 1, 2, 3, 4, 24, 48, and 72 h.In order to eliminate any potential influence from the media components, the collected samples were diluted with acetonitrile and subsequently underwent a 15 min centrifugation at a speed of 2000 rpm. Afterward, the supernatant was collected and mixed with isopropyl alcohol to break the niosomes apart and retrieve the remaining drug trapped inside them. This allowed us to obtain a suitable concentration for later analysis using HPLC. HPLC analysis quantified the non-penetrated drug amount, allowing for the calculation of drug penetration by subtracting this quantity from the initially added 100% of the drugs.5 cells/mL in 2% FBS media. After 24 h, a uniform scratch wound was created using a 200 µL pipette tip, and the cells were washed to remove any debris.The anti-migratory properties of various formulations were assessed using the wound healing assay. Briefly, the breast cancer cell line MCF-7 was cultured in a 24-well plate at a seeding density of 8 × 1010 concentrations of MET (0.3 mM) and CXB (1.0 µM) individually, as well as a combination of MET and CXB at concentrations of (0.3 mM and 1.0 µM), respectively. Additionally, MET NP (0.2 mM), CXB NP (0.7 µM), and a combination of MET NP and CXB NP 0.2 mM and 0.7 µM, respectively, were examined. Hydrogen peroxide (H2O2) was introduced at a concentration of 200 µM to enhance cell migration and mimic wound healing conditions [2O2 (200 µM) alone as a positive control. Images of the wound area were captured at 0 and 48 h using an OPTIKA® Inverted Microscope IM-3 . ImageJ 154a software was used to quantify and measure the wound’s free surface area at two different time points. To assess cell migration, we determined the percentage of free surface area after migration using the following formula:The treatment groups consisted of MCF-7 cells exposed to ICnditions . Controlt-test, and p-values were computed to evaluate the statistical significance of the findings. In this study, statistical significance was represented by an asterisk (*) when the p-value was less than 0.05 and by two asterisks (**) when the p-value was less than 0.01.The statistical analysis was conducted using either GraphPad Prism software version 9.0.0 or Microsoft Excel. The studies were performed in triplicate, and the findings are reported as means ± standard deviation (SD). The means were compared using a two-tailed Student’s The thin film hydration approach was successfully employed to prepare several formulations of either niosomal formulations loaded with MET or CXB. The morphology of the developed niosomes was validated using TEM, as depicted in p < 0.01) and M3 (p < 0.01). In contrast, the CXB NP formulations demonstrated PS values ranging from 96.7 ± 0.7 nm to 159.1 ± 1.7 nm. Interestingly, O2 displayed the smallest particle size, with statistical significance compared to O1 (p < 0.01) and O3 (p < 0.01).Regarding particle size (PS), the MET NP formulations displayed a size range from 110.6 ± 0.6 nm to 129.5 ± 3.1 nm. Notably, M2 exhibited the smallest particle size, with statistical significance observed compared to both M1  and Samed et al. (2018) [−1 and 736 cm−1 were identified, as documented by Kenechukwu et al. [The infrared spectra exhibited the primary absorption bands for MET, which included two typical bands at 3291.5 cm. (2018) ,22. Furtu et al. .−1 and 3230.5 cm−1, indicating the stretching of N-H2 bonds. Additionally, a band at 1346 cm−1 was observed, corresponding to the stretching of S=O bonds. Two further bands were identified at 1274.5 cm−1 and 1229 cm−1, which can be attributed to the stretching vibrations of C–F bonds. Lastly, another band associated with the stretching of S=O bonds was detected at 1156 cm−1, as previously reported by Lakshmi et al. and Vijayakumar et al. [In the case of CXB, the primary peaks were observed at 3336 cmr et al. ,25.−1 as a result of OH stretching, 2917 cm−1 suggesting -CH stretching, and a notable peak at 1736 cm−1, signifying the robust C=O ester bond. Farmoudeh et al. and Ur Rehman et al. have reported the presence of supplementary peaks at 1174 cm−1, which can be attributed to the stretching vibrations of C–O and C–C bonds [−1, which belongs to C–C connections. In contrast, cholesterol exhibits distinct spectral peaks at specific wave numbers. Notably, a peak is observed at 3430.5 cm−1, which corresponds to the stretching of O-H bonds. Additionally, peaks at 2931 cm−1 and 2867 cm−1 are attributed to the stretching of C-H bonds. Furthermore, vibrations associated with C-O bending are evident at 1055 cm−1. Finally, two peaks at 958.5 cm−1 and 840.5 cm−1 are observed, which can be attributed to aromatic substitutions, as presented by Samed et al. and Zaid Alkilani et al. [The primary spectral peaks of Span 60 were detected at around 3400 cm–C bonds ,27. Furti et al. .−1 in MET, CXB, and blank niosomes. This signal arises from the OH stretching vibration of cholesterol and Span 60, indicating their interaction and bonding in the formation of the niosomal structure. Interestingly, none of the characteristic peaks for CXB are observed in its corresponding niosomal formulations. Conversely, in MET niosomes, only minor shifts from 736 cm−1 to 742 cm−1 are evident, signifying an interaction between MET and the formulated niosomes through bond formation. This absence of most of the peaks in the case of MET can be attributed to the entrapment of the drugs within the core of the niosomes. No free drug molecules are detected on the surface of the niosomes due to the optimized washing process designed to completely eliminate unentrapped molecules. Additionally, the weight of the drug loaded, whether MET or CXB, is minimal compared to the total formula weight , which explains the absence of all CXB peaks.In −1 to 2937 cm−1, from 2867.5 cm−1 to 2870.5 cm−1, and from 481.5 cm−1 to 844.5 cm−1. Similarly, Span 60 characteristic peaks shifted from 3400 cm−1 to 3391 cm−1, from 1736 cm−1 to 1739.5 cm−1, and from 1174.5 cm−1 to 1177.5 cm−1.To further confirm the niosomal formulation in M2 niosomes, characteristic cholesterol peaks shifted from 2931 cm−1 to 2871.5 cm−1 and from 840.5 cm−1 to 844.5 cm−1, while Span 60 characteristic peaks shifted from 3400 cm−1 to 3391.5 cm−1 and from 1735.5 cm−1 to 1738.5 cm−1.For the confirmation of niosomal formulation in O2 niosomes, characteristic cholesterol peaks shifted from 2867.5 cmThis study aimed to evaluate the release profiles of MET NP (M2) and CXB NP (O2) under two distinct pH conditions: an acidic pH of 5.1 and a physiological pH of 7.4. These pH values were deliberately chosen to enable a comparative analysis of drug release behavior. This choice stemmed from previous research indicating that cancer cells typically exhibit a lower pH environment compared to the typical pH of 7.4 found in healthy cells . It is iThese specific formulations, namely M2 and O2, were chosen due to their higher percentage of EE, indicating their greater ability to retain the drugs. The release characteristics of both medications over a 72 h span at pH 5.1 are shown in It has been previously shown that niosomes containing MET release the drug at a slower rate compared to the free drug solution . The relThe behavior of nanocarriers in vitro and in vivo is influenced by their stability . Hence, p < 0.05). In contrast, the O2 niosomal formulation exhibited negligible leakage, as evidenced by the lack of a significant disparity in EE from 94.54 ± 2.09 to 90.73 ± 3.26 (p > 0.05) between the freshly prepared formulation and the one stored for three months  of M2 had a modest reduction from 68.94 ± 1.28 to 57.12 ± 0.95 . Similarly, for MDA-MB-231 cells, free MET and MET NP displayed concentration-dependent effects on cell viability (p < 0.05).Both free MET and MET NP showed a concentration-dependent decrease in MCF-7 cell viability a. Howeveiability b. Consisp < 0.05).In regard to CXB treatments, in MCF-7 cells a, both fp < 0.05). These results imply that the effect of nanoparticle delivery on MET and CXB efficacy might vary between different breast cancer cell lines.Similarly, for MDA 231 cells, free CXB and CXB NP displayed concentration-dependent effects on cell viability b. Furthe10, IC20, and IC50 values, which are vital for potential combination therapies. The outcomes revealed that the nanoparticle formulations (MET NP and CXB NP) demonstrated higher cytotoxicity at lower concentrations compared to the free drugs , 60.86% (at IC20 of MET and CXB) (p < 0.01), and 25.22% (at IC50 of MET and CXB) (p < 0.01) (p < 0.05), 62.52% (at IC20 of MET and CXB) (p < 0.05), and 31.21% (at IC50 of MET and CXB) (p < 0.01)  a. In the < 0.01) b. The fiDespite promising results in 2D preclinical studies, several drugs have encountered significant failures when translated into clinical settings . Therefo® 3D cell assay, viability was calculated relative to untreated control spheroids, and dose-response curves were constructed to determine the IC10, IC20, and IC50 values , CXB (5–200 µM), MET NP (2.5–200 mM), and CXB NP (5–200 µM) for 72 h. On day 6, using the Cell Titer-Glo0 values a,b.50 values for MET as a free drug were significantly higher in MCF-7 3D spheroids (158.2 mM) compared to 2D monolayer cultures (16.1 mM), representing almost nine times the concentration required to achieve the same effect in 2D  loaded into niosomes reduced viability to 34.6%. A similar pattern was observed for CXB, with 50 µM free CXB reducing viability to 70.4% on average, whereas niosomes loaded with the same concentration (50 µM) reduced viability to 45.2%. These findings highlight the enhanced efficacy of drug-loaded niosomes in the 3D model.p < 0.01). The combination of MET NP (IC50 of 24.35 mM) and CXB NP (IC50 of 32.01 µM) exhibited the greatest inhibition of survival, reaching 84%. This combination demonstrated superior efficacy compared to the individual drugs employed alone.Given the enhanced response observed in 2D cells when combining MET NP and CXB NP, we evaluated whether this combination would also elicit an improved response in the 3D model . SimilarTo assess the degree of drug penetration, we employed HPLC to measure the residual drug concentration in the media. This allowed us to indirectly determine the quantity of the drug that had entered the spheroids and accumulated within them. Specifically, we evaluated the penetration and accumulation of MET, MET-NP, CXB, and CXB NP within the spheroids c.At the initiation of the experiment (Time 0), it was observed that all drugs displayed negligible penetration, providing evidence for the integrity of the spheroid models. In the subsequent hours, both MET NP and CXB NP consistently and significantly exhibited penetration. Specifically, MET NP achieved a penetration rate of 65.93% at the 3 h mark, surpassing the penetration rate of MET, which reached 34.47%. In a comparable manner, CXB NP demonstrated a penetration rate of 47.48% after 3 h, surpassing the 15.92% penetration rate attained by CXB. The aforementioned results emphasize the complicated dynamics of drug penetration and the impact of nanoparticle formulations, providing insights into their prospective utilization in spheroid-focused investigations and drug administration approaches. Furthermore, the sustained and prolonged penetration was observed over 72 h, with MET NP reaching an 85.26% penetration rate compared to METs at 61.50%, and CXB NP demonstrated a 71.08% penetration rate versus 31.29% for CXB, highlighting the therapeutic promise of niosomes for enhanced tissue penetration, which is a crucial aspect to consider in the fields of cancer drug development.2O2 is well-known to induce cell migration at concentrations below (200 µM). Accordingly, we used 200 µM of H2O2 in the current experiment [We conducted an investigation to assess the impact of various formulations on the migration of MCF-7 cells. To ensure the specificity of the treatment’s influence on migration rather than cell death, we chose IC10 for the migration assay. Furthermore, Hperiment .2O2 for 48 h led to a significant increase in MCF-7 cell migration. However, when treated with individual concentrations of MET IC10 (0.3 mM) and CXB IC10 (1 µM), as well as their combination, cell migration induced by H2O2 was reduced. The reduction in migration was substantial, and the free surface area after treatment was 78%, 81%, and 97%, respectively, compared to 1.7% of the control cells containing H2O2 , while CXB NP reduced migration by 67%. Combining both niosomal formulations resulted in an 80% decrease in cell migration. These findings indicate that both MET and CXB, along with their niosomal formulations, effectively inhibit H2O2-induced cell migration in MCF-7 cells.Additionally, we explored the effects of niosomal formulations of MET NP (0.2 mM) and CXB NP (0.7 µM), both individually and in combination, on cell migration. The results demonstrated that MET NP reduced migration by 84% compared to cells treated with HHowever, it is worth noting that the combined niosomal formulations did not show superior results compared to the combination of free drugs. This difference can be attributed primarily to the slower release of CXB from the niosomes. The release data indicated that approximately 73% of encapsulated CXB was released within 72 h, while only 48.7% of CXB was released during the 48 h timeframe of the migration assay.This study represents the initial application of MET and CXB in combination for breast cancer treatment. Additionally, it is the first instance of assessing their niosomal formulation in the context of cancer research. Recently, niosomes have attracted significant interest as a promising option for drug delivery systems . These nThe properties of niosomes are significantly influenced by their morphology , and this is crucial for determining their applications  . Hence, The thin-film hydration approach was selected as it was shown in prior studies to consistently generate spherical niosomal particles . The appThe TEM image of the M2 niosomal formulation showed an average particle size of 111.03 nm, which closely matched the average particle size of 110.6 ± 0.6 nm determined by the particle size analyzer. This is likely due to the low PDI value of 0.139 ± 0.017. The TEM image of the O2 niosomal formulation showed a particle size of 72.06 nm. This size was slightly smaller than the measurement obtained from the particle size analyzer (96.7 ± 0.7 nm) but was still consistent with previous studies ,38. The Moreover, the significance of HLB values in determining PS is readily evident . SpecifiIt is noteworthy that the MET niosomal formulation exhibited a particle size that was relatively greater than the CXB formulations, despite the fact that both formulations contained the same components. This could be explained by the fact that the interaction between hydrophobic drugs and niosome vesicles typically reduces vesicle size. This phenomenon occurs as a result of hydrophobic drugs helping to compact the niosomal structure or promoting the formation of smaller vesicles during preparation .The measurement of ZP is crucial in assessing the stability of niosomes . In thisOne notable attribute of niosomes is their capacity to effectively encapsulate drug agents . In our The correlation between %EE and the observed molecular interactions within our niosomes is crucial. The ATR-FTIR analysis revealed the structural properties of our niosomes and their interaction with the niosomal components. Notably, the absence of characteristic peaks for CXB in its niosomal formulations and the minor shifts observed for MET niosomes, as described earlier, indicate effective encapsulation of the drug within the niosomal core rather than the surface as reported, respectively, by Aguilar-Jiménez et al. and Alkilani et al. ,51.In addition, our research results show that 89.2% of MET and 77.80% of CXB were released from their respective niosomes under acidic pH conditions compared to 66.8% of MET and 33.9% of CXB at physiological pH. This suggests that there is a possibility of a significant increase in drug release from niosomes when they reach tumor sites, as the lower pH environment facilitates this process . This coThe findings of our study demonstrated that the application of MET and CXB resulted in a decrease in cell viability in MCF-7 and MDA-MB-231 breast cancer cells. The nanoparticle formulations exhibited superior performance compared to free drugs. In addition, the IC10, IC20, and IC50 values obtained from the analysis indicated that MET NP and CXB NP demonstrated enhanced cytotoxicity at lower concentrations in comparison to their unencapsulated counterparts. This study holds significant importance as it indicates that the utilization of these niosomal formulations could decrease the necessary drug concentrations and, subsequently, mitigate the accompanying adverse effects. The co-administration of MET NP and CXB NP in the 2D cell culture model has led to a notable reduction in cell viability percentages compared to the separate administration of either drug. This suggests the possibility of employing these niosomal formulations in the context of combination therapy for the treatment of breast cancer.In order to more accurately replicate the tumor environment in vivo, we conducted an assessment of the NPs using a 3D spheroid model. Our observations revealed a reduction in cytotoxicity on the spheroids to both free drugs and niosomal formulations compared to 2D monolayer cultures. This observed expected decrease in sensitivity can be attributed to the limited penetration of drugs into the tightly packed spheroids, hindering their access to the interior regions. Nevertheless, the niosomal formulations demonstrated increased sensitivity in the 3D model in comparison to the free drugs due to enhanced penetration. MET NP exhibited an 85.26% penetration rate, surpassing MET’s 61.50%, and CXB NP demonstrated a 71.08% penetration rate compared to 31.29% for CXB. These results suggest that employing niosomal formulations holds promise in overcoming penetration challenges in 3D culture models, thereby enhancing their effectiveness in mimicking physiological settings.2O2 in MCF-7 cells. The co-administration of MET and CXB, whether in their free or niosomal formulations, demonstrated significant inhibition of cellular migration. This observation implies that these therapeutic interventions not only affect the survival of cells but also impede the migratory potential of cancer cells, which is a critical factor in the prevention of metastasis.In addition to evaluating cellular survival, our study aimed to examine the effects of MET, CXB, MET NP, and CXB NP on cell migration, a critical determinant of cancer advancement. The treatments exhibited a reduction in cell migration mediated by HIn conclusion, the results of our study emphasize the potential of utilizing niosomal formulations of MET and CXB to augment their effectiveness in the treatment of breast cancer for the first time. These formulations have been shown to enhance drug penetration, increase cytotoxicity, and inhibit cell migration, particularly in 3D culture settings. The utilization of MET NP in conjunction with CXB NP exhibits potential as a therapeutic approach in breast cancer. The findings presented in this study provide significant contributions toward the advancement of more efficient and focused treatment strategies for breast cancer patients, harnessing the potential of clinically established drugs.In addition, our research employing the 3D spheroid model has uncovered the inherent constraints of traditional 2D culture models in accurately forecasting the effectiveness of drugs. This study presented evidence of enhanced response and cytotoxicity when MET NP and CXB NP were combined, as opposed to using the medications individually. This finding underscores the importance of including 3D culture models in drug assessment and combination approaches, as they provide a more precise evaluation of therapeutic efficacy. Our findings pave the way for further investigations in an in vivo setting. Future work should focus on transitioning these promising niosomal formulations into preclinical and clinical trials to assess their safety and efficacy in living organisms. Ultimately, this research could lead to the development of more effective and targeted treatment options for breast cancer patients to improve their clinical outcomes and a better quality of life.


Mycobacterium tuberculosis (Mtb) strains and antibiotic-resistant mutants. We collect over 20,000 single-cell Raman spectra from isogenic mycobacterial strains each resistant to one of the four mainstay anti-TB drugs  and train a machine-learning model on these spectra. On dried TB samples, we achieve > 98% classification accuracy of the antibiotic resistance profile, without the need for antibiotic co-incubation; in dried patient sputum, we achieve average classification accuracies of ~ 79%. We also develop a low-cost, portable Raman microscope suitable for field-deployment of this method in TB-endemic regions.Tuberculosis (TB) is the world’s deadliest infectious disease, with 1.5 million annual deaths and half a million annual infections. Rapid TB diagnosis and antibiotic susceptibility testing (AST) are critical to improve patient treatment and to reduce the rise of new drug resistance. Here, we develop a rapid, label-free approach to identify  Raman spectroscopy and machine learning can be used to rapidly identify and differentiate strains of antibiotic-resistant tuberculosis (TB) with high accuracy, on a low-cost platform suitable for deployment in TB-endemic regions. At that time, TB had become curable and was considered to be on a path toward elimination. However, by the end of the 20th century, TB re-emerged as a leading cause of death globally, in part due to challenges in diagnosis and its evolved resistance to antibiotics. While growth in liquid culture is considered the gold standard for organism identification and determination of antibiotic susceptibility, the causative organism, Mycobacterium tuberculosis (Mtb) can take up to 40 days to culture. As antibiotic susceptibility testing requires pathogen growth, definitive determination of Mtb antibiotic resistance using traditional antibiotic susceptibility testing (AST) can take additional weeks. In the delay between diagnosis and AST results, many patients are treated with only partially active antibiotic regimens, giving rise to resistant strains. In 2021 alone, more than half a million cases of multidrug resistant TB infections were reported  is to improve surveillance of antibiotic-resistant infections, and to promote the appropriate use of quality medicines . To addr−1, with lipids, proteins, and nucleic acids exhibiting fingerprints between 1000–1700 cm−1, 1200–1660 cm−1, and 600–1100 cm−1, respectively , we achieve >98% resistant versus susceptible classification accuracy across all 5 BCG strains. In addition, we show that various genetic mutants can be accurately classified according to their antibiotic resistance. Using feature recognition algorithms, we also identify the subsets of wavenumbers which most strongly influence antibiotic resistance classification. These algorithms show that antibiotic resistance behavior is primarily reflected in certain vibrational modes – particularly those of mycolic acid - that underlie the molecular-level antibiotic response of the bacteria. These algorithms also show that targeting the entire spectral range of scientific grade Raman instruments is not necessary for TB AST. To this end, we develop a low-cost portable Raman setup that targets select wavenumber bands, for point-of-care applications. Our instrument costs <$5000, and with inclusion of plasmonic nanoantennas for surface-enhanced Raman spectroscopy (SERS), achieves an average accuracy of 89% across all 5 BCG strains. In dried patient sputum samples, our instrument achieves ~79% classification accuracy – providing a foundation for rapid, low-cost, and portable AST testing in resource-limited regions.Here, we demonstrate a rapid, culture-free, and antibiotic co-incubation free antibiotic susceptibility test for TB, based on the integration of Raman spectroscopy and machine learning. Particularly, we classify the antibiotic resistance profile of 5 isogenic Bacillus Calmette-Guérin (BCG) strains using a convolutional neural network. We collect Raman spectra from over 20,000 individual cells resistant to one of the four mainstay anti-TB drugs  and a pan-susceptible wild type strain. Using only 15-second integration times and Raman features from across the entire spectral range . −1 bands between wildtype and four resistant strains; the majority of these vibrational modes correspond to cell-wall components such as mycolic acid and proteins. Therefore, the differences between the spectra are most likely due to differences in cell-wall composition and structure, discussed later in the text. We performed t-distributed stochastic neighbor embedding (t-SNE) which shows significant clustering for each of the 5 strains , mixed the sample with gold nanorods, and collected a total of ~5300 spectra from the BCG strains. Using only 0.3 second integration times and 3 accumulations per sample, we obtain an average classification accuracy of ~79% for the 5 strains across two separate sputum samples . This spWe note that the spectra differ from those without using AuNPs, which is as expected as now the Raman signatures primarily originate from the surface-bound molecules on the bacteria, due to the electric field localization of the AuNPs. This intraclass spectral variation is evident in the milder, less intense clustering of the 5 strains in the t-SNE in In summary, we demonstrate a rapid and accurate TB AST methodology that is antibiotic co-incubation free and culture-free. Unlike current culture-based and nucleic acid amplification-based testing (NAATs) platforms, our Raman approach does not require lengthy culturing, expensive reagents, or thermal cycling equipment, and is robust to single nucleotide point mutations. Our approach can accurately classify resistance across a variety of antibiotics, and across a variety of genetic mutations. In addition, our machine learning models not only provide accurate AST classification, but also interpretability to the Raman spectra. Indeed, as highlighted by our feature-recognition algorithms, resistance behavior is primarily reflected in certain vibrational modes, particularly those of mycolic acid, that underlie the molecular-level antibiotic response of the bacteria. By focusing on the wavenumbers most important for Mtb AST, we developed a low-cost portable Raman microscope, capable of high Mtb classification accuracy on dried samples and in dried sputum. On-going work is aimed at testing our approach in the field on Mtb-positive patients. Such clinical work will elucidate the intrinsic differences between strain types, well beyond sputum-to-sputum variations. If successful, this clinical work will provide a new and needed approach to enable the TB surveillance and diagnostic milestones set by the WHO.Supplement 1


We present a patient with IgG4-related disease (IgG4-RD) that developed after receiving extra-periosteal paraffin-embedded therapy for the treatment of pulmonary tuberculosis. The patient showed clinicopathological features consistent with IgG4-RD, including the enlargement of affected organs , elevation of serum IgG4 levels, and infiltration of IgG4-positive plasma cells. The presence of reactive granulomas with foreign body giant cells (FBGCs) surrounding the paraffin-filled site suggested a type 2 helper T (Th2)-dominant immune response induced by the implanted biomaterial. Furthermore, paraffin, known to act as an adjuvant, may have played a role in activating the immune response and inducing IgG4-RD-like symptoms. This case highlights the potential relationship between foreign substances and the development of autoimmune diseases such as IgG4-RD. IgG4-related disease (IgG4-RD) is an immune-mediated, chronic fibro-inflammatory disease characterized by the enlargement of the affected organs, elevation of serum IgG4, and significant infiltration of IgG4-positive plasma cells in the affected organs . While tA 75-year-old male visited his family physician after having difficulty moving his left fingers for one month. He had undergone extra-periosteal paraffin plombage therapy 54 years earlier for pulmonary tuberculosis. Chest computed tomography (CT) revealed an enlarged plombage space with surrounding soft tissue density in his left chest cavity and bilateral axillary lymphadenopathy. He was referred to our hospital for concerns of tuberculous granuloma, sarcoma, or pyothorax-associated lymphoma.18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)-CT showed the abnormal accumulation of FDG in the soft tissue mass around the plombage, enlarged salivary glands, cervical and axillary lymphadenopathy, and a retroperitoneal soft tissue mass surrounding the abdominal aorta  surrounding the site of paraffin plombage. A foreign body granuloma is a Th2-predominant immune response triggered by implanted biomaterials in the body . BrieflyIn addition, paraffin is known to act as an adjuvant, which is a substance that boosts antigen-specific immune responses and triggers a characteristic inflammatory cascade in vivo . To our The etiology of IgG4-RD remains to be elucidated, and various factors, including genetic predisposition and environmental triggers, may contribute to the pathogenesis of this disease. The clinicopathological alterations consistent with IgG4-RD observed in this case might have been influenced by a persistent immune response to the implanted paraffin. Despite significant changes in the treatment of pulmonary tuberculosis over the past 50 years, paraffin plombage in the body may have remained a foreign substance and continued to chronically stimulate the systemic immune system. Our case may provide insight into the autoimmune developmental process of IgG4-RD triggered by foreign substances.


Acute otitis media (AOM) in children is one of the leading causes of health care visits and antibiotic prescriptions worldwide. The overall aim of the current study is twofold: 1. to analyze and discuss the antibiotic prescription patterns in AOM in children without complications or risk factors and 2. to assess to what extent the watchful-waiting approach is a real practice or a mere desideratum. We performed an electronic search in the PubMed and Embase databases from 2013 to 2023 to capture original research studies investigating antibiotic prescribing patterns for AOM in children. Among the 12 papers included in the analysis, the antibiotic prescription rate ranged from 44.8% to 98%. Our study reveals similarities regarding the use of amoxicillin as a first-line antibiotic in pediatric AOM, but also discrepancies in the watchful-waiting approach attitude and in the choice of second or third-line antimicrobial agents. The proportion of cases managed with the watchful-waiting approach ranged from 7.5%  to 55.2% (Finland). Denmark was the only country reporting penicillin V as a first-choice regimen for children with AOM, which fulfils the guidelines’ recommendations. The most unsatisfying rate of amoxicillin use was recorded in Japan, contrary to the recommendations of local guidelines. The use of quinolones was reported in two out of twelve studies, with the highest proportion in Japan, where tosufloxacin was used in 21.4% of the total number of cases. The duration of the antibiotic regimens was analyzed in three out of twelve papers. Since global antibiotic overuse contributes to the emergence of antibiotic resistant bacteria, new strategies are needed to increase the rate of watchful waiting and to promote the judicious use of antibiotics. Currently, antibiotic overuse is still an increasing threat towards bacterial resistance, leading to avoidable short- and long-lasting side effects for the population, such as higher medical costs, prolonged hospital stays, and increased mortality . Acute oA meta-analysis performed by Venekamp et al.  revealedThe overall aim of the current study is twofold: 1. to analyze and discuss the antibiotic prescription patterns in AOM in children without complications or risk factors and 2. to assess to what extent the watchful-waiting approach is a real practice or a mere desideratum.We performed an electronic search in the PubMed and Embase databases from 2013 to 2023, to capture original research studies investigating antibiotic prescribing patterns for acute otitis media in children. We used the following search terms: [“acute otitis media”] AND [(“treatment”) OR (“therapy”) OR (“management”) OR (“antibiotics”) OR (“watchful-waiting” OR “wait and see”)] AND [(“children”) OR (“pediatric”) OR (“paediatric”)]. Our results were limited to humans and restricted to the English and French language. All of the significant articles were manually searched to further identify any additional eligible studies. We focused only on original studies reporting the approach to antibiotic treatment and/or watchful waiting in nonrecurrent and uncomplicated AOM in children (0–18 years old) without comorbidities which might influence the decision of antibiotherapy.The literature search retrieved 1964 references . After rThe main characteristics of the included studies are summarized in The antibiotic prescription rate ranged from 44.8% to 98%. Seven out of the twelve papers included in this analysis report amoxicillin as the first-choice antibiotic throughout the study period. The highest rate of amoxicillin use in an analysis of the antibiotic prescription patterns in children aged 2 years or older was 81.2% . AmoxiciCephalosporins were the third most used antibiotic for AOM after amoxicillin and amoxicillin/clavulanic acid in five out of twelve studies. Specific antibiotics belonging to the first three generations of cephalosporins were preferred in studies from Australia  , Spain  . In AustOnly three of the twelve papers ,20,21 anFive of the twelve studies depict the trends in time regarding antibiotic treatment in AOM, bringing valuable information about the changes in preferred therapeutic agents in France, Finland, Italy, Korea, and Japan. The studies reveal an improvement in the use of amoxicillin and amoxicillin/clavulanic acid, with the most striking increasing rate of amoxicillin use reported by Levy et al. in France (2.6% in 2009 versus 66.1% in 2012) . YamagucThe watchful-waiting approach was mentioned in nine of the twelve studies. The proportion of cases managed with watchful waiting ranged from 7.5%  to 55.2%In order to provide an accurate insight into medical practices regarding the best choice of antibiotic regimen in AOM, we further analyzed the local guidelines  as geogrThe high rate of cephalosporins use reported by Park et al. in Korea  is not jAmoxicillin was the most frequent choice of antibiotic by the clinicians in both studies reporting AOM cases from the United States ,19. HoweIn the reports from Spain  and DenmThe report from Israel  revealedThe study from Italy  reportedThe most unsatisfying rate of amoxicillin use was recorded in Japan , contrarIn the study by Levy et al. (France), an increasing rate of amoxicillin use was observed after the introduction of the 2011 French guidelines .The lowest proportion of antibiotic prescriptions in children <2 years old was reported by Csonka et al. in Finland, which is contrary to our expectations, as children under 6 months old are recommended to undergo immediate antibiotherapy according to ten out of eleven guidelines .All the guidelines analyzed in Streptococcus pneumoniae, beta-lactamase-negative Haemophilus influenzae, and Streptococcus pyogenes [Currently, the most encouraged strategy in the guidelines on the management of AOM is watchful waiting, while the use of antibiotics has only limited indications . Accordipyogenes .resistant Streptococcus pneumoniae strains (82.85%), as well as high resistance rates to trimethoprim/sulfamethoxazole, macrolides, and lincosamides were reported in the same hospital [In Romania, we have analyzed the medical records of 3103 pediatric patients aged 2–18 years admitted and diagnosed with AOM over a 10-year period (from July 2010 to December 2021) in a tertiary hospital. Overall, clinicians recommended immediate antibiotic treatment in 98.03% of cases. The preferred antimicrobial agent used in 42.12% of cases was a third-generation cephalosporin (ceftriaxone), followed by a second-generation cephalosporin (cefuroxime) in 15.79% of cases. Amoxicillin was recommended in only 1.16% of cases, whereas amoxicillin/clavulanate was used in only 3.93% of cases. In this context, it is not surprising that worrying proportions of multidrug-hospital .Streptococcus pneumoniae and Haemophilus influenzae. Amoxicillin, although widely recommended, is not totally harmless. The use of high-dose amoxicillin had the second-highest rates of diarrhea (13.8%) and the highest rates of a generalized rash (6.5%) in a meta-analysis including 82 papers [Pseudomonas aeruginosa [Haemophylus influenzae [Antibiotic treatment in AOM in children is prescribed accordingly to the most common microorganisms isolated from the middle ear fluids. The spectrum of activity should cover, at least, 2 papers . However2 papers . The fluruginosa . Levofloruginosa . Approvefluenzae . Howeverfluenzae , due to fluenzae . Furtherfluenzae , recent fluenzae .Our analysis reveals that further improvements are needed in antibiotic stewardship in AOM in children. The studies included in our review are incontestable proof that effective strategies for guideline implementation are needed in order to encourage the watchful-waiting approach and to provide suitable antibiotic treatment, by avoiding broad-spectrum antibiotics. Lautenbach et al.  reportedThe barriers for adopting guideline recommendations may be due to a lack of guideline knowledge and current practice inertia, but also due to parental pressure and the fear of severe complications . In studThe watchful-waiting approach is highly encouraged nowadays in uncomplicated cases of AOM , as in hThe judicious use of antibiotics may decrease the danger of high antibiotic resistance rates. Clinicians should be encouraged to delay antibiotics prescriptions and to overcome the fear of rare complications in children with AOM. Although important improvements have been made in the stewardship of antibiotics in AOM, increased efforts are still needed to improve the adherence to guidelines.Our study reveals similarities regarding the use of amoxicillin as a first-line antibiotic in pediatric AOM, but also discrepancies in the watchful-waiting approach attitude and in the choice of second or third-line antimicrobial agents. Among the 12 papers included in the analysis, the antibiotic prescription rate ranged from 44.8% to 98%. Denmark was the only country reporting penicillin V as a first-choice regimen for children with AOM, which fulfils the guidelines’ recommendations. The most unsatisfying rate of amoxicillin use was recorded in Japan, contrary to the guidelines’ recommendations. The use of quinolones was reported in only two of the twelve studies, with the highest proportion in Japan, where tosufloxacin was used in 21.4% of the total number of cases. The proportion of cases managed with the watchful-waiting approach ranged from 7.5%  to 55.2% (Finland). Since global antibiotic overuse contributes to the emergence of antibiotic resistant bacteria, new strategies are needed to increase the rate of watchful waiting and to promote the judicious use of antibiotics.


Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress  promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.Despite being considered fragile and fastidious,  Among the routes of human infection, chicken meat represents the main source  state after exposure to various stresses, including low temperature, presence of oxygen, salt treatment and reduced nutritional intake   induce b. jejuni strains, characterise morphological aspects, determine their adaptive and invasive potential and perform comparative metabolomic evaluation, considering the possible risk to the consumer.Given the evidence, we were instigated to qualitatively and quantitatively estimate the time required for the complete acquisition of the VBNC form in 27 C22.1C. jejuni strains, cryopreserved at -80°C and from chicken carcasses, isolated by ®). The strains evaluated were previously explored by phenotypic antimicrobial resistance, virulence profile and RAPD-PCR and were considered different strains in a previous study (®) and incubated for 48 hours at 37°C in microaerophilic conditions, followed by centrifugation for 5 minutes at 10,000 rpm at 4°C. The pellet was washed three times in 0.9% saline, and the resulting suspension was standardized to an OD600 of approximately 0.4 in sterile saline equivalent to about 7–8 log CFU/mL (PGInstruments®), as confirmed by plate count and used as the standard inoculum for obtaining the VBNC form. All assays were done in three replicates and three independent replicates, and the standard IAL 2383 strain of C. jejuni was used as a control for viability and culturability.Twenty-seven phylogenetically distinct us study . They we2.2The strains in saline suspension were incubated at 4°C for up to 60 days in conditions of oxygen presence, simulating thermal, nutritional and oxidative stress to induce the VBNC state, according to The method used to evaluate culturability was conventional counting on CCDA agar plates, accompanied by morphological evaluation in light microscopy, performed every four days  in three replicates per strain, in three repetitions. In parallel, BacLight biovolume analysis was used for one of the replicates. Day 0 corresponded to the cells (VT form) before exposure to the stress conditions and was used in the comparison. On each assay day, the tubes with samples to be tested were removed and vortexed briefly, and then representative aliquots of 10 serial dilutions were taken for each test, followed by returning the tubes to 4°C. For assessment of culturability, 1 mL of the dilutions were transferred to mCCDA plates. The plates were incubated micro-aerobically at 37°C for 48 hours for further quantification.2.3The LIVE/DEAD BacLight staining assay (Thermo Fischer) was used to obtain total cell counts in one of the replicates for the 27 strains. A 5 mL aliquot of each sample was stained with the fluorescent dyes propidium iodide  and SYTO 9 (3.34 mM in dimethyl sulfoxide) in a 2:1 ratio. Samples were incubated with 15 μL of dye mixture at 37°C in the dark for 15 minutes and then immediately transferred a volume of 200 μL to 96-well microplates in triplicate. Results were read using fluorescence spectrophotometry (Biotec), according to the protocol described by 2.4C. jejuni VT and VNBC strains in suspension were randomly selected, subjected to washing and centrifugation at 10,000 g for 5 minutes at 4°C. 100 μL of the pellet was pipetted onto a 1 cm2 polyurethane slide coated with a 0.22 μm pore size cellulose membrane (Millipore®). Slides were dried at room temperature for 15 minutes and fixed with 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M PBS buffer (pH 7.4) overnight at 4°C. The fixative was removed, and the samples were washed three times with PBS buffer. Slides were post-fixed with 1% osmium tetroxide for two hours and washed three times with PBS buffer. The slides were then dehydrated in a series of ethanol solutions  for 15 minutes for each step. The samples were dried in a CPD  , using liquid carbon dioxide as the transition fluid. The coating was made with a 20 nm thick layer of gold  and visualized on a VP Zeiss Supra 55 FEG SEM operating at 20 kV.The preparation of the material for analysis in SEM was done, according to 2.5C. jejuni strains in VT and VNBC forms in suspension were randomly selected. Initially, the samples were fixed in Karnovsky’s solution  for 1 hour, centrifuged  to decant the cells for 5 minutes and then the supernatant was discarded. Then, the samples were extensively washed in sodium cacodylate buffer (0.2 M) for 6 hours, centrifuged and included in gelatin (0.5%).For the evaluation of internal morphology, three The material was post-fixed in a 1% osmium tetroxide solution for 1 hour and subjected to dehydration in an increasing series of alcohol at 50%, 70%, 80%, 90%, 95%, 100%, 100% and 100% for five minutes in the first five baths and ten minutes in the last three. For the final dehydration, three 10-minute baths in propylene oxide were applied. Subsequently, the material was placed in a solution of Epon resin and propylene oxide in the proportions 2:1 and 1:1 for 12 and 6 hours, respectively. After this period, the solution containing the material was placed in an oven at 37°C for 12–24 hours.After evaporation of all propylene oxide, the blocks were embedded in pure resin and kept for two days in an oven at 60°C. Finally, ultrathin sections were obtained using the ultramicrotome, which were contrasted with uranyl acetate and lead nitrate and analyzed in the transmission electron microscope (TEM)  .2.6®) was added and homogenised by vortexing (Phoenix®). Subsequently, 200 μL of chloroform (Sigma Aldrich®) was added and the vortexing homogenisation procedure was repeated, followed by centrifugation at 12,000g for 15 minutes at 4°C. The aqueous portion formed was transferred to a new microtube, to which 500 μL of isopropanol (Sigma Aldrich®) was added, homogenised and centrifuged again at 12,000g for 10 minutes at 4°C. To the pellet formed, 1 mL of 75% ethanol (Sigma Aldrich®) was added and then homogenised and centrifuged at 7,500 g at 4°C for 5 minutes; the supernatant obtained was discarded. The RNA pellets were dried at room temperature to be diluted in 20 μL of DEPC water (Invitrogen®). The RNA concentration used was 200 ng/μL, which was quantified in a NanoDrop spectrophotometer (Thermo Scientific®). All RNA was treated with TURBO™ DNase (Invitrogen®), according to the manufacturer’s recommendations.Strains were evaluated for the ability to produce transcripts in typical and VBNC forms. For this purpose, RNA extraction was performed using the Trizol method as described by ®) and 1 μL of RNA; all were kept at 37°C for one hour to obtain complementary DNA (cDNA). Subsequently, 3 μL of cDNA was used for amplification in a 25 μL reaction volume comprising: 0.625U of Taq DNA polymerase, 5 mM MgCl2, 200 μM of dNTPs and 4 pmoles of each primer (®). Amplification and electrophoresis were performed as described by Reverse transcription was performed with 10 U of RNase inhibitor, 40U of MMLV-RT, 1X of MMLV-RT buffer, 200 μM of dNTPs , 126 random hexamer oligonucleotide holes as primers, 20 μL of DEPC water .We removed the liver from five chicken embryos at 14 days of incubation. Then, to remove the red blood cells, we intensely cut the liver, washed and discarded the supernatant (3 times) and added 0.25% trypsin to promote enzyme breakdown on shaking for 10 minutes. We then removed the supernatant from the decant and added 199 (Gibco™) media plus 20% fetal bovine serum (FBS), which was centrifuged at 900 X g for 10 minutes. The pellet was resuspended in 199 media plus 10% FBS and amphotericin B antibiotic (amphotericin B (2.5 uG/mL), gentamicin (50 uG/mL), streptomycin (100 mcg/mL) and penicillin (100 U/mL). Finally, the medium containing the cells was filtered and homogenised, and we counted the cells in the Newbauer Chamber using an optical microscope  of two strains of C. jejuni in VT and VBNC forms in triplicate. A negative control group  alone) and a positive control group (cells treated with the viable forms of C. jejuni IAL8323) were added.The cell suspensions were seeded on a glass coverslip inside a 12-well plate at 1.43 × 104 cells/well density and cultured at 41.5°C and 5% COAfter a period of 24 hours, the cells were washed three times with PBS and treated with Yo Pro-01 (YP) and propidium iodate (PI) (1:1000 each) (Invitrogen) for 30 minutes at room temperature to stain the cells in the apoptosis and necrosis phase. Then, we washed the cells three times and fixed them with 4% formalin for 10 minutes. After that, the cells were treated with Hoechst (Sigma) to stain the DNA of the cell. We washed the slides and arranged them on a ProLong (Invitrogen) anti-smearing slide. We counted the cells under a fluorescence microscope , evaluating four fields per slide. We considered the DAP-labelled cells as the total number of cells. The apoptotic cell index was calculated by the ratio between the number of cells stained only by green and blue colour.2.8C. jejuni strains kept in suspension in VT form and VBNC form until freeze-drying. For extraction of metabolites, 1000 µL of spectroscopic grade methanol was added to the lyophilised material and homogenised in a vortex for 5 minutes. The material was centrifuged for 15 minutes at 13,000 g, and the supernatant was transferred to another Eppendorf, which was subjected to vacuum concentrator for 30 minutes. The material was resuspended in 400 µL of methanol and filtered through a 0.22 micrometre filter.The test was performed with one of the -1. The following GC conditions were used: the initial column temperature was maintained at 60°C for 2 minutes, then increased to 280°C at 5°C min-1 and then maintained for 30 minutes. The mass spectrometre was operated in full scan mode from 50 to 550 m/z. The transfer line to the mass spectrometre was heated to 240°C and the quadrupole to 150°C. The injection volume was 6 µL.Metabolite analyses were performed using an Agilent 7890B GC System/5977B GC/MSD technologies instrument. Separation was provided by a capillary column (Agilent DB-5HT 30 m x 250 µm x 0.25 µm) with high purity helium at a constant flow rate of 1.2 mL minMassHunter Qualitative v. 10.0 software was used to process the raw data. A ‘Molecular feature extraction (MFE)’ tool was used for extraction of the mass spectra and conversion to.CEF extension. Agilent Mass Profiler Professional (MPP) software v. B.13.1.1 was used to filter and analyze the extracted molecule compounds. The filters used were minimum absolute abundance = 5,000 counts and all allowable charges. The analysis parameters were retention time tolerance of 0.15 min; mass window 15 ppm + 2 mDa. Molecular compounds considered were those present in 100% of at least one group.2.9The results were subjected to an initial percentage descriptive analysis. For comparative tests, the normality of the data was verified, followed by the application of Fischer’s exact test for comparison of two variables and one-way ANOVA or Kruskal-Wallis when comparing three or more variables.The percentage of necrotic cells was calculated by the ratio between the number of red and blue stained cells. We analyzed the total number of cells by the average number of cells stained in blue. Thus, we used ANOVA followed by Tukey’s test, comparing each group with the negative control group. In addition, we used chi-square and Fisher’s test followed by the binomial between two proportions for analyses of apoptosis and necrosis, comparing each group with the negative or positive group or the VT and VBNC. We evaluated our data with a 95% confidence level using GraphPad Prism 9.0.2 transformed values. The Mann-Whitney test was applied with p-value < 0.05 and fold change equal to or greater than 2.00. The metabolites were identified using the METLIN 2019 database (included in MPP).The statistical analyses of the data generated in the metabolomic analysis were performed with log33.17 CFU/mL. The results showed that all strains maintained culturability until day eight and then all were induced to the VBNC state over the time of stress exposure.The assays to evaluate the viability and acquisition of the VBNC form were conducted under nutritional, thermal and oxidative stress conditions from an average amount of 7.8 ± 0.4 log CFU/mL of the bacteria in its VT form, equivalent to 9.7x10C. jejuni to become VBNC was 26 days. Overall, most strains (16/27 - 59.3%) took 20 and 24 days to acquire the VBNC form. In a discriminated way we had four strains that acquired the VBNC form at 12 days, nine strains at 20 days, seven strains at 24 days, three strains at 32 days and four strains at 56 days. For all strains we observed, the change from the spiral to the coccoid form happened over time, with the concomitant presence of both morphologies in the last eight and four days prior to the complete acquisition of the VBNC form for all strains, at which time the average colony growth was restricted to an average of 0.5 log CFU/mL, equivalent to 3.0 CFU/mL. This shows that the transition is a gradual process, when considering the same strain  on day 0 and 67.7% (with a maximum of 71.6% and minimum of 61.4%) on day 60 Figure 1The behavior of the strains was similar throughout the first eight days under stress, with the fourth day determining the most significant mean reduction of the culturable form of 3.2 log CFU/mL, in order to maintain the %live without significant changes until day 12. For the other days, this reduction was constant, with an average value of 0.33 log CFU/mL. Starting on day 12, we observed distinct counts of the culturable form among the strains in the same period, which determined the variations in the time of acquisition of the VBNC form. The same was observed for %live from day 16. From day 20 to 60, there was no significant difference in the counts for the strains that maintained their culturability, as well as in viability from day 36 onwards, with an average of 70.8 ± 1.9% Figure 1The VT form demonstrated the expected morphological pattern with the helical and spiral structure, typical of the species, with size variations of 0.5–5 µm in length by 0.2–0.8 µm in width, with a flagella at one or both ends . For the ciaB gene, the transcript was only complete for the VT of C. jejuni and in 16/27 (59.3%) strains in the VBNC form, indicating inactivation of virulence ability (ciaB) in 11/27 (40.7%) and maintenance of adaptive ability under stress (p19) in the VBNC form with the presence of transcripts in 100% of the strains. 13/16 (81.2%) strains that produced transcripts for ciaB in the VBNC form represent the 13 strains that acquired this stage most rapidly (12 and 20 days), and for 3/16 (18.8%), this acquisition occurred at the last detectable time of our study (56 days) and represent 3/4 strains belonging to this group. Paradoxically, the faster acquisition of loss of cultivation capacity maintained the invasive potential of the strains, as well as strains that remained viable and cultivable for long periods also transcribed ciaB when VBNC form.We detected the presence of transcripts for C. jejuni VBNC. For the VT, the ciaB transcript was significantly higher (mean 54.2 ng/uL) compared to p19 (mean 43.9 ng/uL); whereas, for the VBNC stage, the means were 15.7 and 11.2 ng/uL of transcripts for the respective genes  ester in the VT and peaks of phthalate derivatives that include 1,2-benzenedicarboxylic acid bis(2-methylpropyl) ester and bis(2-ethylhexyl) phthalate in the VBNC form.C. jejuni VBNC and concern squalene, 2,4-di-tert-butylphenol, acetic acid, cyclotetrasiloxane octamethyl, palmitic acid and palmitoleic acid. Similarly, some precursors of volatile organic compounds, belonging to the bacterial volatilome, such as octadecanoid acid, hexanoic acid and tetra decanoic acid, represented an indicative of disruption of the normal metabolism of the cell in the VBNC form.Metabolites associated with protective mechanisms were found at high levels in C. jejuni, we can highlight the high levels of dodecane, 9-octadecenamide and di(2-propylpentyl) ester, consistent with the exponential and stationary growth phases representing the metabolism of the bacterium in this life form.In the VT form of 44.1Campylobacter spp. are difficult to grow, and when they are subjected to stress situations, such as food refrigeration or freezing, a morphological transition to the coccoid form occurs, especially in the stationary phase of growth.Obtaining the VBNC form identified in all strains in our study indicates danger to public health because the food may be released as safe, harboring the pathogen that can resume its spiral form when inside the host. According to C. jejuni strain, isolated from clinical samples from humans in Thailand, determined the need for 38 days for the acquisition of the VBNC form. Our results coupled with those of 8 CFU/mL . Stress resistance and progression to the VBNC state is known to vary between different strains of C. jejuni, due to the level of stress tolerance  and p19 (iron uptake) – and the importance verified in the literature.  in the VBNC form. Nutritional, thermal and oxidative stress induced the decrease of virulence gene transcription. And, in contrast, p19 transcription was 100% in the VBNC form, maintaining the potential found in the VT form. Therefore, VBNC bacteria regulate and control the transcription of virulence genes that are unnecessary for survival in a stressful environment and prioritise the transcription of genes linked to adaptive processes, such as p19. This same behavioural parameter was identified by C. jejuni grown at 5°C for up to 7 days, finding a negative regulation in the expression of flagellin genes (flaA and flaB), linked to virulence. In contrast, many genes involved in energy metabolism increased at 5°C, indicating that C. jejuni may require higher energy to survive under low temperatures.The transcription of the 4.3Strain VT1 is probably more pathogenic since the number of cells stained by DAP was lower than the negative control . AlthougCampylobacter jejuni  more pathogenic in low doses than a control strain isolated from humans (IAL), which is routinely used in our research. The IAL strain was probably not pathogenic in our study because we used a very low infection dose (2.8 log CFU/well). Furthermore, our results indicate that VBNC forms can be active in the cell culture of hepatocytes. These forms represent a key point in studying the epidemiology of 4.4Adaptation of bacterial metabolism is crucial for the survival of the microorganism in different environments. Pathogens alter their metabolism to use available resources in the most efficient way and to escape adverse conditions .Campylobacter jejuni is considered a microorganism with restricted metabolic capabilities, which justifies the reduced number of compounds found, distinct from what has already been identified for other bacteria, such as P. aeruginosa. It is also worth noting that C. jejuni exhibits strain-dependent metabolic heterogeneity, with some strains possessing genomic loci that confer different substrate utilization  was identified by the cyclotetrasiloxane octamethyl- metabolite most expressed in the VT form of lus spp. . It is pr jejuni .Bacillus spp. and now in C. jejuni VT form.Volatile amide 9-octadecenamide (oleamide) were identified by C. jejuni in the typical form. PAEs have also been detected in other bacterial species such as H. pylori and B. mcbrellneri  ester, a phthalic acid ester (PAE), was overtly identified in rellneri . In parasporters .C. jejuni VBNC reveals the intense antioxidant activity in this life form. The compound is an alkylbenzene member of the phenol class that carries two tert-butyl substituents at positions 2 and 4. In vitro bioassays have demonstrated the ability of this compound to significantly reduce DNA damage produced by the presence of oxygen free radicals, such as those derived from hydrogen peroxide (H2O2)  , which aal cells , which mC. jejuni. It is worth noting the relevance of volatile compounds that comprise the bacterial volatilome (C. jejuni is consistent with the fact that it is a metabolite produced during the exponential growth phase or in the stationary growth phase, with a key role associated with signaling or virulence of the pathogen. The importance of this metabolite as a marker of diarrheal infection by C. difficile and respiratory infection by A. baumanni has already been described in the literature (A wide diversity of metabolites derived from alkane hydrocarbons, associated with other alcoholic or acidic radicals, were identified in both life forms of latilome . One sucterature .C. jejuni (C. difficile infection (C. jejuni VBNC may be due to a disruption of metabolism resulting from environmental stimuli maintaining precursors at doses higher than those identified in the VT form.In a parallel manner, we identified the higher production of other volatile compound derivatives in the VBNC form. A derivative of octadecanoid acid, 1-Octen-3-ol, identified in our study was determined to be a marker found in patients infected with . jejuni , as wellnfection . The higP. aeruginosa and E. coli (C. jejuni (High levels of acetic acid have also been identified in blood cultures of  E. coli . In the . jejuni , resultiC. jejuni.Despite reports in the literature regarding the composition of the volatilome of numerous bacterial species, it is still not possible to state the specificity of these metabolites since, besides strain-dependent variation, there is also the involvement of niche, nutrient availability for these pathogens of the storage conditions of the samples and the bacterial growth phase , which mC. jejuni and describes possible form biomarkers for this bacterium. The description of such biomarkers is of great epidemiological relevance as it forms the basis for research and innovation studies, especially in the construction of diagnostic methods for the VBNC life form of C. jejuni that is not identified by conventional methods.However, our work opens doors for better phenotypic understanding of VBNC forms of 5C. jejuni assumes the VBNC state by thermal, nutritional and oxidative stress after an average of 26 days, with strain-dependent variations. The VBNC form remains potentially virulent, but mainly adapted to stress, as well as regaining its invasive potential in host cells. Image analysis coupled with metabolomics brought explanations about changes in the volatilome and the mechanisms of oxidative protection and cell wall integrity that enhance the maintenance of viability of this life form. These results serve as a public health warning of the potential risk of maintaining this latent form.The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.DR, BF, MB and RM contributed to conception and design of the study. LS, RB, MG-T, RA, HA-S, LM, PS, JS organized the database and experimental analyses. RM, HA-S, BF performed the statistical analysis. RM, BF, HA-S wrote the first draft of the manuscript. DR, RB, MG-T wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


During the COVID-19 pandemic, historic gains made in antibiotic stewardship were challenged as antibiotic utilization for patients with COVID-19 increased despite low co-infection rates, as evidenced by a 15% increase in resistant infections 2019 - 2020. Studies demonstrate that input of an infectious diseases (ID) specialist can improve appropriate antibiotic use without increasing length of stay or mortality. But, there is limited data on this among COVID-19 patients. We evaluated the impact of an ID fellow rounding in the Medical ICU (MICU) and medical intermediate unit (MIU) on antibiotic prescribing among COVID-19 patients admitted to a tertiary care center in eastern North Carolina.An ID fellow rounded with the MICU and MIU teams twice weekly to provide antibiotic guidance from 3/21-6/21. The intervention group comprised patients admitted with COVID-19, prescribed antibiotics, and receiving antibiotic guidance. Concurrent (3/21 - 6/21) and retrospective (11/20 – 1/21) controls met the same criteria but without ID fellow antibiotic guidance. Antibiotic and other data were obtained from chart review. Prescribing rates  were calculated and compared using Poisson regression to estimate rate ratios while controlling for covariates.Analysis included 48 intervention patients, and 38 concurrent and 64 retrospective controls. The intervention group had higher BMI, longer hospital stay, and more frequent ICU stay than either control group (Table 1). The median prescribing rate in the intervention group  was lower than the retrospective controls but higher than the concurrent controls . Upon adjusting for covariates, the intervention group had a 32% lower antibiotic prescribing rate than retrospective controls . A similar result was seen when compared to the concurrent controls (Table 2).Table 1Demographic, clinical, and hospital stay characteristics, by intervention group, among those prescribed any antibioticsFigure 1Median antibiotic days per 1,000 hospital days, by intervention groupTable 2Adjusted analysis of antibiotic prescribing rate  1: Adjusted for age, BMI, and spending time in the ICU (Yes/No) 2: Restricted to only the Concurrent Control and Intervention group, Adjusted for age, BMI, and spending time in the ICU (Yes/No)We found a significant reduction in antibiotic prescribing among COVID-19 patients when an ID fellow provided antibiotic guidance. Our study contributes to the growing body of data that ID providers are critical to the fight against antimicrobial resistance and are valuable resources in determining the need for antimicrobials.All Authors: No reported disclosures


Regulatory processes in biology can be re-conceptualized in terms of logic gates, analogous to those in computer science. Frequently, biological systems need to respond to multiple, sometimes conflicting, inputs to provide the correct output. The language of logic gates can then be used to model complex signal transduction and metabolic processes. Advances in synthetic biology in turn can be used to construct new logic gates, which find a variety of biotechnology applications including in the production of high value chemicals, biosensing and drug delivery. In this review, we focus on advances in the construction of logic gates that take advantage of biological catalysts, including both protein-based and nucleic acid-based enzymes. These catalyst-based biomolecular logic gates can read a variety of molecular inputs and provide chemical, optical and electrical outputs, allowing them to interface with other types of biomolecular logic gates or even extend to inorganic systems. Continued advances in molecular modeling and engineering will facilitate the construction of new logic gates, further expanding the utility of biomolecular computing. For example, fundamental cellular processes including transcription and translation are highly regulated. Protein function, especially enzyme function, is often modulated by the concentrations of metabolites and signaling molecules. The activity of many enzymes depends not just on the concentration of substrate, but on other metabolites that bind to allosteric sites, allowing for more complex regulation. The interconnected web of the reactions and regulation involved in metabolism can be conceptualized as a circuit of logic gates, analogous to a computer, where each individual reaction is a logic gate that produces output molecules in response to input molecules. In the case of an enzyme allosterically regulated by metabolites, the “decision” to produce more or less of a given metabolite depends on the concentration of the metabolites that control the activity of the enzyme. As such, biological regulation can be re-conceptualized in terms of logic gates, which then has a wide variety of biotechnology applications, including in synthetic biology for optimized production of high value chemicals e.g. ,2), for , for 2])1.1Logic gates are the basic building blocks of computers. In electronic Boolean logic gates, a low voltage/current is 0 and a high voltage/current is 1. This concept can be expanded to chemical reactions, where a low signal is 0 and a high signal is 1. Although the inputs and outputs of a molecular logic gate are not truly binary, molecular circuits have been designed to behave closer to binary logic gates with a sigmoidal response to inputs –12 thougA logic gate returns an output based on one or more inputs . The sim1.22+ and Mg2+ dependence of malate dehydrogenase activity )tabolism . Bindingtabolism . Allostetabolism ,56  and maltose binding protein (MBP) that relies on a large conformational change induced by binding of maltodextrins to activate β-lactamase –59. The 2+ as inputs [2+ are always present so it effectively behaves as a YES gate for the analyte.Another clever system that has been engineered to behave as a logic gate is pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) fusion proteins, in which PQQ-GDH is fused to a reporter domain that binds a given ligand. GDH activity is allosterically regulated by ligand binding to the reporter domain. An advantage of using PQQ-GDH is that PQQ-GDH catalyzes a redox reaction, allowing the signal to be easily incorporated into an electronic circuit. Guo and colleagues  convertes inputs , althougSubsequent work by the Katz group  used theDokholyan’s group constructed an OR gate in live cells by engineering focal adhesion kinase (FAK) to respond to chemical and optical inputs . FAK is Ostermeier’s group  used a p2.32O2) in response to a logic gate made of a four-level enzyme cascade to release molecules from an Fe3+ cross-linked alginate hydrogel. Prior to this work, only two enzymatic reactions were able to be carried out in sequence with the enzymes immobilized on the hydrogel [+ to NADH. The next enzyme was lactate dehydrogenase (LDH), which catalyzes the reduction of pyruvate into lactate with NADH as a cofactor. The final enzyme was lactate oxidase (LOx), which catalyzes the oxidation of lactate by O2 to produce pyruvate and H2O2. The hydrogen peroxide then diffuses into the hydrogel and destroys it via a free radical mechanism, releasing the reporter molecule. The reporter molecule used was a fluorescent DNA, which in principle could be used as an input to DNA logic gates (e.g. see below) or could be a small molecule drug or input for downstream enzymatic reactions.The chemical reactions catalyzed by a series of different enzymes can also be constructed to act as logic gates e.g. see . For exahydrogel . Their sKatz’s group also created a reversible CNOT logic gate using two enzymatic reactions resulting in pH changes in opposite directions . First, + and HK consumes glucose only in the presence of ATP. The second set of enzymes consisted of alcohol oxidase (AOx) and glucose oxidase (GOx). AOx uses EtOH to produce H2O2 and GOx uses glucose to produce H2O2. Production of H2O2 is considered the output, as it is used by horseradish peroxidase to produce a spectrophotometric signal. The system used two inputs: 1) EtOH and ATP and 2) glucose and NAD+. The key concept is that in the presence of both inputs, EtOH and glucose are both fully consumed by the first enzyme set so that no H2O2 is produced after addition of the second enzyme set. When only one input is added, the enzyme that would consume either EtOH or glucose in the first set does not have its cofactor and so the EtOH or glucose will be present in solution for the second enzyme set to use to produce H2O2. In the absence of both inputs, there is no glucose or EtOH so no H2O2 is produced.Katz’s group created two other enzyme-based XOR gates. The first system consisted of two sets of enzymes, with the second set of enzymes added after the first set of reactions was given enough time to reach completion  Figure . The fir2O2 [+ (cofactor for GluDH) and glucose (substrate for GDH and GluDH). If H2O2 is produced at the surface of the hydrogel i.e. by GOx or GluOx, the H2O2 decomposes the hydrogel to release a nanoparticle. In the absence of any input, the nanoparticle is not released from the hydrogel because no H2O2 is formed. When just NAD+ and glucose were present, the glucose could not be reduced by GDH without PMS, so it was oxidized by GOx, producing H2O2 and releasing the nanoparticle. When just PMS and glutamate were present, glutamate was oxidized by GluOx, again resulting in production H2O2 and nanoparticle release. When both inputs were present , glucose was reduced by the GDH in solution before it could be oxidized by GOx on the hydrogel surface, preventing production of H2O2. Similarly, the glutamate was reduced by GluDH before it could be oxidized by GluOx, preventing production of H2O2. This system is a notable advancement over previous work, as time-dependent addition of enzyme is not required. The nanoparticle released from this system can in principle be used for colorimetric sensing, as an input for further logic circuits, or for targeted drug delivery.In later work, Katz’ group constructed a different XOR gate to release nanoparticles from an alginate hydrogel by production of H2O2 . Two enz2.4A number of biomolecular logic circuits have made use of proteases to control protein-protein interactions and transcription. As these systems have been extensively reviewed in recent articles –79, we wSimilar to the CHOMP system, split-protease-cleavable-orthogonal-coiled-coiled (SPOC) based logic circuits were developed, in which split proteases are reconstituted in human cells by the formation of coiled-coils regulated by orthogonal proteases. In the absence of protease, an autoinhibitory coil is covalently linked to a target coil that is linked to the protein of interest . Adding protease to cleave off the autoinhibitory coil allows for a different coiled-coil to displace the autoinhibitory coil. When this displacing coil contains the other half of a split protease, the protease is reconstituted and can be used to cleave the linker of another coil, allowing for logic cascades. Previously, this framework was used to construct an AND gate in an in vitro translation system by using a protease to cleave between the target and autoinhibitory helices  Figure . Fink et3.3.1While most catalysts in biological systems are protein enzymes, nucleic acids can also catalyze reactions and undergo allosteric regulation, allowing for the construction of logic gates. A common foundation for constructing logic gates with molecules of DNA in bulk solution is toehold-mediated strand displacement. In this method, signals are carried by single-stranded DNA with two recognition domains that can hybridize with the two strands of the gate that the signal is associated with. Each gate consists of double-stranded DNA, with one strand having a short overhang called a toehold in addition to the displacement domain. Both domains are complimentary to an input strand. The input strand is single-stranded DNA that is complimentary to the toehold and the displacement domain will preferentially displace one of the strands of the gate by first hybridizing with the toehold before proceeding by branch migration. After hybridizing with the toehold, the input strand hybridizes with the displacement domain, displacing the other strand that was initially part of the double-stranded gate. The displaced strand, which contains a sequence complimentary to a different toehold than the displacing input strand then proceeds as an input for the next strand displacement reaction at the next gate in the circuit. Two-input AND gates can be created by using displacement of a strand by the first input to reveal a toehold that the second input can use to displace the output strand . OR gateAn extension of the toehold-mediated strand displacement method is the use of toehold exchange and “Seesaw” gates . “Seesaw3.2Instead of the toehold-mediated strand displacement mechanism described above, logic gates can also be constructed by using strand displacement to allosterically regulate the catalytic activity of DNAzymes –99 Figu. Early w3.32O2, oxidizing a substrate to produce a colored product or chemiluminescence analogous to horseradish peroxidase [G-quadruplexes, stacks of four planar guanine bases associated by Hoogsteen base pairing stabilized by a metal ion that are found in G-rich sequences of nucleic acids, have also been used as the basis for DNA-based logic gates. There is a large body of work on using G-quadruplexes that respond to input DNA oligonucleotides ,118–121 roxidase . These croxidase –127, smaroxidase , and speroxidase  or formeroxidase ,118 or iroxidase . A notabroxidase  (Figure 4.The logic gates presented here that depend on protein-level regulation can be compared against those that regulate at the level of transcription. In general, logic gates that depend on protein-level regulation are faster than transcription-based logic gates, occurring on the timescale of minutes rather than hours to days ,132. ThiLogic gates that rely on the detection of specific molecules, such as those presented in 2O2. These logic circuits could be combined with H2O2-releasing hydrogels that have been developed for therapeutic applications such as wound healing [2O2-releasing hydrogels release H2O2 from the oxidation of glucose catalyzed by glucose oxidase. By using protein-based logic gates to regulate glucose oxidase activity, such as in the work described in 2O2 could be used as alternatives to glucose oxidase in these systems.Some of the logic circuits in this review ultimately release molecules from hydrogels by production of H healing ,135. CurDNA-based logic gates have several advantages over protein enzyme-based logic gates for in vitro computations. One advantage is that they are easier to scale up due to the relative simplicity of Watson-Crick base pairing, as complimentary DNA sequences with well-understood displacement kinetics can be designed and synthesized much more easily than allosterically regulated proteins or protein-protein interaction interfaces . An imprWhile there are many examples of protein-based enzymatic logic gates that have been implemented in living cells ,80,132, Biomolecular logic gates are likely most useful for diagnostics, while therapeutic applications are more challenging. For DNA-based biosensors, a logic circuit with inputs based on aptamer binding and an electronic output that relies on G-quadruplex formation as the output like that used by Yu et al.  is the m5.The logic gate framework can be used to conceptualize, and in some cases simplify, complex regulatory networks in biology. In metabolic engineering, this framework can then be used to optimize production of high value chemicals ,2. Advan