Rxrα expression in mononuclear phagocytes on cardiac remodeling after myocardial infarction (MI) has not been investigated to date. Here, we use a temporally controlled deletion of Rxrα in monocytes and macrophages to determine its role in ischemia-reperfusion injury. We show that reduced expression of Rxrα in mononuclear phagocytes leads to a decreased phagocytic activity and an accumulation of apoptotic cells in the myocardium, reduces angiogenesis and cardiac macrophage proliferation in the infarct border zone/infarct area, and has an impact on monocyte/macrophage subset composition. These changes are associated with a greater myocardial defect 30 days after ischemia/reperfusion injury. Overall, the reduction of Rxrα levels in monocytes and macrophages negatively impacts cardiac remodeling after myocardial infarction. Thus, RXRα might represent a therapeutic target to regulate the immune response after MI in order to improve cardiac remodeling.Retinoid X receptors (RXRs), as members of the steroid/thyroid hormone superfamily of nuclear receptors, are crucial regulators of immune response during health and disease. RXR subtype expression is dependent on tissue and cell type, RXRα being the relevant isoform in monocytes and macrophages. Previous studies have assessed different functions of RXRs and positive implications of RXR agonists on outcomes after ischemic injuries have been described. However, the impact of a reduced  Cx3cr1-Cre mice to modulate Rxrα expression in mononuclear phagocytes.Myocardial infarction (MI) is a common and potentially lethal condition caused by the disruption of coronary artery blood flow leading to myocardial ischemia. Technical and conceptual advances over the last decades have facilitated early diagnosis and therapy of MI; however, a considerable number of patients still develop heart failure after an ischemic event . Thus, nRetinoid X Receptors (RXRs) are part of the steroid/thyroid hormone superfamily of nuclear receptors operating as transcription factors. The three subtypes  are differently expressed in cells and tissues ,8, with Rxrα is associated with an increase in infarct size during the later stages of MI. Mechanistically, we identified a reduced mononuclear phagocytic activity paralleled by higher amounts of apoptotic cells in the myocardium, a lower vessel density in the infarct border zone, an impaired proliferation of cardiac macrophages, and alterations in monocyte and macrophage populations in the blood and myocardium in mice with a reduced Rxrα expression in mononuclear phagocytes. Together, RXRα modulates the cardiac immune response and impacts the outcome after ischemia-perfusion injury.In this study we aimed to decipher the role of RXRα in mononuclear phagocytes on cardiac remodeling after MI using a cell-specific conditional deletion model. We provide evidence for the involvement of RXRα in monocytes and macrophages in cardiac remodeling after MI. Our results indicate that a reduced expression of Rxrαflox/flox mice have been previously described  FDG was provided by the company PET Net GmbH. Mice were narcotized using isoflurane 2.0% p.i. and the tracer was injected in a lateral tail vein. After 20 min, acquisition was started using an Inveon P120 μPET Scanner. An emission scan was performed for 15 min, followed by a transmission scan for 7 min. The software ‘Inveon acquisition and research workplace’ was used for technical processing. Evaluation of the reconstructed images was performed using the commercially available software QPS/QGS , as described previously [Small-animal imaging was performed in cooperation with the Department of Nuclear Medicine of the LMU Munich. The tracer [eviously .n < 15) and the Welch′s t-test for n > 15. p-value < 0.05 was considered significant. All results are shown as mean ± standard deviation.Statistical analysis was performed using the software ‘GraphPad PRISM’  and ‘Microsoft Excel 2016′ . The Fisher–Pitman test was used for small animal numbers (Rxrα in monocytes and macrophages on cardiac remodeling after MI has not been assessed so far. We used a tamoxifen-inducible Cre-lox-system specific for mononuclear phagocytes (Cre-ert2Cx3cr1) to delete Rxrα (flox/floxRxrα) and combined it with an I/R-model to study the function of RXRα. Using this depletion model, we excluded influences of Rxrα depletion during developmental stages as well as deletion in other cell types.RXRs are involved in the regulation of different genes, and various studies on the function of RXRs during health and disease have been previously performed ,8. HowevCre-ert2-YFPRxrαflox/floxCx3cr1 mice, deletion of exon 4 of the Rxrα gene (encoding parts of the DNA-binding domain) by the Cre-enzyme leads to the expression of RXRα molecules that cannot maintain their physiological function [Rxrα wildtype and Rxrα L- allele in different tissues  after 4 weeks of tamoxifen chow (40mg/kg per day) in Cre/+  and control mice. In the control mice, by contrast, only the expression of the Rxrα wildtype allele could be detected, Cx3cr1ΔRxrα mice expressed the Rxrα L- allele as well as the Rxrα wildtype allele  in blood monocytes using qPCR. Rxrα expression in blood monocytes of Cx3cr1ΔRxrα mice was significantly reduced compared to the control animals  and on d1 after MI  measured by the total perfusion deficit (TPD) using FDG-PET. Infarct size was assessed on d6 and d30 after MI and compared between Cx3cr1ΔRxrα and control mice  we performed broad characterization of cardiac cytokine- and chemokine profiles in the infarcted area on d1 after MI and compared the expression in the ischemic tissue of Cx3cr1ΔRxrα and control mice. Comparison of 110 cytokines, chemokines, and growth factors did not yield significant differences between the two groups (RXRs are known to contribute to the regulation of chemokine and cytokine expression in the course of inflammation ,11. Antio groups .Rxrα expression in myeloid cells does not seem to have a substantial impact on cytokine and chemokine levels in the cardiac tissue after acute MI.Thus, based on our studies, reduction of Rxrα expression in myeloid cells on cardiac remodeling after MI has not been investigated to date [Advances in treatment of ischemic heart disease over recent years have improved outcomes and reduced mortality after MI. However, a considerable number of patients develop heart failure after an ischemic event, and ischemic heart disease is still a leading cause of death worldwide ,32. The  to date ,14,18,29Rxrα expression in myeloid cells with a tamoxifen-induced Cre-lox-system, we were able to show that a reduction of RXRα levels in monocytes and macrophages negatively impacts cardiac remodeling after myocardial infarction. Lower expression of Rxrα promotes apoptotic cell accumulation in the myocardium, reduces cardiac macrophage proliferation and angiogenesis in the infarct border zone/infarct area, impacts monocyte/macrophage subset composition, and is associated with a higher total perfusion deficit on day 30 after MI.By specifically reducing Cx3cr1ΔRxrα in comparison to control mice, pointing towards a relevant function of RXRα in myeloid cells during remodeling processes after an acute ischemic injury.Positive implications of RXR agonists on outcomes after ischemic injuries have been previously described. Application of bexarotene after ischemic stroke decreased the infarct size and was associated with improved post-stroke recovery ,29. AccoCx3cr1ΔRxrα mice on d30 after MI in relation to controls. These findings are consistent with former studies reporting an increase in proangiogenic molecules and in vessel formation in the infarct area, whereas expression of VEGF-A in the non-infarcted myocardium remained unchanged [Cx3cr1ΔRxrα compared to control mice.Angiogenesis is crucial for physiological remodeling after myocardial infarction . Especianchanged . In contnchanged , we coulRxrα-KO mice in kidney disease. Impaired phagocytosis leading to a reduced cell uptake was associated with a higher number of apoptotic cells in the kidney [Cx3cr1ΔRxrα mice, and we could detect higher numbers of apoptotic cells in the infarct area of Cx3cr1ΔRxrα compared to the control mice on d1 after MI as an indicator of immune dysregulation after MI associated with an increased infarct size.Phagocytosis and apoptosis are important components of a regulated immune response. Uptake of cell debris by macrophages triggers an anti-inflammatory response and maintains self-tolerance ,37,38. Re kidney . In lineCx3cr1ΔRxrα in relation to control mice during the acute state after MI. Cardiac macrophages are an important part of the physiological immune response after cardiac injury, which is emphasized by the fact that depletion of cardiac macrophages leads to worse outcomes [Cx3cr1ΔRxrα mice, no changes in overall cardiac macrophage count could be detected. As we did not differentiate between resident cardiac macrophages and monocyte-derived macrophages, it is conceivable that a reduction in proliferating resident macrophages is associated with increased infiltration of monocyte derived macrophages, which would be in line with the reduced proportion of Ly6chi monocytes in the blood of Cx3cr1ΔRxrα mice. Based on previous studies, Ly6chi monocytes can contribute to different cardiac macrophage subsets and a regulated biphasic monocyte, and macrophage immune response after MI is crucial for adequate cardiac remodeling [Rxrα expression has an effect on the proportion of resident and monocyte-derived macrophages after MI, leading to a dysregulated immune response contributing to an enlargement of the infarct area.Cardiac macrophages have the ability to proliferate and self-maintain locally . While toutcomes . Even thmodeling ,3,27,39.hi macrophages could be detected in Cx3cr1ΔRxrα animals after MI. The MHCII complex is involved in antigen presentation and immune cell activation during acute inflammation; thus, differences in MHCII expression could have an impact on the course of the acute immune response after MI [hiLy6clo macrophages in the heart of Cx3cr1ΔRxrα compared to control mice could indicate increased monocyte-to-macrophage differentiation due to excessive inflammation. Taken together, future studies will be necessary to investigate the effect of the shift in monocyte and macrophages subpopulations on acute inflammation and on outcomes after MI.Moreover, FACS analysis revealed changes in cardiac macrophage populations in mice expressing reduced levels of RXRα. Increased proportions of MHCIIRxrα expression in myeloid cells by around 50%. Moreover, the missing differentiation between resident cardiac macrophages and bone marrow-derived macrophages, as well as between classical and non-classical monocytes, are other drawbacks. However, we show for the first time that reduced Rxrα expression in myeloid cells impacts cardiac remodeling after myocardial infarction and could identify an association with immune dysregulation leading to an increase in infarct size. Overall, further investigations with a more efficient reduction in Rxrα expression and future studies assessing the benefit of RXRα agonists in the course of acute MI will be necessary.The main limitation of our study is that we only achieved a reduction of Rxrα expression in monocytes and macrophages negatively impacts cardiac remodeling after myocardial infarction as indicated by an increase in infarct size. Thus, RXRα might be a promising target for regulating the immune response after MI in order to improve outcomes.In summary, RXRα in myeloid cells is involved in immune regulation in the course of acute inflammation after MI by modulation of phagocytosis, cell apoptosis, angiogenesis, macrophage proliferation, and monocyte/macrophage subset composition. Reduction of


In knee osteoarthritis, progressive degeneration of the articular cartilage surface produces disability and chronic pain. Intra-articular injections of stromal vascular fraction (SVF) could be an innovative approach to manage patients with early knee osteoarthritis.Between June 2019 and November 2020, 123 patients were recruited to receive intra-articular injection of SVF. Radiographic evidence of degenerative joint disease was classified according to Kellgren and Lawrence grades. Knee injury and osteoarthritis outcome score (KOOS) and visual analog scale (VAS) were collected preoperatively, at 1 month, and after 6 months from injection.p < 0.05). The mean KOOS before injection was 51.4 ± 16.5, after 1 month it was 75.5 ± 15.8, and at 6 months it was 87.6 ± 7.7. Stratifying the mean KOOS according to Kellgren–Lawrence Grades, the difference remained statistically significant (p < 0.05). The patients’ mean VAS before injection was 6.5, after 1 month it was 3.5, and after 6 months it was 2.4. No complications were observed.There was a statistically significant improvement of KOOS and VAS of all patients to 6 months (.Intra-articular knee injection of SVF is safe and effective to ameliorate the clinical and functional scores in patients with early knee osteoarthritis for 6 months The knee is the weight-bearing joint most frequently plagued by osteoarthritis (OA). Conservative management of OA includes physical exercises, body weight reduction, drugs, hyaluronic acid injections with or without corticosteroid, and platelet-rich plasma (PRP) injection. Nonsteroidal anti-inflammatory drugs (NSAIDs) provide short-term benefits, while corticosteroids may worsen the clinical picture in the long term , 2. HyalIntra-articular cell therapies have recently emerged as a method to manage the early stages of knee osteoarthritis (KOA). Mesenchymal stem cells (MSCs) can be considered a potential biological approach to articular cartilage restoration given their properties of multi-lineage differentiation potential, self-renewal, and immunomodulatory capacity . MSCs maMSCs can be collected from different tissue sources. Autologous cells induce no rejection, carry no risk of disease transmission, and are less tumorigenic than embryonic stem cells . Bone maAlthough MSCs have shown efficacy in clinical studies, evidence on their efficacy remains unclear in KOA, given cell heterogeneity and concomitant procedures.This study analyses the short-term clinical and functional results of patients with early KOA treated with one single intra-articular injection of adipose-derived stem cells without expansion or enzymatic treatment.Between June 2019 and November 2020, 123 patients undergoing intra-articular injection of adipose stem cells were recruited. The patients’ age, sex, body mass index (BMI), previous surgery on the affected knee, and medical comorbidities were recorded at pre-operative assessment. Selection criteria were clinical findings of KOA with radiographic evidence of degenerative joint disease on standing anteroposterior and lateral radiographies. Exclusion criteria were age over 75 years, body mass index < 18 or ≥ 30, patients with a severe (> 10°) varus or valgus deformity, severe Kellgren–Lawrence grade (IV) on radiographs, recent trauma with acute involvement of ligaments and/or menisci, and infectious or inflammatory joint disease. Patients who received both MSCs harvesting and knee arthroscopy at the same time were excluded. The Kellgren and Lawrence classification was used to grade the severity of osteoarthritis: grade 0 is absence of radiographic signs of osteoarthritis; grade 1 is characterized by doubtful joint space narrowing and possible osteophytic lipping; in grade 2, there are osteophytes and possible joint space narrowing; grade 3 is defined by moderate multiple osteophytes, definite narrowing of joint space and some sclerosis and possible deformity of bone ends; grade 4 is a severe condition with large osteophytes, marked joint space narrowing, severe sclerosis, and definite deformity of bone ends. The subcutaneous adipose tissue on the abdomen was evaluated at the first consultation because the “relative” absence of fat and the presence of abdominal scars could complicate the harvesting. Medical history, Knee Injury and Osteoarthritis Outcome Score (KOOS), and visual analog scale (VAS) were collected by two orthopedic surgeons (CA and LG) for all patients before lipoaspiration and at the first and sixth month from injection. The Knee Injury and Osteoarthritis Outcome Score (KOOS) evaluates the course of knee injury and treatment outcomes. It assesses 42 items in 5 separately scored subscales: Pain (nine items), Symptoms (seven items), Function in daily living (ADL) (17 items), Sport and Recreation Function (five items) and Quality of Life (QOL) (four items). Scores are transformed to a 0–100 scale, with zero representing extreme knee problems and 100 representing no knee problems. The visual analog scale (VAS) is used to classify knee pain; it ranges from no pain (0) to an extreme amount of pain (10). Informed consent was obtained from all individual participants included in the study.Patients were placed supine; the abdomen was prepared in a standard fashion with betadine and chlorhexidine. The surgical field was prepared, and 1 ml of Lidocaine 2% was injected at the site of skin incision. All procedures were performed by two fully trained surgeons (DN and AZ). The preferable harvesting area is the iliac and lumbar abdominal area or the periumbilical area. After performing a small skin incision, an infiltration fenestrated blunt cannula (16G) connected to 50 ml syringes was used to inject Klein solution homogenously. This solution contains 250 ml of NaCl 0.9%, 20 ml of Lidocaine 2%, and 0.5 ml of Epinephrine 1 mg/ml. Transverse movements with the cannulas should be avoided, and the solution should be injected during retrograde movements of the cannula. The distribution of 150–200 ml of the Klein solution in the subcutaneous layers is facilitated by digital manipulation of the abdomen  connected to a self-blocking syringe is introduced in the subcutaneous fat and lipoaspiration can start. The block system produces negative pressure inside the syringe, allowing to harvest the lipoaspirate from the previously infiltrated areas just moving the plunger back . The lipoaspirate is transferred into the Lipocell device where it is dialyzed with a filter and washed with 500 ml of Ringer lactate or NaCl 0.9%. When the lipoaspirate becomes yellow and the outflow irrigation is almost clear of blood, it is ready to be collected using a 10-ml syringe and to be injected (10–15 ml) intra-articularly. The choice of injection portal may be either superolateral under the patella with the knee extended or through the inferomedial or inferolateral soft part of the knee with the knee flexed to 90°. After the injection, the knee is flexed and extended several times to diffuse the product in the joint  than males  with an average body mass index (BMI) of 27. The STROBE diagram shows the various phases of the study  had medial meniscus surgery, 4 patients (3%) had undergone anterior cruciate ligament reconstruction, 32 patients (26%) had tried hyaluronic acid injections, 2 patients (1%) had undergone tibial osteotomy, and 67 patients (54%) had not received other treatments before the index procedure (Table p < 0.05). The Student’s t test showed statistically significant difference between the mean values of KOOS before and at 1 and 6 months after injection (p < 0.05). Stratifying mean KOOS on Kellgren–Lawrence Grades, the difference between values remains statistically significant (p < 0.05) (Table Radiographic signs of grade I of Kellgren–Lawrence were evident in 52 patients (42%), 61 patients (49%) showed grade II, and 10 patients (8%) showed grade III. The mean KOOS of all patients before injection was 51.4 ± 16.5, at the first month from injection it was 75.5 ± 15.8, and at the sixth month from injection it was 87.6 ± 7.7  were 67, while “not-naïve” patients were 56. Table t test showed statistically significant difference between the mean values of KOOS for naïve patients before and at 1 and 6 months after injection (p < 0.05). The same results were obtained with not naïve patients.The Student’s t test showed statistically significant difference between the mean values of VAS before and at 1 and 6 months after injection (p < 0.05). No complications were observed in the patients treated.The patients’ mean VAS before injection was 6.5, after 1 month it was 3.5, and after 6 months it was 2.4. The Student’s Recently, clinicians and researchers are focusing on prevention of progression of KOA through the use of mesenchymal stromal stem cells (MSCs). These cells, with their chondrogenic and anti-inflammatory properties, may well reverse the first stages of KOA, reducing synovitis, cartilage degeneration, and osteophyte formation . AdiposeMSCs may have limited clinical efficacy in high grade KOA, and more studies with high-level evidence  recommenThe area of fat harvesting and patient age may influence the stem cell yield ; furtherAutologous adipose stem cells show beneficial effects when compared to other MSCs to relieve pain and improve function, possibly because of the visco-supplementation effect of fat tissue on cartilage surfaces . The mosThis study did not evidence any adverse events. Therefore, intra-articular administration of adipose tissue derivatives can be considered safe and less invasive than other approaches. No patient needed any additional surgical treatment within the follow-up period, and a second injection of adipose stem cells might be possible when the improvement of the first injection becomes ineffective. Biological treatments are obviously more expensive than other conservative treatment, but traditional treatments are often only palliative. Biological treatments offer the opportunity to regenerate tissues and delay the progression of KOA . To dateThe principal limitation of this study was that the potential biological effects of the adipose stem cells on the joint surface were not evaluated through MRI or arthroscopy, and we can only base our considerations on patients’ symptoms. Furthermore, there was no comparison with other conservative therapies; to assess the efficacy of adipose stem cells, a control group treated only with NSAIDS, or PRP, or HA or other conservative modalities would be needed.Another important limitation of this study was that the follow-up (6 months) was short, but we point out that we wanted only to assess the short-term efficacy, and long-term efficacy will be the object of future studies.Isolated intra-articular knee injection of SVF is safe and effective to ameliorate the clinical and functional scores in patients with early KOA for 6 months. It can be considered an innovative approach able to prevent OA progression and delay total knee arthroplasty.


The programmable synthesis of rationally engineered crystal architectures for the precise arrangement of molecular species is a foundational goal in nanotechnology, and DNA has become one of the most prominent molecules for the construction of these materials. In particular, branched DNA junctions have been used as the central building block for the assembly of 3D lattices. Here, crystallography is used to probe the effect of all 36 immobile Holliday junction sequences on self-assembling DNA crystals. Contrary to the established paradigm in the field, most junctions yield crystals, with some enhancing the resolution or resulting in unique crystal symmetries. Unexpectedly, even the sequence adjacent to the junction has a significant effect on the crystal assemblies. Six of the immobile junction sequences are completely resistant to crystallization and thus deemed “fatal,” and molecular dynamics simulations reveal that these junctions invariably lack two discrete ion binding sites that are pivotal for crystal formation. The structures and dynamics detailed here could be used to inform future designs of both crystals and DNA nanostructures more broadly, and have potential implications for the molecular engineering of applied nanoelectronics, nanophotonics, and catalysis within the crystalline context. Engineered crystal architectures from DNA have become a foundational goal for nanotechnological precise arrangement. Here, the authors systematically investigate the structures of 36 immobile Holliday junction sequences and identify the features allowing the crystallisation of most of them, while 6 are considered fatal. A variety of methodologies that allow for programmable self-assembly have been developed, including the attachment of DNA linkers to the nanoparticle (NP) surfaces3 for the construction of 3D lattices with user-defined colloidal NP crystal configurations8. With the advent of DNA origami9, 3D superlattices of nanoparticle cluster shapes have been described11 along with 3D origami lattices displaying customizable geometry to host guest species12. Rationally designed crystals based upon the “tensegrity” motif13 which self-assemble with designed four-arm junction crossover points and “sticky end” cohesion have also been shown to be capable of being converted into nanodevices14. Recently, several unique motifs with distinctive crystal symmetries, improved resolution, and in one case, a unique junction nucleotide sequence, have been reported18.The fabrication of highly customizable 3D DNA-based architectures for the precise organization of nanoscale materials was originally conceptualized by Seeman in 198219. Holliday junctions have been extensively structurally characterized29, and emerged as a key motif for rationally designed nanoscale assemblies and devices in structural DNA nanotechnology30 Naturally occurring HJs can “slide” and change the length of their arms32, a process known as branch migration33. Introducing asymmetric sequences at the branching point, however, effectively immobilizes the junction and allows its use in constructing well-defined nanostructures34. Although a variety of multi-branch junctions have been employed41, the four-arm HJ remains the most popular.The application of four-way junctions for 3D DNA crystals was inspired by genetic recombination, whereby an unstable branched intermediate termed a Holliday junction (HJ) is created and subsequently undergoes a dynamic reconfiguration to facilitate recombination during cell division43, and has been used nearly exclusively in the construction of self-assembled 3D crystals17 with a single exception where “J10” was used18. Some early work explored the sequence influence on packing of stacked-X junctions using theoretical45 or experimental methods47, but to our knowledge, no systematic study of the immobile HJs has been performed. In addition, the relationship between ion concentration and the transition of the HJ from the open to the stacked conformation is well known51; however, studies of sequence effects on the ability to bind ions are limited, making the elucidation of the structural parameters that influence crystallization and symmetry a desirable route for exploration.Theoretically, there are 36 base pair combinations of immobile sequences. “J1” was the earliest to be designedIn this work, we exhaustively probe the ability of all 36 immobile HJ sequences to form DNA crystals in two different designed systems. Our work revealed that a large majority of immobile HJs enable crystallization, with some yielding higher resolution structures and a variety of symmetries. The symmetry is also highly sensitive to the sequence of the junction arms (stems) further away from the branching point, as we demonstrate using scrambled stem sequences. Unique ion binding sites were also observed at two conserved positions within the structures. By performing molecular dynamics (MD) simulations of all 36 HJs in solution, we show that these sites are pivotal for crystallization, with the universally non-crystallizing “fatal” junctions showing zero ability to bind ions in this fashion. Although limited by sampling, the modeling used in this work provides a reliable picture of sequence-dependent flexibility and solvent effects of all 36 immobile HJs as they relate to self-assembled DNA crystal lattices. Overall, the work provides a rigorous and complete description of how different immobile HJ sequences, flanking sequence modifications, and the ability of the junction to capture ions profoundly affect the rational design of self-assembled systems that span a multitude of designer DNA architectures.15 and “4 × 6”16 designs (collectively referred to as the 4 × N systems), and a third construct with a “scrambled” sequence variant of the 4 × 6 lattices. Self-assembly was mediated by three constituent oligonucleotides  codes Fig. . Each as codes FiP3221, P32, and R3) that are described in detail below  and 56.59° (σ = 1.50) for the P32 and P3221, respectively , with the majority preferring slightly basic pH conditions in cacodylic acid (Supplementary Table P32) to rhombohedral (R3). Furthermore, J4 and J36 crystallized exclusively in R3, but J5, 31, 33 exhibited the ability to crystallize in both P32 and R3 , with P32 crystals were a = b = 68.29 Å c = 55.68 Å. The c-axis, in particular, was ~5 Å shorter, and had a much larger degree of variability (σ = 1.97), than those that crystallized using the 4 × 5 motif . The cavity volumes were also markedly different from the P32 cavity volumes (~614.7  nm3) compared with ~532.1 nm3 in the more densely packed R3 structure  and 58.37° (σ = 2.3) for the P32 and R3 symmetries, respectively , along with the R3 systems, also shared a preference for low salt conditions, unlike the original P32 crystals. This change in buffer preference suggests that global sequence content can indeed influence self-assembly behavior. Further, we observed modest improvements in resolution, reaching as high as 2.7 Å in J36. Notably, all junctions preventing crystallization compared to its original counterpart remained fatal  and 58.05° (σ = 1.39), respectively. Although the average angle in the R3 crystals is nearly 3° higher than those from the native sequence , compared with 54.60° , a less accurate average due to a small sample size, so we posit that 54.60° more closely reflects the average observed angles in the 4 × 6 systems.The role of longer-range sequence effects on junction angles remains a largely open question, but the effect in our crystal system is significant. There appeared to be only a marginal difference in the average cell lengths in the 15 the presence of arsenic ions at two opposing positions  and 10 mM MgCl2, and 50 mM HEPES pH = 7.5 with 20 mM MgCl2, respectively. Only cobalt could account for the different peaks in the Fo–Fc maps in J25, whereas in J34, and J22 and J23 in the 4 × 6 and 4 × 6 scramble systems, respectively, contained Mg2+. The modeled ions were readily superimposable, and well-coordinated at Pos1 & 2 . In over 224 μs of simulations, the force-field performance was deemed satisfactory, as stable base pairing and B-form helical topologies were observed in agreement with our crystallographic experiments and earlier studiesσ = 0.28), suggesting that the ability to capture ions is pivotal to the ability to crystallize the immobile HJs. We speculate that the branching-point ion-binding site could be stabilizing the DNA strand exchange during the formation of the lattice and thus facilitate the crystal growth. This hypothesis is supported by the fact that the junctions unable to form this ion-binding site, or those which do so to a lesser degree, are consequently among those that either do not grow crystals at all, or that are more sensitive to the crystallization conditions. The sole exception was J7 which, despite possessing an appropriate branching point sequence to do so, did not form the ion-binding site in simulations or experiments, but was still able to crystallize.The most significant difference between the individual junctions observed in MD simulations was their ability to form distinct potassium ion binding sites near the junction branching point. In all cases, the ion formed a bridge between the phosphate right at the branching point and one or two closest bases. These sites were in good agreement with the Pos1 and Pos2 sites observed in the experimental crystal structures Figs.  and 5a. + cations  should bind at this location. This result is consistent with the highly negative molecular interaction potential  with cacodylate at this site. Cacodylate anions are known to form interactions with negatively charged segments of nucleic acids in this fashion60. Lastly, there could be multiple Na+ ions (the counterion of cacodylate) present around the anion. Due to the nominal resolution of our structures, which is in the ~3 Å range, it is not possible to unambiguously identify all the interacting species, nor is it possible to fully describe the exact coordination, such as the involvement of water molecules and Na+. The binding of Na+ could compensate or even over-compensate the cacodylate negative charge, effectively re-creating the common cation-binding site seen in simulations and other experimental structures. The Na+ ions are often fluctuating and their coordination requirements are pliable, rendering them entirely invisible to us. Furthermore, as suggested by simulations, there could be significant local dynamics that obscure the densities with averaging. Past MD simulations reported highly variable Na+ binding sites, from stiff to dynamic ones, around nucleic acids61, and it is entirely possible that invisible Na+ cations can bridge the cacodylate anion with the DNA carbonyl and phosphate groups. While the inability to describe the interaction site in greater detail is a limitation of the current work, it is abundantly clear that accommodation of ions at this location is a strict requirement for the junction to stabilize, and to effectively crystallize.Initially, there might be an apparent contradiction associated with the experimentally detected presence of arsenic from a cacodylate anion in the region where the simulations localize Kons Fig. . HoweverIn this work, we have reported a systematic study of all 36 immobile HJ sequences across two different crystal systems, findings that can, in principle, be extended to any 3D DNA crystal and potentially other types of DNA architectures. We show that J1 (or any other junction) should not be considered a privileged option for designing self-assembled lattices. Rather, a host of other sequence combinations discussed herein should be explored, with many of them providing potentially superior performance. We make the essential observation that several junctions—including J11, 12, 13, 17, 18, and 27—are universally fatal to crystallization and should be avoided in future crystal designs, unless perhaps alternate isomers are considered. Another major observation is the importance of stem region sequences outside the HJ, given that this sequence can control crystal symmetry and lattice architecture, and improve resolution in non-obvious ways. We also elucidated the key role of ion coordination in driving these effects. Scaling up DNA nanostructures to larger assemblies—for DNA crystals, as well as 1D and 2D lattices, and potentially DNA origami systems—will likely require taking into account sequence-dependent effects both at the level of stem and HJ geometries. Finally, the angle distributions obtained from our MD studies of junctions can improve accuracy of coarse-grained models and DNA nanotechnology design tools to more accurately represent nanostructures.In summary, our experimental results are corroborated by the largest scale MD (224-μs-aggregate) simulation performed to date on HJs. The simulations elucidated the role of sequence-dependent flexibility and solvent effects on HJ conformations. This work also demonstrates that HJ sequence plays a non-trivial role in the ability of DNA crystals to form, and can dramatically influence crystal symmetry. Furthermore, we have provided a systematic and comprehensive sequence-structure study on self-assembled DNA crystal systems using all 36 junctions. Lastly, the study unexpectedly revealed specific molecular interactions (ion binding) using both experimental and modeling parameters, that has never been performed on this scale.2O and washed 5× using 3 kDa molecular weight cut off filters (Amicon) to remove any remaining salt. A stock of the three-component strands (S1:S2:S3) was made with final concentrations of 30:120:120 µM for each construct. The sitting drop vapor diffusion method was performed with Cryschem plates (Hampton Research) using an adaptation of a discontinued DNA crystallization screen from a commercial vendor (Sigma Aldrich) containing a sparse matrix of 48 conditions  and purified using 14% denaturing polyacrylamide gel electrophoresis (PAGE) or HPLC. Following purification, the pelleted DNA was resuspended in nanopure H62, and the initial phases were calculated using molecular replacement in Phaser63 from the PHENIX64 suite of programs with either the J1 4 × 5 structures 5KEK and 6X8C, as the initial search models for the duplex and junction structures, respectively, and the J1 4 × 6 structures 5VY6 and 6XNA for the duplex and junction structures containing P32 symmetry in that system, respectively. However, with few exceptions, the majority of the 4 × 6 R3 symmetry crystals required another R3 model of one of its counterparts as the ideal search model. Multiple rounds of model building were performed in Coot, with the initial model first treated as a single rigid body, followed by subsequent iterative rounds using restrained refinement in REFMAC65 from CCP466, along with real space, and XYZ coordinate calculation in phenix.refine. All ions were modeled into regions of Fo–Fc difference density with a contour level ≥ σ = 3.0 and refined. Atom occupancies and B-factor calculations were then refined, along with simulated annealing to conclude refinement. All refined models used an Rfree set containing 5–10% of the unique reflections for each structure. The coordinates and structure factors, totaling 134 unique structures, were deposited in the Protein Data Bank (PDB), and the corresponding accession codes are listed in Supplementary Table 67.All diffraction data were processed in HKL200015 as the starting structure for all simulations. Each arm of the HJ was extended to contain at least eight base pairs, with the extended parts of the helices taken from the adjacent cells of the crystal lattice. The resulting construct contained 64 nucleotides and was used as the initial structure for simulations of all the 36 immobile junction sequences, by substituting the branching point base pairs accordingly. We have used xLeap module of AMBER1868 to prepare the topology and coordinate files. The latest AMBER OL15 DNA force field69 was used in all reported simulations. During the revision, we also tested the alternative parmbsc1 DNA force field70 for a subset of the HJs; however, with bsc1 we detected a significant population of non-native and possibly spurious β/γ g + /t backbone conformations Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting summaryPeer Review File


Vibrio species are classified as potent hazards because of their tendency to effect serious diseases like cholera and other gastrointestinal ailments in humans, as well as vibriosis in fish. A total of 144 freshwater samples were aseptically collected monthly across four rivers  over a 12-month period from which Vibrio spp. were isolated using culture procedures, confirmed by means of biochemical test as well as Polymerase Chain Reaction (PCR) assay and further characterized for their phenotypic antibiotic susceptibilities and relevant antimicrobial resistant determinants by PCR. Three hundred and fifteen (58%) isolates confirmed across the sampled sites  showed high resistance against erythromycin—95%, Sulphamethoxazole—94%, rifampicin—92%, doxycycline—82%, tetracycline—75%, amoxicillin—45%, cephalothin—43% and varied susceptibilities to other antibiotics. The multiple antibiotic resistance indices of 97% of the Vibrio isolates were above the 0.2 threshold limit with MAR phenotype pattern E-SUL-RF-TET-DOX (0.38) found to be the most prevalent pattern among the isolates. The distributions of resistance determinant of the tested antibiotics were revealed as follows: sulII 33%, sulI 19% (sulfonamides); blaOXA 27%, ampC 39%, blapse 11% (beta-lactams); tetA 28%, tetE 20%, tet39 8%, (tetracyclines) and strA 39%. aacC2 24%, aphA1 14% (aminoglycosides). Strong positive associations were observed among tetA, sulI, tetE and sulII. This study raises concerns as these selected rivers may contribute to the environmental spread of waterborne diseases and antibiotic resistance genes. Therefore, we recommend environmental context-tailored strategies for monitoring and surveillance of resistance genes so as to safeguard the environment from becoming reservoirs of virulent and infectious Vibrio species. Vibrio species are aquatic bacteria of ecological significance belonging to Vibrionaceae family. It is phenotypically characterized as Gram-positive, oxidase positive, motile rods, halophilic and facultatively anaerobic in its metabolism. They are commonly regarded as autochthonous to marine environment and its detection in freshwater ecosystem has been documented globally. There are over 140 different species of Vibrios documented currently1, however, 12 Vibrio species are classified as infectious to humans with V. cholerae recognized as the causative agent of cholera disease in humans. Other non-cholerae Vibrio spp. are common causative agents of foodborne related infection in humans arising from contaminated water, undercooked or raw seafood consumption2. The common symptom of Vibrio infection is gastroenteritis and diarrhea which may sometimes be associated with virulence factors like shiga-like cytotoxin, toxin-coregulated pilus, cytotoxins, siderophores, hemolysins, proteases and heat-stable enterotoxin4.Vibrio associated diseases in human and animal is increasingly being reported globally. This bacterium has developed to inhabit diverse settings within ecosystems due to their complex mode of living5. They have been isolated from diverse environments including crustacean guts, fishes in sea hydrothermal vent, rivers, wastewater, plants etc. and they carry out important role of nutrient cycling in aquatic milieus8.The incidence of Vibrio infections are commonly treated with tetracyclines, third-generation cephalosporin, fluoroquinolone, trimethoprim-sulfamethoxazole as well as aminoglycoside. Tetracyclines and fluoroquinolones are mostly used in protracted or severe cases of Vibrio illnesses as they are known to be associated with lesser mortality rates4. However, in recent decades, many bacteria including Vibrio have emerged with unprecedented resistance to many antimicrobials owing to abusive use in agricultural and human systems9.10 and their resistance genes can be transferred to other pristine microorganisms by means of mobile genetic elements and horizontal gene transfer thereby causing changes in the development and adaptation of microbial residents of aquatic environment11.Antibiotics is among a prominent member of the list of “contaminants of emerging concerns” circulating in different environments. Bacteria that are exposed to antibiotics in aquatic environments can become antibiotic resistance13. Likewise unregulated and ease of access to antibiotics in developing nations like Nigeria can contribute to persistent exposure of humans to antibiotics via consumption of water and food. Thus, polluted aquatic environments have now been identified as significant reservoirs of antibiotic resistance genes requiring more investigation14. Some resistance genes contribute to the virulence expression in Vibrio infections in human and animal setting and this includes beta lactam genes , sulfonamides genes (sul1 and sul2), quinoline genes  etc.Evidence has shown that anthropogenic activities resulting in agricultural, municipal and aquaculture wastes as well as water run-off can introduce multiple antibiotic resistance organism and their resistance factors into the environment and water bodies15. Hence, monitoring antibiotic resistance patterns of pathogenic bacteria in anthropogenically impacted environment is a relatively cost effective substitute where a national surveillance activity is absent16.In most developing countries, identification of new and emerging threats, practices of good treatment of patients, and effective control of antibiotic resistance is not effective due to non-existence of national antimicrobial resistance surveillance programs. Even where surveillance programs do exist, implementation strategies are lacking due to poor infrastructural and equipment supportVibrio spp. and clinically relevant antibiotic resistance genes. Therefore, we document the first assessment of the prevalence of 11 resistance determinants of Vibrio isolates obtained from four selected freshwater bodies in South-western region of Nigeria.Moreover, if the dynamics of antibiotic resistance in pathogenic organisms and diversity of their resistance genes are to be accurately evaluated; there is a need to assess the freshwater environment for contamination with pathogens such as The sampled rivers selected for this study includes: Asejire—SS1 (N7° 21′ 46.9″ E4° 07′ 51.5″), Dandaru—SS2 (N7° 26′ 25.0″ E3° 49′ 57.5″), Eleyele—SS3 (N7° 26′ 17.1″ E3° 52′ 42.2″), Erinle—SS4 (N7° 46′ 23.7″ E4° 27′ 58.3″); located in three towns in Southwest Nigeria   and were thereafter transferred into agar plates of TCBS  and then incubated overnight at 37 °C. Yellow and green characteristic colonies were quantified as colony forming units (CFU) per 100 mL of samples and 3–5 colonies each were randomly selected, Gram-stained and subjected to oxidase test. Afterwards, 315 presumptive Vibrio species that were Gram-negative and oxidase positive were purified on a non-selective agar and stored for molecular analysis.Pre-sterilized 1 L plastic bottles were used for water sample collection. After the bottles were triple rinsed with sample water, actual samplings were done at approximately 20 cm below the water surface. Samples were subsequently transported in ice coolers and processed according to the recommendation of the American Public Health Association6. Specifically, 18–24 h old pure colonies of presumptive Vibrio spp. were suspended in sterile 200 μl distilled water, then lysed by heating for 15 min at 100 °C temperature using an AccuBlock (Labnet). A Mini Spin microcentrifuge was used to decant cell debris by centrifugation for 10 min at rate of 15,000 rpm. The DNA template from the recovered supernatants was employed in subsequent PCR tests.The isolates' genomic DNA extraction was carried out using the boiling techniqueVibrio was established by PCR using the Vibrio genus primers (F: CGG GAAATGCGTAGAGAT R: TACTAGCGATTCCGAGTTC)17 to amplify a 663 bp sequence of the 16s RNA genes of Vibrio.Genotypic confirmation of The reaction mixture contains PCR Master Mix (12.5 μl), (Thermo Scientific), oligonucleotide primer (1 μl each) , DNA template (5 μl), and, nuclease free water (5.5 μl), to make up 25 μl. reaction volume. The PCR protocols used comprises: Initial denaturation for 15 min at 93 °C followed by 35 cycles of denaturation—annealing—elongation steps at 92 °C for 40 s; 57 °C for 1 min and 72 °C for 1.5 min respectively followed by 7 min final extension at 72 °C. Electrophoresis was performed by loading 5 μl amplicons, 3 ul 100-bp ladder (Thermo Scientific), on 2% (w/v) agarose gel (Merck) pre-stained with 5 μl ethidium bromide (Sigma-Aldrich). The electrophoresis process was carried out at 90 V for 50 min and visualized under an ultra-violet trans-illuminator system (Alliance 4.7).18, Fresh cultures (18–22 h old) of all isolates that have been positively identified were introduced into 5 mL of 0.85% sterile saline with the suspension turbidity set to 0.5 McFarland standards. MH agar plates were inoculated uniformly with sterile swabs after which discs were aseptically added and incubated for 18–24 h at 37 °C. Measured Zones of inhibition were interpreted as resistant (R), susceptible (S), intermediate (I), using the zone diameter interpretation according to CLSI guideline18.Standards disc diffusion technique with Mueller–Hinton agar (MHA) was used for antimicrobial susceptibility testingVibrio infection treatment selected for this assay are: Amikacin—AMK (30 μg), Streptomycin—S (300 μg), Gentamycin—G (10 μg), Cefotaxime—CEF (30 μg), Imipenem—IMI (10 μg), Ciprofloxacin—CIP (5 μg), Meropenem—MEM (10 μg), Ampicillin—AP (10 μg), Sulphamethoxazole—SUL (25 μg), Chloramphenicol—C (30 μg), Tetracycline—TET (30 μg), Erythromycin—E (15 μg), Trimethoprim + Sulphamethoxazole—TS (25 μg), Amoxycillin—AMC (25 μg), Norfloxacin—NOR (30 μg), Doxycycline—DOX (30 μg), Cephalothin—CEP (30 μg) and Rifampin—RF (5 µg).Eighteen (18) antibiotics which comprise the Centre for disease Control (CDC) recommended antibiotics for Vibrio spp. was obtained and those that presented resistance to three and more antibiotics were “phenotyped” to indicate the multiple antibiotic resistances (MAR) phenotypes expressed as:20.The antibiotic resistance profile of the confirmed 21. Pattern of antibiotic resistance (ARPA) was likewise evaluated22.MAR index value ≥ 0.2 threshold suggests high-risk pollution, possibly by uncontrolled use of antimicrobials around the sample sites, which may lead to development of antibiotic resistanceVibrio isolates in the selected freshwaters were determined by calculating their resistance quotients23. This determines probable ecological-risk presented by antibiotics tested on the confirmed Vibrio isolates. The RQs were determined according to the equation24:Possible variations in the phenotypes of antimicrobial-resistant Vibrio isolates were evaluated for their antibiotic resistant genes using specific primer pairs and PCR conditions , tetracycline genes , β-lactamases genes , and aminoglycoside genes . The PCR assay and electrophoresis procedure is as described in “Vibrio isolates harboring ≥ 2 resistance genes were determined.Phenotypically resistance The data obtained were statistically analyzed using IBM (SPSS) version 22.Vibrio isolates were evaluated with Pearson’s chi-square exact test at P = 0.05. A positive relationship between two genes indicates that the genes were associated together, while a negative correlation shows there is no association between the genes.The associations between the resistance genes found in Vibrio confirmed isolates across the samples freshwaters ranges between 72 CFU/100 ml at SS3 and 87 CFU/100ML at SS2  whereas resistance to doxycycline were recorded in 72% Vibrio isolates in Erinle.In total, 315 confirmed Vibrio tested varied and follows the order: ciprofloxacin—93%, norfloxacin—98%, meropenem—91%, amikacin—76%, cefotaxime—88%, Trimethoprim + Sulphamethoxazole—75%, gentamicin—74%, ampicillin—54%. Although, high resistance to erythromycin—95%, sulfamethoxazole—94%, rifampicin—92%, doxycycline—82%, tetracycline—75% were observed as well as mild resistance to amoxicillin—45% and cephalothin—43% respectively  isolate at SS1 (Erinle).Vibrio isolates across the sampling sites ranged between 0.11 and 0.72 with MAR indexes less than 0.2 threshold limit obtained in SS2 (n = 2), SS3 (n = 4) and SS4 (n = 3). Highest (0.72) MAR index was recorded in SS3 with resistance to 13 of the tested antibiotics. Abundance of resistance pattern observed across the sample sites showed that SS1 had the lowest resistance pattern with ARPA of 0.106 while the remaining 3 sampled sites had 0.126 ARPA respectively. MAR phenotype pattern E-SUL-RF-TET-DOX with index of 0.38 was found to be the most common pattern to all the isolates across the sampling sites having being recorded in 8, 4,10 and 5 isolates in SS1 (Asejire), SS2 (Dandaru), SS3 (Eleyele) and SS4 (Erinle) respectively.MAR indexes of index of 97% of Vibrios from all the four rivers sampled were above 0.2 threshold value , tetE-tet39 (3), tetA-tet39 (1) and tetA-tetE-tet39 (1)  were examined for probable detection of blaOXA and blaPSE genes. Both genes were spotted in 27% and 11% of the isolates respectively with dual blaPSE-blaOXA resistant pattern observed in 8 of the isolates  and Dandaru river (SS2) exhibiting the highest prevalence. Occurrence of high Vibrio prevalence in Erinle river suggested the presence of healthy Vibrio carriers who shed Vibrio into the environment on a regular basis. Although severe rainfall modifies the physical and chemical parameters in the aquatic environment, the subsequent influx of flood water together with organic waste  can aid Vibrio species proliferation in the chosen rivers. This finding is consistent with earlier studies that recorded high incidence of Vibrio species28. Furthermore, prior research has confirmed the link between Vibrio species and the aquatic environment25. The incidence of Vibrio spp. did not differ significantly between the sampled sites, according to statistical analysis.Pollution of water bodies also influence the microbial population and species of organisms found in them. Vibrio species and antimicrobials29. Although, Vibrios are largely considered to be very susceptible to most antimicrobials recommended for medical treatment30, resistance among environmental isolates of Vibrio is increasingly being recorded in many countries.Water ecosystems have become a well-known hotspot for environmental diffusion of both Vibrio isolates against some endorsed and frequently used antibiotics in Vibrio-associated infection treatment which correlated with previous reports31. Remarkable resistance against erythromycin and sulfamethoxazole observed in this study is comparable with previous reports on clinical and environmental Vibrio isolates33. Likewise, relative resistance against the two tested β-lactams drugs (Amoxicillin 45% and amikacin 30%) recorded in this study is similar to that documented in environmental Vibrio isolates elsewhere36. The observed resistance of Vibrios to tested β-lactams in this study maybe related with the isolation source as bacteria such as Vibrio, recovered from water environment have been documented to show high resistance towards β-lactams class of antibiotics38. The resistance to amoxicillin and amikacin observed in this study is not surprising as these drugs enjoys widespread usage across different settings. Moreover, the frequency of antibiotics use over time has been hypothesized by researchers to contribute to development of resistance to antibiotics among bacteria39. Resistance to imipenem (18%) was also recorded in this study which corresponds to studies on Vibrio spp. recovered from shellfish and fish in Egypt33 and marine environment in Norway40. The recorded resistance to imipenem in this present study raises public health concerns as this drug is one of the major drugs of last resort for medical treatment of multiple drug-resistance Vibrio infections. Moreso, carbapenemases responsible for resistance to carbapenems has been associated with mobile genetic elements which can perhaps be transferred to humans through food interface41. Resistance recorded against tetracycline and cephalothin showed comparable profile with those stated in various studies across other countries44 and signifies public health threat as these are prescribed drugs in the treatment of humans infections45. High susceptibility to norfloxacin (98%), ciprofloxacin (93%), meropenem (91%), cefotaxime (88%), amikacin (76%), trimethoprim + sulfamethoxazole (75%), gentamicin (74%) recorded in this study is comparable to studies elsewhere46. Generally, varied resistance among antibiotics belonging to the sulfonamides, β-lactams, aminoglycosides, macrolides and tetracycline classes obtained in this study is similar to other findings47 in which similar findings of multiple antibiotic resistance of Vibrio spp. recovered from aquatic habitat have also been reported.Eighteen antibiotics from 9 different classes were examined in this investigation. Multiple antibiotics resistance was observed in the recovered Vibrio isolate resistance to many of the antibiotics examined across all sample sites in this present study suggests a probable misuse or overuse of antimicrobials around the study area for a variety of purposes beyond what the environment can absorb, thereby resulting in the maintenance of unabsorbable residue in the water bodies. Such practices have been suggested to impact the likelihood of bacteria to develop resistance to commonly used antibiotics26. Furthermore, it has been hypothesized that the ability of microbes to share their genetic material in fresh and aquatic environments is a contributing factor to the rise in antibiotic resistance48. In light of the possible influence on treatment outcomes, the continued development of antimicrobial drug resistance to the standard oral treatments for Vibrio-associated diseases raises public health concerns6.The high level of The degree of exposure of all the four rivers to contamination by antibiotics and its possible human health risk was assessed by the multiple antibiotic resistance index (MARI) which shows that the indexes range from 0.06 to 0.72 across all the sampled sites.Vibrio isolates recovered from SS2 (n = 2), SS3 (n = 4) and SS4 (n = 3) indicates low risk of environmental contamination. However, higher MARI values obtained across the sampling sites with highest (0.72) recorded in SS3 having exhibited resistance to 13 of the examined antibiotics indicates the Vibrio isolates have a high ability to contaminate the water sources; this is in accord with results documented in other findings43. MAR phenotype pattern E-SMX-RP-T-DXT with index of 0.38 was found to be the most common pattern among the isolates concurs with a study on Vibrio parahaemolyticus from shellfish in Selangor Malaysia in which resistance to five antibiotics was found prevalent among the isolates49. it was observed in this study that MARindex of 98% of Vibrio isolates from all the four rivers sampled greater than the threshold value. Similar findings of high MAR index of Vibrio spp. has been reported in aquatic environment elsewhere52. The high multiple resistance observed signifies a possible treat public health in the area of bacterial infection control and managements as these antibiotics are still the drug of choice in managing Vibrio related infection in both human and animal. According to many studies, the role of wastewater run-off in diffusion of antibiotic resistance in intestinal bacteria harboring resistant plasmids cannot be over-emphasized53.MARI of 0.06, 0.11 and 0.16 recorded in few of the 54. In this present study, 11 different resistant genes were assayed on phenotypically resistant Vibrio isolates. Among the sulfonamide and tetracycline resistance genes tested, tetA (28%) and sulII (33%) had the highest frequency of occurrence. This outcome is similar to reports by55 in which tetA (64.28%) was among the highest detected genes in Vibrio parahaemolyticus isolated from Malaysian seawater and fishes56 and with high detection of sulII (53.8%) genes in Vibrio cholerae recovered from Hilsha fish.Different samples recovered from environmental sources have been found to harbor diverse antimicrobial genes that encodes resistance to antibiotics such as aminoglycosides, β-lactams, tetracycline, chloramphenicol sulfonamides and trimethoprimsulI gene is linked with class1 integron while sulll are typically found on large infectious multi-resistant or small non-conjugative plasmids which may contribute to the commonness of sulll detection in water environment. The detection of tetA and sulII genes advocates that they are extensively used antibiotics in animal and human therapy in this region. This is because tetracyclines and sulfonamides have broad spectrum of activities and are relatively cheap57. They are commonly incorporated into livestock feed as growth promoters in aquaculture. Likewise, the two classes of antibiotics are used in treatment of Vibrio associated infections in humans, as sulfonamide drugs are used as combination drug for cholera treatment in both children and adults58. This findings match reports of other studies where sulII gene detection was reported as the most profuse sulfonamide resistance conferring gene in freshwater environments62. Likewise, high tetA genes prevalence is consistent with64 reports in which the ribosomal efflux gene, tetA, have been found to be the most commonly detected resistance genes in Vibrio spp. from fresh environment attesting that tetA gene can be easily harbored by different class of bacteria due to their affinity for diverse host range.Specifically, blaOXA, blaPSE and ampC were examined. However, ampC had the highest frequency of recovery among all tested isolates. As stated in earlier studies, diverse pools of β-lactamases are existing in many environmental isolates of Vibrio species signifying that such acquirement of β-lactamase genes is not primarily due to β-lactamase treatment's selective pressure65. Some gram-negative environmental isolates have been found to naturally have low levels of β-lactam antibiotic resistance. It has been stated also that some intrinsic ampC resistance gene found in Enterobacteriaceae are positioned on mobile genetic element with the ability to transmit between diverse bacteria. Some other studies have also recorded high prevalence of ampC gene in gram negative bacteria from freshwater environment66.Beta-lactams, an extensively used antibiotics in the management of several infections are usually distributed in freshwater environment and consequently pose substantial risk to environmental health. Three β-lactams genes str genes that bestows streptomycin phosphoryl transferase resistance, had more strA prevalence than others. These findings agree with similar studies in which high occurrence of strA detection were reported in aquatic bacteria68. Several means of acquiring resistance to aminoglycosides has been described; modification of enzymes such as neomycin phosphor-transferase, alteration of chromosome binding sites and altered aminoglycoside uptake/efflux pumps which bestows cross- resistance and change in cell membrane.Aminoglycosides are a type of antibiotic that has become increasingly significant in the management and prevention of severe bacterial infections in both animals and humans since resistance to diverse classes of antibiotics are increasing in gram negative bacteria. Among the different aminoglycosides genes that were examined, the Vibrio isolates to some of the antibiotics conventionally used in treatment of cholera calls for concern as this could have direct influence on the treatment of cholera cases in Southwestern Nigeria and other parts of the country where the organism could spread.The correlation between the emergence of aminoglycoside resistance and the widespread use of antibiotics for cholera prevention and treatment as well as other usage is well known. The expression of multiple drug resistant by non-cholera tetA and sulII had the highest frequency of genotypic resistance occurrence while erythromycin, rifampin and sulphamethoxazole showed the most prevalent phenotypic resistance. Reasons for the observed discrepancy between genotypic and the phenotypic antibiotic resistance might be presence of expression profiles that were not subjected to testing due to limitations in selecting genes in this study as reported elsewhere69. Also, there is possibility that some of the ARGs in these Vibrios are unexpressed genes61. Consequently, these genes might be possible source of risk in the environment because apart from contributing to the resistance reservoir, they could also be horizontally transferred under suitable conditions. This suggests complexity of the linkage between phenotypes and genetic constructions. The results obtained were put through a statistical test to see if there was any correlation between the various resistance genes investigated, and high correlations were found among some of the resistance determinants. Notable comparable association between the resistance genes indicates tetA was strongly associated with sulI and both genes were the most detected among the three tetracycline and sulfonamide resistance genes tested. High proliferation of sulII than sulI genes in freshwater bacterial isolates is well documented70. This observation further affirms that resistance to antibiotics is triggered by more than two genes.This study reiterated that antibiotic susceptibility and PCR assays are complementary. The PCR assay shows the existence of genes linked with resistance in many of the phenotypically resistant strains and absence of resistance conferring genes in some other strains that shows phenotypic resistance. As observed, 71 horizontal gene transfer predispositions for genetic mutations, recombination events66 and the dissemination of antibiotic-resistant bacteria from human and veterinary medicine72. These mechanisms assist microorganisms to acclimatize to varying environments and contribute to the widely reported antibiotic resistance patterns observed in aquatic habitats. Furthermore, the genotypic resistance information can be conveyed via mobile genetic elements  from a particular species to different species 74. Also, antibiotics in the aquatic environment can lead to transformation in the ecological roles of microbes75 apart from encouraging the development and spreading of antibiotic resistant microorganisms.Antibiotic resistance development and persistent in the freshwater environment has been established to be initiated by various resistance mechanism such as selective pressure of antimicrobial substancesThe high concentrations of antibiotic resistant organisms and antibiotic resistant genes in the rivers examined indicated the rivers are polluted and highly impacted by waste discharge and other anthropogenic activities around the rivers. It is anticipated that unregulated waste disposal into the rivers and usage of antimicrobials in agriculture, clinical and domestic setting will keep on generating pools of resistance genes within the surface waters. Also, this can create a selective pressure in the immediate environment that would lead to genetic mutations in both allochthonous and autochthonous microorganisms thereby enabling them to persistently thrive and multiply. These observations therefore buttress the importance of environmental context-tailored strategies for continuous monitoring and surveillance of the aquatic environments for the presence of emerging contaminants such as, antibiotic resistance bacteria and their resistance determinant in order to evaluate their potential effects on environmental and human health.Supplementary Information.


Cicadellidae) that feed exclusively on a plant’s xylem sap, a nutrient-deficit source (95% water). To eliminate their high-volume excreta, these insects exploit droplet superpropulsion, a phenomenon in which an elastic projectile can achieve higher velocity than the underlying actuator through temporal tuning. We combine coupled-oscillator models, computational fluid dynamics, and biophysical experiments to show that these insects temporally tune the frequency of their anal stylus to the Rayleigh frequency of their surface tension-dominated elastic drops as a single-shot resonance mechanism. Our model predicts that for these tiny insects, the superpropulsion of droplets is energetically cheaper than forming jets, enabling them to survive on an extreme energy-constrained xylem-sap diet. The principles and limits of superpropulsion outlined here can inform designs of energy-efficient self-cleaning structures and soft engines to generate ballistic motions.Food consumption and waste elimination are vital functions for living systems. Although how feeding impacts animal form and function has been studied for more than a century since Darwin, how its obligate partner, excretion, controls and constrains animal behavior, size, and energetics remains largely unexplored. Here we study millimeter-scale sharpshooter insects ( Sharpshooters can catapult their droplet excreta with a speed faster than their own movement speed. Challita et al. find that superpropulsion is achieved by the temporal tuning between the droplet and the stylus. Specifically, we focus on how excretion influences small-bodied animals’ behavior, morphology, and energetics since they face unique challenges due to their high metabolic rate3 and physical limits set by the natural world4. Millimeter-sized xylem-feeding insects exemplified here with sharpshooter insects (Cicadellidea) face dual fluid dynamic challenges of surface tension due to their small size and energy constraints due to their xylem sap diet. Plant’s xylem sap is very poor in nutritional compounds (95% water5) and energetically costly to pump out since it is under negative tension6 (≤ − 1 MPa). To survive on this frugal diet, sharpshooter insects use large cibarial muscles and an efficient digestive system (filter chamber) to extract and filter large volumes of the xylem fluid (up to 300 × body weight/day8 compared to ~1/40 × body weight/day for humans). Subsequently, sharpshooters must constantly and efficiently excrete their fluidic waste , contributing to their role as plant disease vectors9. Here, we ask: what are the fluidic, energetic, and biomechanical principles that enable tiny xylem sap-feeding insects to survive on a nutrient-sparse diet?Consumption of nutrients and subsequent waste elimination are hallmark functionalities of a living organism. Although fluid feeding in insects  has received considerable attention since Darwin’s timeHomalodisca vitripennis, n = 5 individuals, N = 22 droplet ejections). Tracking the angle θ between the anal stylus and the axis along the body of the insect reveals three consecutive phases: droplet formation, spring loading, and droplet ejection  gradually grows in the normal direction to the stylus reaching a diameter of Do = 725 ± 188 μm (Mean of mean ± SD) in a formation time (τd) of about 80 ms while the stylus remains at a quasi-constant angle  of 0.23 ± 0.07 m/s. Notably, the catapulted water droplets are ~40% faster speeds (Vd = 0.32 ± 0.1 m/s) than the stylus. Calculating the speed ratio λ = Vd/Vs reveals that λ > 1, suggesting a superpropulsive regime  than the maximum speed of a superhydrophobic vibrating plate (i.e. λ > 1)10. Such counterintuitive propulsion is achieved only in elastic projectiles (such as a water droplet) by carefully tuning the underlying actuator’s vibrational frequency to the projectile’s natural frequency. The superpropulsion principle may be exploited to enhance the throwing of rigid projectiles through the elastic tuning offered by compliant substrates15. By adding a soft layer with specific geometrical properties to a rigid projectile, the reaction force between the thrower and the projectile might be delayed to increase the initial take-off speed11.The physics of elastic projectiles had been previously explored in the context of throwing or hitting sports ballsDo < 1 mm is the diameter of the droplet and g is the gravitational constant) indicate that surface tension forces dominate the ejection dynamics over bulk forces. As a result, the droplets visibly deform into oblate spheroids reaching maximum compression at tc before extending, supporting elastic energy storage for superpropulsive principles  represents the droplet and the lower mass-spring  represents the stylus and resilin  and resilin are modeled as an elastic Hookean spring19  ranging from 0 (low dissipation) to 0.75 (high dissipation). We calculate the frequencies fo and f, where λth = Vd,th/Vs,th is extracted for various values of the frequency ratio fo/f and ζ, where Vd,th is equal to the maximum speed of the upper mass and Vs,th is equal to the maximum speed of the lower mass. The superpropulsion regime is highlighted by λth > 1 whereas the subpropulsion regime is represented by λth < 1 (and λth = 1 corresponds to a rigid-projectile-like behavior).We model the droplet ejection mechanism using a reduced-order mathematical model that consists of a 1D two-spring oscillator where the upper mass-spring-damper  regime for fo/f < 1.16, suggesting that at these frequency ratios, the elasticity of the projectile undermines its kinematic performance during take-off , blue-green sharpshooter Graphocephala atropunctata  and red-banded sharpshooters Graphocephala coccina . We calculate the frequency of droplet vibrations (fo) as the second mode of the Rayleigh frequency 23 using high-speed footage, and the frequency of stylus (f) using the kinematics analysis of the stylus /T, where T is the period of the engine oscillations. The two-spring model, CFD, and plate experiments show that the superpropulsion peak λmax occurs when the maximum compression of the projectile coincides with the maximum velocity of the engine at τ = 0  is the force on the upper mass and the λ is maximum (λ = 1.76) and τ = 0   at higher speeds for their stylus p < 0.02)  located at the apex of the anal stylus of glassy-winged sharpshooters   and synthetic substrates25. This parahydrophobicity is evidenced by both the constant contact area S during droplet growth and observations that the droplets do not roll off due to gravity or stylus orientation  of ~2 cm/s without detaching the droplet 26. We assume that droplet ejection occurs when the stylus kinetic energy A is the amplitude of the arclength set by the stylus tip and Es ∝ Sγ(1 + cosθa) Next, we consider the effect of the surface properties of the stylus on the dynamics of droplet ejection. Detaching a droplet efficiently from a rotating substrate requires two competing demands: low surface energy for easy detachment and sufficient adhesion, so the droplet does not roll off prematurely. We observe that the stylus is parahydrophobic, having both a high apparent contact angle ηx is the energy content of nutrients in xylem sap, ηf is the energy per unit volume expended by the cibarial pump during feeding, and ηex is estimated from the energy required during excretion. Maintaining net positive energy gain (η) from feeding on xylem fluid is critical for the insects’ functioning and survival30. Andersen et al.5 estimated the net energy of xylem sap and cibarial pumping at negative pressures for sharpshooters, i.e. ηin = (ηx − ηf) as 2 × 105 − 8.2 × 106 J/m3 while considering factors such as xylem tension, time of the day, metabolic cost, the efficiency of the cibarial pump and nutrient concentration in xylem fluid. This range sets the upper limit for ηex since any higher energy expenditure on excretion will place the organism in an energy deficit.We finally explore if superpropulsion could be energetically advantageous for these insects with a nutrient-limited food source. The net energy gain per unit volume (Energy Density) per feeding-excretion cycle for the organism can be estimated as:ηex, we model the excretion of fluid through the anal tube as a pressure-driven flow of liquid water through a straight and circular cylinder having a diameter d and lengthlestimated from microCT  using the energy balance equation to pump water at a steady state with a flow speed u across the hindgut and form a droplet on the stylus as following31 , which is lower than ηin ensuring a net positive energy gain to meet other biological functionalities . The average kinetic energy per unit fluid volume Re), the Bond number (Bo), and the Weber number (We)  rely on inertial forces for repeatable fluidic ejection by forming jets or large sheets of liquids during urination31 or hunting34. Smaller organisms, however, living in a surface-tension-dominated world (Bo < < 1 and We < 1) develop ingenious mechanisms to overcome capillary adhesion.Lastly, we discuss how the fluidic excretion mechanism of sharpshooters compares with the other organisms excreting fluid through an orifice Fig. d. The meWe) Fig. d. Animal35. Not all xylem-feeders employ droplet ejection to eliminate their fluidic waste. Cicadas (Hemiptera: Cicadidae) form powerful jets to remove their excreta because they are ~2–7 × larger than an adult sharpshooter  use their specialized resilin-powered and tuned anal stylus to transfer kinetic energy to the droplets. A similar droplet catapulting strategy is also observed in phloem fluid-feeding aphids (Hemiptera: Aphididae) that kick their sugar-rich excrement (with either their legs or caudas). Aphids also manipulate surface tension forces by coating their excrement with powdery wax, transforming them into a non-sticky liquid marbleter Fig. d36 and dηex ∝ d−2, suggesting a fundamental limit on the minimum body size of xylem-sap feeders38. In addition to energetic advantages, flinging droplets to large distances could reduce the chances of detection by predators such as the parasitic wasp Cosmocomoidea, which may be attracted to chemical cues in the accumulation of sharpshooters excreta39  leads to higher energetic losses during fluidic pumping due to viscous and surface tension forces 2. For instance, frass-shooting skipper larvae employ biological latches on their anal plates coupled with a hydrostatic blood pressure buildup to propel solid pellets up to 38 times their body length with a speed larger than 1.5 m/s. Some species of noctuids violently shake their abdomen as they release their frass pellets, and some geometrid larvae use their thoracic legs to kick away their frass pellets2. Insects, in particular, exhibit a wide range of ingenious waste disposal strategies determined by their environment, diet, and generalized lifestyle. Thus, by conducting a detailed analysis of excretion dynamics in the sharpshooter insect, our work reinforces the importance of studying waste elimination to reveal a holistic perspective of organismal behavior.The need to create a considerable distance between insects and their waste is mainly observed in shelter-dwelling or site-faithful insects, which are typically pressured to maintain hygiene at the site to avoid the growth of pathogens and reduce chemical cues for potential predators. The ballistic ejection of excrement is not uncommon among insects. Many insect species may be described as ‘frass-shooters,’ ‘butt-flickers,’ and ‘turd-hurlers’ that innovated unusual strategies to launch away both liquid and solid excrements10. For physical systems dominated by surface tension forces, superpropulsion offers an ingenious mechanism through which this impeding force can be harnessed as a spring, albeit with tight temporal tuning for a full advantage. However, this superpropulsion mechanism may not be unique to surface tension-dominated systems. It may be exploited by other small power amplified and impulsive biological systems that rely on elastic structures to overcome the power limits of striated muscles16. Our work takes these insights from nature and provides a fundamental framework to implement an energy-efficient superpropulsion mechanism for manipulating elastic objects in synthetic systems ranging from pick and place nano- and micro-fluidic devices to smart wearable electronics and soft, elastic robotic engines40.Finally, our study of the droplet catapulting mechanism in sharpshooters offers a first observation and quantification of superpropulsion in a living organism. At its core, superpropulsion provides a gateway to propel an elastic projectile faster than the maximum speed of its actuator through temporal tuning and can be viewed as a single-shot resonance system10, chained spring systems15 and bouncing model28 . The upper mass-spring-damper represents the water droplet, and the lower mass and spring represent the resilin compression at the stylus. We ignore dissipation in the resilin-based spring due to the high efficiency of resilin19.We describe the dynamics of the stylus and the droplet with a minimalist mathematical model based on a coupled double mass-spring-damper inspired by the wave modelγ = 72 mN/m, Density: ρw = 996 kg/m3), the Weber number is We ~ 10−1 indicating the dominance of the surface forces. Also, since the acceleration of the actuating substrate is relatively low , the droplet remains in the linear droplet deformation regime and does not form water puddles14. Under these conditions, water droplets (Bo < 1) deform from a spherical cap into an oblate spheroid and behave as a quasi-Hookean spring with a stiffness kd = 32πγ/3 = 2.41 N/m18.Elastic deformation in water droplets is mediated by surface tension. We consider the Weber number 19. Natural resilin has an elastic modulus (Er) ranging between 0.1–3 MPa41. During the spring loading phase, the stylus bends and deforms the resilin blob into a complex shape. To estimate the effective spring constant ks, the tiny resilin blob is assumed to obey Hooke’s law of elasticity throughout compression and extension cycles. For simplicity, we also assume that the resilin blob is a sphere with an area of contact with the stylus Sr ~ 0.25–1 × 10−8 m2 estimated from video analysis and close-up microscopic imaging. Based on these simplifying assumptions, the effective spring constant of the resilin is calculated using ks = SrEr/d ~ 2.5–600 N/m, where d is the maximum compression of that blob, assumed to range ~50–100 × 10−6 m. We note that the ratio of kd/ks ~ 0.004–1 indicates little to no coupling between the two springs. In addition, the effective mass of the stylus combines the droplet and the stylus meff = md + ms > ms indicating no inertial coupling.Resilin is a super elastic protein known for its high efficiency (~97% efficiency) in loading and releasing energy in insect movementz-direction as follows:We write the 1D coupled equations of motion in the upward dzs below the equilibrium point (replicating the deformation of resilin during the spring loading phase). At t = 0, the compressed lower spring is released. The spatiotemporal dynamics of the two springs are obtained by numerically solving the system of equations of motion using the 4th-order Runga-Kutta method in MATLAB.The simulations start by compressing the lower spring with a displacement 22. The effect of dissipation is gauged by running simulations for different values of damping ratio ζ = 0 (no dissipation) to ζ = 1  . We observe that as damping increases, the effect of elastic advantage decrease where the elastic projectile gradually behaves like a rigid projectile . In addition, increasing the damping coefficient ζ shifts τ since the frequency of the upper spring now corresponds to the damped Dissipation within sessile droplets is modeled as a simple linear damper. Origin of damping includes bulk viscous dissipation, interfacial boundary layer damping, and moving contact line∘–180∘. For 2D simulations, we fixed the area of the droplet corresponding to the droplet’s diameter of 1 mm, forming a perfect circle and using this same area to equilibrate droplets under gravity at different contact angles. Given no coupling between the upper and lower springs . The experimental data capture the overall shape of the theoretical and computational curves over a wider range fo/fs.We analyze the ejection of 1 Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary video 1Supplementary video 2Supplementary video 3Reporting Summary