International crude oil prices has become one of the important factors that affect exchange rate fluctuation. By combining TVP-VAR model and DY time-varying spillover index, this paper measures the two-way fluctuation spillover effect between international crude oil prices and RMB exchange rate from 2011 to 2022. The research results show that: (1) There are obvious time-varying characteristics among international crude oil prices, onshore RMB exchange rate and offshore RMB exchange rate, and they are strengthened when major events occur at home and abroad; (2) The spillover and absorption level among international crude oil prices, onshore RMB exchange rate and offshore RMB exchange rate is relatively limited; (3) After the “exchange rate reform on August 11”, the market participation of RMB exchange rate has increased year by year, and the offshore RMB is mainly net spillover, while the onshore RMB is mainly reflected as a recipient; (4) The offshore RMB exchange rate has a driving effect on the onshore RMB exchange rate. Based on this, it is necessary to orderly promote the development and utilization of new energy, actively promote the market-oriented reform of RMB exchange rate, play the leading role of offshore market, and accelerate the construction of crude oil-RMB settlement system. As the first crude oil importer, the sharp fluctuation of international crude oil prices, the excessive dependence of China’s economy on crude oil and the basic resource role of crude oil have become important factors affecting macroeconomic stability of China . AccordiIn light of this, the article employs a TVP-VAR-DY model to construct a time-varying spillover index that incorporates international crude oil prices and the RMB exchange rate. This model not only adequately accounts for extreme volatility but also offers the advantage of objectively and flexibly measuring the nonlinear relationship between the two variables. This is in line with the research objectives of this paper.Both crude oil prices and exchange rate are the important basis and guarantee of the macroeconomic stability of a country. The existing research has rich and detailed materials on the mutual influence and interaction between them. By sorting out the content related to the research objective of this paper, it can be divided into the following three categories.From the results of foreign studies, it’s widely believed that there is an obvious linkage relationship between crude oil prices and exchange rate, but there is no consensus on the research conclusions. Krugman  was a scWith the acceleration of China’s opening up and the continuous improvement of the market-oriented level of exchange rate, domestic scholars have also begun to pay attention to the fluctuation of RMB exchange rate under the fluctuation of crude oil prices. The most research results show that there is a negative relationship between crude oil prices and RMB, such as Zhang Qingjun , Ding XuDue to the presence of time-varying issues and highly volatile research subjects, the research in this area primarily focuses on securities markets, exchange rate markets, and interest-rate policies. Many scholars have used GARCH models and VAR models to construct empirical testing models. For instance, Jia et al.  employedAfter reviewing the existing research, the research experience of domestic and foreign scholars on the relationship between crude oil prices and exchange rate fluctuation provides strong theoretical support for this paper. However, the above researches still have room for further improvement: Firstly, the research method is a linkage effect analysis mainly based on the GARCH model and VAR model. Although the interrelationships among the variables have been effectively measured, however, above model has shortcomings, such as difficulty in accurately grasping non-linear relationships, requirements for sample data volume, and difficulty in understanding the interaction of real variables, which cannot meet the research needs and objectives of this paper. Secondly, most current studies focus on the RMB/US dollar exchange rate or real effective exchange rate, and few studies take the special situation of “one market, two exchange rates” in China into account. Thirdly, few studies focus on the different impact of between onshore RMB exchange rate and offshore RMB exchange rate on crude oil prices. Therefore, the following two aspects of the tentative research were carried out in this paper: (1) expanded the research methods. Through the TVP-VAR-DY model, the application of this model can effectively overcome the deficiencies of the above model. Not only can the model autonomously select the window to ensure the objectivity of the analysis results, but the full use of the whole sample data can effectively capture the changes under different economic and financial conditions. More importantly, it can realize the different changes of the overall and individual variables, establishing a comprehensive relationship framework; (2) Increased the dimension of the research content. RMB exchange rate was decomposed into onshore RMB exchange rate and offshore RMB exchange rate, which improved the accuracy of the research results.From the perspective of theoretical mechanism, the transmission process of international crude oil prices to RMB exchange rate is complicated and long-term. As the most important industrial raw material, crude oil is closely related to economic life. When the price of crude oil changes, it will play a role in the RMB exchange rate through different mechanisms , 19, 20.This paper takes the rise of international crude oil prices as an example. The change of international oil price mainly affects RMB exchange rate market through two paths of international balance of payments and domestic economy, and the relationship between the two is negative in general. The details are shown in Due to the existence of the oil-dollar system, when the price of crude oil denominated in dollars rises, China needs to pay more dollars to complete the transaction settlement, and the demand for dollars increases rapidly in short term, causing additional foreign exchange payment. Other things being equal, increased demand for dollars will result in the relative depreciation of RMB. The rise of crude oil prices will also bring pressure on the import cost of crude oil for relevant industries and sectors. The rise of product cost will make sales price rise simultaneously, which will not help Chinese enterprises to maintain their price advantages and competitiveness in international trade. The decline of export may result in trade deficit and reduce the real demand for RMB in the international market. Of course, the degree and size of changes in import demand mainly depend on the elasticity of crude oil import demand. It is worth noting that once the rise of crude oil prices forms market expectation, this expectation will intensify the preference of domestic market for foreign currencies, especially for US dollar, which will trigger short-term capital outflows, resulting in pressure on RMB.Change of international crude oil prices will have an impact on important domestic economic indicators, and change of macroeconomic fundamentals will also have an impact on RMB exchange rate, and such impact will last further. When the international crude oil prices rise, it will have an adverse impact on related industries and alternative industries based on crude oil, and the increase of crude oil prices will be passed from upstream industries to downstream industries, resulting in the rise of producer price index (PPI) and consumer price index (CPI), and imported inflation will reduce the real purchasing power of RMB. Furthermore, the rise of crude oil prices will prompt relevant domestic enterprises to seek resources that can replace crude oil, such as coal, hydropower and natural gas, etc., which will increase the market price of energy and cause the overall price level of the society to increase. In this case, the consumption capacity and willingness of domestic residents will decline under the influence of prices, and the reduction of total social demand will not be conducive to domestic economic development. The superposition of three factors, including decline of export scale, decline of consumer expenditure and high price level, will inevitably result in the decline of the willingness of domestic enterprises to make new investment and reinvestment, and RMB depreciation will be expected to increase.In the 1970s, the impact of the war on international crude oil prices was increasing sharply, the United States took the opportunity to sign an oil-dollar agreement with the Saudi government to determine use the US dollar as the main means of payment. In recent years, with the rise of China’s economy and the promotion of the international status of RMB, the steady appreciation of RMB can improve the purchasing power of China in the international crude oil market. However, as the import scale is relatively limited compared with the world import scale and the US dollar is still the main currency of international commodity pricing transactions, it is difficult for RMB exchange rate fluctuations to have a significant impact on international crude oil prices in the short term.From the above analysis on transmission path, international crude oil prices change mainly have an impact on RMB exchange rate currently, and the transmission mechanism includes foreign exchange expenditure, current account, capital account, price level and consumption level, etc. The rise of crude oil prices has a negative effect on the RMB.By virtue of the research idea of Antonakakis et al. , this paj to variable i. In addition to the total spillover index, Eqs   ϕ˜ij,t is positive, it means that the spillover level of variable This paper selects the daily data of international crude oil prices, onshore Renminbi exchange rate, and offshore Renminbi exchange rate from January 4, 2011 to September 30, 2022 as the research objects, and the corresponding data are the spot price of Brent crude oil, the central parity rate of RMB against the US dollar and the non-deliverable forward exchange rate of RMB. All data with intersection in retention time totally get 2675 observed values. After the outbreak of the international financial crisis in 2008, the People’s Bank of China returned to the Renminbi-US dollar peg system to prevent excessive fluctuations during the crisis, which could not reflect the actual economic situation. In June 2010, the marketization reform of the Renminbi exchange rate was put back on the agenda. Considering the lagging effect of policy implementation, this paper sets the starting point of the study in 2011.All data is from the WIND database. Before using the TVP-VAR-DY model for estimation, it is necessary to make sure that all data can meet the stationarity requirements. Therefore, this paper draws on the existing research experience and processes the data according to the formula to get the volatility.From Based on the SBIC principle, this paper determines that the optimal lag order of this model is 2, and the decomposed number of stages in predicting error variance is consistent with the selection of Guo Na and Zhang Jun . Static The average value of spillover index of international crude oil prices, onshore RMB exchange rate and offshore RMB exchange rate are reported in The content in Decision of the CPC Central Committee on Several Issues Concerning the Establishment of a Socialist Market Economic System, a single, managed floating exchange rate system based on market supply and demand was realized to set the main tone for the development of the exchange rate formation mechanism, which became an important milestone in the market-oriented reform of the exchange rate. In order to adapt to the economic development situation and actual demand better, the People’s Bank of China began to implement a managed exchange rate system with “in reference to a basket of currencies” as the core in July 2005. In August 2015, the People’s Bank of China further strengthened the role of market pricing mechanism, laying more emphasis on and emphasizing the development trend of the two-way fluctuation of RMB exchange rate and its ability to depreciate or rise. In Firstly, the implementation of market-oriented reform of domestic exchange rate is conductive to improve the total spillover level. The market-oriented reform of RMB exchange rate started in 1994. With the Secondly, international economic and financial events will also increase the total spillover level. In The trend of the total spillover index represents the overall characteristics of all variables. In order to better understand and study the influential effect of a single variable on all other variables and a single variable influenced by all other variables, it is also necessary to investigate and analyze the spillover level (To) and absorption level (From) of a single variable.TVP-VAR-DY model also provides the results of net spillover level of each variable itself, and its established core is the net value obtained from the variable spillover level excluding the absorption level. In By combining the trends of international crude oil prices, onshore RMB exchange rate and offshore RMB exchange rate in Figs To ensure the robustness of the research conclusions, the model was tested by adding variables in this paper. According to Beckmann et al. , stock pTVP-VAR-DY model is used in this paper to study the fluctuation spillover effects between international crude oil prices, onshore RMB exchange rate and offshore RMB exchange rate from 2011 to 2022. Through empirical test, the following conclusions are made: Overall, there is a significant time-varying relationship between international crude oil prices, onshore RMB exchange rate and offshore RMB exchange rate, and this total spillover relationship will be enhanced under the influence of domestic policy reform and international economic and financial emergencies. From the specific performance of each variable, the spillover and absorption levels between international crude oil prices, onshore exchange rate and offshore RMB exchange rate are not particularly significant. The exchange rate results show that the net spillover level of offshore RMB exchange rate is significantly higher than that of onshore RMB exchange rate, and RMB exchange rate spillover is mainly driven by offshore RMB exchange rate to a large extent.Based on the above research conclusions, the following policy recommendations are put forward in this paper.With more complex international economic situation, the trend of large fluctuation in international crude oil prices still exists due to uncertainty under the imbalance between supply and demand. In consideration of China’s share in the crude oil reserve and consumption market, it is necessary to proactively invest in clean energy and renewable energy, fully harness the advantages of wind energy in desert and barren areas, utilize regions abundant in solar energy for photovoltaic power generation, promote comprehensive utilization of nuclear energy, and prepare for pumped storage power stations. These measures aim to achieve high-quality and sustainable energy utilization. Alongside the process of energy transformation and upgrading, efforts should be made to shift the role of crude oil from primary energy to a more adjustable energy source.In order to ensure economic stability, it is important to gradually increase the fluctuation range of the RMB exchange rate in an orderly manner, forming an awareness and expectation of two-way fluctuations, and enabling the RMB exchange rate to serve as a shock absorber for absorbing internal and external impacts. At the same time, it is necessary to fully utilize the international platforms provided by Shanghai and Hong Kong as international financial centers, offering more exchange channels and meeting the demands of domestic and foreign enterprises and individuals, thus achieving synchronized development of onshore and offshore markets. It should be noted that with the increased level of RMB marketization, the effects of cross-border capital flows, two-way capital movement, and external risk shocks may be magnified. Therefore, it is important to conduct early risk monitoring and management.The establishment of a crude oil-RMB settlement payment system is fundamental to mitigating the impact of international crude oil price fluctuations. Actively promoting the regionalization of the RMB and implementing RMB settlements in trade activities along the Belt and Road countries, ASEAN, and BRICS nations will lay the foundation for the construction of the crude oil-RMB system. Additionally, maintaining good diplomatic relations with major oil-producing countries such as Iran, the United Arab Emirates, and Russia, will help drive the scale of crude oil-RMB settlements and establish a closed-loop circulation system for crude oil-RMB.Overall, there is a certain degree of linkage between international crude oil prices and the RMB exchange rate. This research conclusion not only provides insights into the potential impact of international crude oil price fluctuations on China’s economy but also explains the mechanisms through which the two exchange rates reflect each other. However, the financial market is a complex and dynamic environment, and with the acceleration of China’s financial opening, future research needs to consider more factors such as stock prices, interest rates, and international balance of payments and their role in this linkage.S1 Appendix(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Salmonella infection has emerged as a global health threat, causing death, disability, and socioeconomic disruption worldwide. The rapid and sensitive detection of Salmonella is of great significance in guaranteeing food safety. Herein, we developed a colorimetric/fluorescent dual-mode method based on a DNA-nanotriangle programmed multivalent aptamer for the sensitive detection of Salmonella. In this system, aptamers are precisely controlled and assembled on a DNA nanotriangle structure to fabricate a multivalent aptamer (NTri-Multi-Apt) with enhanced binding affinity and specificity toward Salmonella. The NTri-Multi-Apt was designed to carry many streptavidin-HRPs for colorimetric read-outs and a large load of Sybr green I in the dsDNA scaffold for the output of a fluorescent signal. Therefore, combined with the magnetic separation of aptamers and the prefabricated NTri-Multi-Apt, the dual-mode approach achieved simple and sensitive detection, with LODs of 316 and 60 CFU/mL for colorimetric and fluorescent detection, respectively. Notably, the fluorescent mode provided a self-calibrated and fivefold-improved sensitivity over colorimetric detection. Systematic results also revealed that the proposed dual-mode method exhibited high specificity and applicability for milk, egg white, and chicken meat samples, serving as a promising tool for real bacterial sample testing. As a result, the innovative dual-mode detection method showed new insights for the detection of other pathogens. Salmonella, as the most widespread foodborne bacterial pathogen, has caused extensive food poisoning, illness, and death. It is reported that worldwide, more than 100 million people are infected and 370,000 people die from Salmonella each year [Salmonella per year [Salmonella is pivotal for effectively mitigating its contamination and curtailing its further propagation within the food supply chain [Salmonella detection strategies are mainly based on microbiological cultures, polymerase chain reaction (PCR)-based methods, and immunological methods. As the gold-standard method, microbiological cultures are time-consuming (lasting 4–7 days) and laborious, which is unsuitable for rapid screening and field applications. The PCR-based nucleic acid analysis exhibits remarkable sensitivity and specificity; however, it involves intricate procedural steps, demands technical expertise, and necessitates specialized equipment [4 CFU/mL) [Salmonella.Foodborne diseases have emerged as a growing global concern . Salmoneach year . The Worper year . The senly chain ,6 Conven CFU/mL) . TherefoSalmonella [Aptamers, called chemical antibodies, have emerged as highly promising recognition element candidates in the field of bacterial detection . These almonella ,13. Howelmonella . Fortunalmonella . Multivalmonella , liposomlmonella , or DNA lmonella , therebylmonella , pentacllmonella , and trilmonella , have beColorimetric detection is one of the most attractive optical assays due to its merits of simple operation, cost-effective analysis, rapid results, and the lack of a need for a complicated apparatus . NeverthSalmonella based on a colorimetric-tag- and fluorescent-tag-labeled DNA-nanotriangle programmed multivalent aptamer. In this study, the aptamer was precisely controlled and assembled on a DNA nanotriangle structure to fabricate a multivalent aptamer (NTri-Multi-Apt) with enhanced binding affinity and specificity toward Salmonella. In addition to labeling the target bacteria with high affinity, the NTri-Multi-Apt can amplify the signal via the tag carried on its scaffold and output a visual colorimetric read-out. Furthermore, the double-stranded DNA nanotriangle loaded a large amount of Sybr green I for the generation of a fluorescence signal which provides self-calibration and an improved sensitivity compared to colorimetric detection. Combined with the magnetic separation of the aptamer, the simple, sensitive, and specific detection of Salmonella in food samples can be achieved based on the DNA-nanotriangle multivalent-aptamer-assisted colorimetric/fluorescent dual-readout method.Herein, we developed a dual-readout method for the detection of 2. The pH of the 1 × TMS buffer was adjusted to 7.5 using glacial acetic acid. All the buffers above were prepared using ultrapure water (>18.25 MΩ cm) from a Milli-Q®® water purification system . All oligonucleotide sequences (Streptavidin-coated magnetic nanoparticles (SA-MNPs) were obtained from Ocean NanoTech . Sybr green I was obtained from Meilun Bio technology Co., Ltd. ; BSA, 4 S GelRed, agarose, TAE buffer, and SA-HRP were sourced from Sangon Biotech Co., Ltd. ; and 2 K Plus DNA Marker was obtained from Tsingke Biotechnology Co., Ltd. . Luria broth (LB) was provided by Hangzhou Microbial Reagent Co., Ltd. . TMB was supplied by Real-Times Biotechnology Co., Ltd.,  and brain heart infusion broth (BHI) was acquired from Qingdao Hope Bio-Technology Co., Ltd. . A 1 × TMS buffer was composed of 10 mM of Tris and 80 mM of MgClequences  were synSalmonella we chose was Salmonella typhimurium (ATCC 14028). Salmonella enteritidis (CICC 10982), E. coli O157:H7 (ATCC 43889), Listeria monocytogenes (CICC 21540), Vibrio parahaemolyticus (CICC 21617), S. aureus (ATCC 25923), Vibrio parahaemolyticus (ATCC 17802), Vibrio alginolyticus (ATCC 17749), and Vibrio vulnificus (ATCC 27562) were used for specific detections and cultured in an LB liquid culture medium for 20 h at 37 °C.The typical representative of 2 to 107 colony-forming units per milliliter (CFU/mL). To determine the bacterial counts per milliliter, the Salmonella strain was plated on agar plates. The colonies were counted to obtain the CFUs per milliliter after incubation. To ensure sterilization, all receptacles used for bacterial handling were subjected to disinfection in an autoclave before usage.Before its utilization, the bacterial solution underwent centrifugation, followed by resuspension in PBS subsequent to a PBST wash. Subsequently, the bacterial solution was subjected to a 10-fold dilution in PBS to yield samples with varying concentrations spanning from 10MNP-Apts were synthesized based on the classical reaction between streptavidin-modified MNPs (SA-MNPs) and biotin-modified aptamers (biotin-Apts). Briefly, 100 μL of 1 mg/mL SA-MNPs and 100 μL of 1 μM biotin-Apts were mixed and incubated at RT for 30 min. The complexes were then supplied with an equal volume of D-biotin (4 μM) at RT for 30 min to block the unbound sites of the SA on the surfaces of the MNPs. Subsequently, the product was separated with the assistance of a magnet and washed with PBST buffer three times. Finally, the obtained MNP-Apts were resuspended in 100 μL of PBS buffer and stored at 4 °C until further use.Before the reaction, all DNA sequences were solubilized in 1 × TMS, leading to a final molarity of 50 μM. The NTri-Multi-Apt was prepared by mixing equimolar amounts of four scaffold clip strands  in 1 × TMS buffer. The solution was heated to 95 °C for 4 min and rapidly cooled to 4 °C to fabricate the NTri-Multi-Apt. Subsequently, the removal of free strands was carried out through ultrafiltration to obtain a pure NTri-Multi-Apt. Then, SA-HRP (0.5 nM) was added to combine the biotin group of aptamers, and 2 × Sybr green I was incubated to embed it in the dsDNA at RT for 25 min. As a result, a dual-mode NTri-Multi-Apt was synthesized and stored at 4 °C for further use. NTri-monovalent and divalent aptamers were prepared using the same method with the selective substitution of specific ssDNA sequences .The NTri-Multi-Apts were characterized via agarose gel electrophoresis in 1 × TAE buffer at 130 V for 30 min. The loading sample was prepared by mixing 5 μL of DNA samples with 1 μL of loading buffer. In addition, the shape of the NTri-Multi-Apt was observed via atomic force microscopy (AFM), using 1 μM of NTri-Multi-Apts as an analytical sample.Salmonella solution at 37 °C for 2 h and blocked with 1% BSA. After washing them with PBST, different concentrations of the NTri-triApt (0.01–0.1 μM) were added to capture the target. After washing out unbound aptamers three times using PBST, SA-HRP was added to combine the aptamers on Salmonella. Then, the color reaction was initiated by adding 50 μL of TMB. Then, the absorbance intensity (450 nm) was measured using a Microplate Reader. Finally, the Kd values were determined via Y = Bmax X/(Kd + X) . Similarly, the affinities of NTri-monoApt and NTri-biApt were also calculated using the same method described above. To gain further insight into the enhanced affinity of the NTri-triApt, we employed confocal microscopy to observe Salmonella labeled with NTri-triApt, NTri-biApt, and NTri-monoApt, all of which were modified via FAM for fluorescence imaging.In this study, the binding affinities of the NTri-Multi-Apts were analyzed via an ELISA. Briefly, the plates were coated with a Salmonella was developed as follows: 10 μL of the prepared MNP-Apt complexes was added the Salmonella solution, followed by incubation at RT for 45 min under gentle shaking. After washing the mixture three times to remove the unbound Salmonella, 25 μL of 0.5 μM NTri-Multi-Apt was added and incubated at RT for 45 min to prepare ‘MNPs-bacteria-multiApt’ sandwich structures. After washing the mixture three times again to remove the unbound NTri-Multi-Apt, the precipitate was dissolved in 25 μL of PBS to measure the fluorescence signal via Blue Light LED using the naked eye. The fluorescence intensity of the mixture could be measured using a = multi-mode microplate reader . Subsequently, an HRP substrate HRP (20 μL TMB) was added, and the reaction was terminated using 20 μL of H2SO4 (2 M) after 15 min. Finally, the resulting solutions were observed via the naked eye, and the optical density at 450 nm was measured.The dual-mode colorimetric/fluorescent for the detection of Salmonella spiking, the supernatant layer was isolated following centrifugation to eliminate impurities. For the eggs, the egg whites were obtained and centrifuged, following the sterilization of the eggs by cleaning the shells with 70% (v/v) ethanol and subsequently breaking them using a sterile fork. Subsequently, the supernatant was diluted 10-fold and set aside for later use. Then, 25 g of chicken meat was added to PBS, followed by homogenization for 2 min. The resulting suspension was then centrifuged at 2000 rpm for 10 min, and the supernatant was collected to serve as the chicken matrix. Finally, the spiked samples were subjected to testing utilizing the proposed detection method.Milk, eggs, and chicken meat were purchased from Ningbo. To prepare the milk for Salmonella is shown in Salmonella was immobilized onto the surfaces of magnetic nanoparticals via the biotin–streptavidin interaction to fabricate aptamer magnetic nanoparticles (MNP-Apts) for the isolation of the bacteria  displayed a significantly higher fluorescence intensity compared to the bacteria treated with the same concentrations of NTri-biApt (middle) and NTri-monoApt (lower), which corresponded to the Kds of the aptamer structures. All these results demonstrated the NTri-triApt possessed an enhanced binding affinity. To further confirm the superior affinity of our planar NTri-triApt over the linear one, a linear trivalent aptamer  of aptamer structures with different valences were determined via a typical ELISA in which the aptamers were used as labeled probes against Salmonella in food samples to reduce the food matrix effect. After the separation, the target bacteria were labeled by the prepared NTri-Multi-Apt for the colorimetric and fluorescent dual-mode detection, and the absorbance value at 450 nm and the fluorescence intensity of Sybr green I were measured. As we can see , subsequently employing this construct for the colorimetric detection of Salmonella. Regrettably, the absorbance value associated with NTriS1-S2-S3 was notably lower in comparison to those attributed to NTriS1-S1-S1 and NTriS2-S2-S2 and even the average of NTriS1-S1-S1, NTriS2-S2-S2, and NTriS3-S3-S3. This unsatisfactory affinity possibly indicates that the binding sites of these three aptamers were not patterned in the shape or size of our proposed triangular DNA nanostructure. Consequently, the multivalent aptamer assembled using S1 with the strongest affinity was adopted for further study.Research has shown that the binding affinity of a multivalent structure is markedly contingent upon the constituent monovalent ligands comprising it . In orde2+, could exert an impact on the binding affinity by modulating the secondary structure of the aptamer [2+ was also subjected to optimization . The limit of detection was calculated to be 316 CFU/mL using the formula 3σ/slope (σ corresponded to the standard deviation of three blank measurements). Likewise, we found a similar enhancement in the fluorescent output via colorimetric detection. The enhanced fluorescence intensity was proportional to the logarithm of the Salmonella concentration among 1.0 × 102–1.0 × 107 CFU/mL, and the linear equation of Y = 0.185 × 106 X − 0.042 × 106 . Additionally, the fluorescence assay displayed a LOD of 60 CFU/mL, which was superior to several other detections  regardless of whether colorimetric or fluorescent detection was used. Therefore, these results illustrate that our proposed method exhibits excellent specificity for Salmonella typhimurium.To verify the specificity of the proposed detection method, some nontarget pathogens including Salmonella detection.The stability of the dual-mode system is crucial for its successful utilization and preservation. Therefore, the stability of the NTri-Multi-Apt complexes under varying storage durations  was evaluated. As evinced by Salmonella-spiked food samples. As shown in Salmonella concentration (102 to 107 CFU/mL) in milk, egg whites, and chicken meat. In addition, the accuracy of the dual-mode detection method was also estimated by measuring the recoveries and coefficients of variation (CVs) of the above-mentioned Salmonella-spiked samples , showing satisfactory accuracy for the analysis of Salmonella. All these results demonstrate that the multivalent-aptamer-based colorimetric/fluorescent dual-mode method developed for the detection of Salmonella in food samples has satisfactory application potential and favorable reproducibility.The practicability of the proposed colorimetric and fluorescent dual-mode detection method was evaluated using  samples . The aveSalmonella was developed via combining aptamer magnetic separation with a bifunctionalized NTri-Multi-Apt. The fabricated NTri-Multi-Apt not only possessed a 1.8-fold enhanced affinity over a monovalent aptamer but was also loaded with numerous colorimetric/fluorescent tags for signal amplification. Due to the dual-functionality of the NTri-Multi-Apt, the proposed method allowed for the simple, stable, and self-calibrated detection of Salmonella with satisfactory sensitivity. Furthermore, the proposed method can detect Salmonella in food samples with high specificity, accuracy, and stability. Finally, by virtue of the versatility of the aptamer, the application range of our NTri-Multi-Apt-based colorimetric/fluorescent dual-mode method can be extended to various targets other than bacteria. Therefore, this method holds great potential for further applications in the detection of other bacteria, food safety monitoring, or clinical diagnostics.In summary, a colorimetric/fluorescent dual-mode for the determination of the pathogen


Background and Objectives: The aim of this study was to investigate under dynamic loading the potential biomechanical benefit of simulated first tarsometatarsal (TMT-1) fusion with low-profile superelastic nitinol staples used as continuous compression implants (CCIs) in two different configurations in comparison to crossed screws and locked plating in a human anatomical model. Materials and Methods: Thirty-two paired human anatomical lower legs were randomized to four groups for TMT-1 treatment via: (1) crossed-screws fixation with two 4.0 mm fully threaded lag screws; (2) plate-and-screw fixation with a 4.0 mm standard fully threaded cortex screw, inserted axially in lag fashion, and a 6-hole TMT-1 Variable-Angle (VA) Fusion Plate 2.4/2.7; (3) CCI fixation with two two-leg staples placed orthogonally to each other; (4) CCI fixation with one two-leg staple and one four-leg staple placed orthogonally to each other. Each specimen was biomechanically tested simulating forefoot weightbearing on the toes and metatarsals. The testing was performed at 35–37 °C under progressively increasing cyclic axial loading until construct failure, accompanied by motion tracking capturing movements in the joints. Results: Combined adduction and dorsiflexion movement of the TMT-1 joint in unloaded foot condition was associated with no significant differences among all pairs of groups (p ≥ 0.128). In contrast, the amplitude of this movement between unloaded and loaded foot conditions within each cycle was significantly bigger for the two CCI fixation techniques compared to both crossed-screws and plate-and-screw techniques (p ≤ 0.041). No significant differences were detected between the two CCI fixation techniques, as well as between the crossed-screws and plate-and-screw techniques (p ≥ 0.493) for this parameter of interest. Furthermore, displacements at the dorsal and plantar aspects of the TMT-1 joint in unloaded foot condition, together with their amplitudes, did not differ significantly among all pairs of groups (p ≥ 0.224). Conclusions: The low-profile superelastic nitinol staples demonstrate comparable biomechanical performance to established crossed-screws and plate-and-screw techniques applied for fusion of the first tarsometatarsal joint. The first tarsometatarsal TMT-1) joint—incorporating a trapezoid-shaped plane—is described as a ‘helicoid’ subtly complex anatomic configuration. Under physiologic conditions, it is well constrained by soft-tissue structures allowing a certain amount of up-and-down and rotational movements  joint—in. HoweverCurrently, the standard TMT-1 fusion technique is fixation with two or three screws inserted in a lag fashion. One of the screws is inserted from the central dorsal aspect of the first metatarsal and routed to the plantar medial aspect of the medial cuneiform. The second screw is inserted from the dorsal aspect of the medial cuneiform and routed laterally to the first screw in the first metatarsal, exiting the plantar cortex. The two screws cross at a point on the longitudinal axis of the first metatarsal. This fixation has the disadvantage of involving 6–8 weeks immobilization . MoreoveOn the other hand, nitinol devices  are partClinically, the ability of the nitinol devices to act as continuous compression implants (CCIs) by applying and maintaining a uniform active compression offers an attractive alternative to conventional screw and plate TMT-1 fusion. Moreover, in contrast to screw fixation that occupies a valuable area across the joint interface, the CCIs could allow full joint coaptation by maximizing the footprint for fusion. Although experience was gained with the use of CCIs in foot surgery ,26,27,28Therefore, the aim of this study was to investigate under dynamic loading the potential biomechanical benefit of simulated TMT-1 fusion with CCIs in two different configurations in comparison to crossed screws and locked plating in a human anatomical model. It was hypothesized that the CCIs would allow more elastic intersegmental movements compared with the other arthrodesis techniques.Sixteen pairs of fresh-frozen (−20 °C) human anatomical lower legs from nine male and seven female donors  with no history of pathology were used in this study carried out in 2019. All donors gave their informed consent within the donation of an anatomical gift statement during their lifetime. The specimens were thawed at room temperature for 24 h prior to preparation and biomechanical testing.Bone mineral density (BMD) was measured in the calcaneus via computed tomography (CT) scanning  at a slice thickness of 0.63 mm with the use of a calibration phantom . The region of interest was a cylinder of 15 mm diameter and 25 mm length, centrally located and oriented longitudinally from the calcaneo-cuboidal joint to the calcaneal tuberosity, at a distance of 10 mm from the latter.n = 8 was chosen based on a priori power analysis with the assumption that the standard deviation in each group is not larger than 70% of the minimum difference in mean values between the groups. The lower legs were well randomized to the four groups, with BMD being not significantly different between them (p = 0.922), as shown in Based on BMD, the specimens were randomized in two clusters of eight pairs each. The lower legs in each cluster were further assigned pairwise for either TMT-1 crossed-screws fixation (Group 1) or plate-and-screw fixation (Group 2), CCI fixation with two two-leg nitinol devices (Group 3) or CCI fixation with one two-leg nitinol device and one four-leg nitinol device Group 4, . Each grAll procedures were performed on intact feet with preparation of the joint articulations and under fluoroscopic control according to the implant manufacturer’s guidelines.A dorsal approach to the midfoot and the metatarsals was performed, taking care not to damage the tibialis anterior and posterior tendons. An experienced surgeon carried out all instrumentations, thus ensuring consistency of the procedures. Provisional reduction was performed with pointed forceps and temporary Kirschner (K-) wires inserted across the joint. All implants were provided by the same manufacturer .Crossed-screws fixation was performed with two 4.0 mm fully threaded stainless steel lag screws inserted in standard fashion, i.e., the first screw from first metatarsal to plantar medial cuneiform and the second screw from medial cuneiform to the plantar base of the first metatarsal. Pilot holes were drilled in a 4.0/2.5 mm combination to achieve lag screw fixation.Plate-and-screw fixation commenced with insertion of a 4.0 mm standard midfoot fully threaded stainless steel cortex screw, inserted cortically in lag fashion, followed by fixation with a 6-hole TMT-1 stainless steel Variable Angle (VA) LCP Fusion Plate 2.4/2.7 of 39 mm standard length. The plate was positioned dorsomedially on the medial cuneiform and the first metatarsal via olive wires. Pilot holes were drilled using dedicated 2.0 mm VA Locking Drill Guides and drill bits of the same dimensions. Drill hole trajectories were chosen individually for optimal screw purchase in the bone. All 6 plate holes were occupied with 2.7 mm VA stainless steel locking screws of sufficient screw length, allowing for additional transfixation of the intermediate cuneiform and second metatarsal bones. All screws were locked at 1.2 Nm torque.CCI simulated fusion with two two-leg nitinol devices was performed with an SE-1818TI DPS BME SPEEDTITAN™ and an SE-2520TI DPS BME SPEEDTITAN™ implant placed at 45° angulation dorsolaterally and dorsomedially, respectively. Pilot holes were drilled in a bicortical fashion using a dedicated drill guide and a 2.65 mm drill bit. The nitinol devices were inserted and released using an insertion device to standardize the insertion process and prevent any deformation of the nitinol devices at the time of insertion.® and an EL-2520S4 DPS BME ELITE® implant placed at 45° angulation dorsolaterally and dorsomedially, respectively. Pilot holes were predrilled in a bicortical fashion using a drill guide and a 3.0 mm drill bit.CCI simulated fusion with a two-leg nitinol device and a four-leg nitinol device was performed with an EL-1818S2 DPS BME ELITEAll specimens were denuded of all soft tissues in the proximal 80 mm of the tibia and fibula and their proximal end was embedded in a 65 mm long cylindrical form of 48 mm diameter using polymethylmethacrylate . Distally to the embedding, a 2 cm wide stripe of soft tissue around the Achilles tendon was removed up to the level of the calcaneus to allow insertion of a metallic eyebolt through the calcaneus, used as an anchor for simulated Achilles tendon fixation. Finally, retro-reflective marker sets were attached to the distal tibia, talus, navicular, medial cuneiform and first metatarsal for motion tracking.Biomechanical testing was performed on a servo-hydraulic material testing system  equipped with a 4 kN load cell.A room temperature of 35–37 °C—the same as in the human body—was ensured with a temperature-sensing fan-heater. The test setup simulated forefoot weightbearing . The proBiomechanical testing commenced with initial quasi-static axial ramped loading from 20 N to 150 N at a rate of 13 N/s, followed by progressively increasing cyclic axial loading at 2 Hz with a physiologic profile of each cycle . WhereasMachine data in terms of axial load and displacement were acquired at 128 Hz. Based on these, initial construct stiffness was calculated from the ascending slope of the load–displacement curve during initial quasi-static ramped loading. Mediolateral X-rays were acquired every 250 cycles under peak loading using a triggered C-arm  for visual examination of any temporal degradation process within the bone–implant interface.Interfragmentary movements were captured at 100 Hz by motion tracking in all six degrees of freedom using five infrared cameras  operating at an accuracy of 1.4–2.4% and a precision of 1.9–3.9 µm . Based ot-test and General Linear Model Repeated Measures with Bonferroni Post Hoc tests for multiple comparisons were conducted to identify significant differences between the fixation techniques. The level of significance was set to 0.05 for all statistical tests.Statistical analysis was performed using SPSS software package . The normality of data distribution was screened and proved using the Shapiro–Wilk test. Paired-Samples The descriptive data of all outcome measures are summarized in p = 0.872).Initial stiffness of the specimens did not differ significantly among the four fixation techniques (p < 0.001); however, no significant differences were observed among all pairs of groups (p ≥ 0.128). In contrast, the amplitude of this deformation between unloaded and loaded foot conditions within each cycle also increased significantly between 500 and 2500 cycles in each group (p < 0.001) but was additionally significantly bigger for the two CCI fixation techniques compared to both crossed-screws and plate-and-screw techniques (p ≤ 0.041). No significant differences were detected between the two CCI techniques, as well as between the crossed-screws and plate-and-screw techniques (p ≥ 0.493) for this parameter of interest.Gap angle—i.e., the combined angular deformation between the two bones of the TMT-1 joint in both adduction and dorsiflexion—in unloaded foot condition increased significantly between 500 and 2500 cycles in each group (p ≤ 0.04) but did not differ significantly among all pairs of groups (p ≥ 0.224).Displacements at the dorsal and plantar aspects of the TMT-1 joint in the unloaded foot condition, together with their amplitudes between unloaded and loaded foot conditions within each cycle, increased significantly between 500 and 2500 cycles in each group (Specimens in all treatment groups failed at the fixation site in approximately 50% of the cases, whereas in the other 50% the site of failure was expressed by either a fixation failure of the calcaneus or general plantar gap opening of the whole foot. At the fixation site, specimens failed by a distinct gap opening and concomitant fragment displacement. This was ascribed either to implant loosening or its cut-through through the bone. Within Group 4, there were two specimens that had a fracture of the medial cuneiform, and one specimen with a fracture of the first metatarsal . No implThe aim of this study was to investigate under dynamic cyclic loading the potential biomechanical benefit of a fixation with nitinol devices in different configurations in comparison to cross-screws and plate-and-screw fixations in a TMT-1 fusion model.The tested hypothesis could only be partially confirmed to the extent that TMT-1 fixation with nitinol devices results in more flexible constructs compared to a conventional technique using either crossed screws or a plate, which was expressed by a larger amplitude of the intersegmental movements between the loaded and unloaded states of the feet. This is a logical consequence of the type of fixation methods. On the one hand, the trajectory of the crossed screws followed a path lying within the bony structures, ensuring a low lever arm relative to the load application point. Furthermore, it allowed joint compression by applying the lag-screw technique, enhancing the resistance against shear movements. As a result, crossed-screws fixation can shield external loads and efficiently protect the construct against bending and shearing. On the other hand, plate fixation uses a relatively strong plate which is attached to the dorsomedial aspect of the TMT-1 joint. This renders the construct resistive against the plantar gap opening due to the high moment of inertia provided by the plate aligned in the bending plane. Consequently, the relatively low-profile nitinol devices inserted on the dorsal aspect of the joint turned out to be the weakest constructs.The part of the tested hypothesis that the nitinol devices would demonstrate lower residual plastic deformations in TMT-1 fusion under unloading conditions over time needed to be rejected. The observation was made that the residual displacements were uniformly distributed across all fixation techniques in terms of statistical significance. Hence, the theory of superelasticity may not be transferred one-to-one in a biomechanical setup using human anatomic preparations. The interpretation of this finding may be multifactorial. Main reasons could be the weak holding strength of the staple legs in the bone, leading to early onset of micro-movements and their accompanying loosening, and the potentially weak spring-effect of the superelasticity feature.The present findings stand somewhat in contradiction to previous investigations using the same type of staples. In their study, Russell et al.  investigDisregarding their main arguments, the authors of all three above-mentioned studies also discuss the alternative use of staples as adjunctive fixation devices to standard fixation methods rather than stand-alone implants to impart compression across the joint. However, the benefit of such a combined use still needs to be demonstrated in vitro.Nitinol devices attracted attention in biomechanical studies striving for alternative fixation techniques in other anatomical body sites, such as for clavicle or patella fractures ,36,37. WOn the other hand, clinical studies using nitinol devices for Lapidus procedures showed promising results in terms of union rates ,29. HoweIn the current study, nitinol devices were placed at the dorsal compression site of the foot to allow continuous compression across the joint while unloading the foot. From this perspective and in alignment with other studies investigating plating of the medial column , althougA main limitation of the current study was the use of anatomical specimens and the associated disabled active stabilizing contribution of muscles and ligaments to the simulated walking cycle. As a compensation for it, the Achilles tendon was passively activated via its attachment to the machine transducer. Although it remains uncertain to what extent this force surrogate is beneficial for stressing the fixation constructs, the observed failure modes render the used setup encouraging. Another limitation of the study was the use of intact specimens instead of specifically ones with deformity indicated for TMT-1 fusion. Collection of such specimens would go beyond the scope of practicability and also induce unknown factors due to variability in foot deformation. Finally, only the propulsion section of a gait cycle could be simulated neglecting the more complex stress on the foot while simulating the full gait cycle. Hence, different results may be expected under different loading modes. Related to this, the study was underpowered within the meaning of high data scattering due to diluted focus on the fixation site for the sake of more physiological gait simulation.The advantage of the present study was the use of precise motion tracking, enabling continuous and accurate measurement of intersegmental deformation during the whole cyclic tests. Furthermore, the selected parameters for stability assessment deemed clinically relevant and were also considered in previous studies ,14,41. FFuture studies shall focus on whether a stand-alone use of staples represents a sufficient treatment or whether the concept must be reconsidered towards their use as adjunctive implants providing continuous compression across the joint, whereas plates and screws take over the main part as external load carriers.From a biomechanical perspective, based on human anatomical setting, the low-profile superelastic nitinol staples, used as continuous compression implants for more elastic stabilization under active compression, demonstrate comparable performance to established crossed-screws and plate-and-screw techniques applied for fusion of the first tarsometatarsal joint and allow full joint coaptation by maximizing the footprint for fusion.


Acinetobacter species exhibit target specificity. They are found, in the same orientation, 5 bp from the XerC binding site of the pdif sites associated with dif modules in Acinetobacter plasmids, and searches revealed they are also found near chromosomal dif sites of Acinetobacter species. These IS are 1.5 kb long, bounded by 24–26 bp imperfect terminal inverted repeats (TIRs) and encode a large transposase of 441–457 aa. They generate 5 bp target site duplications (TSDs). Structural predictions of the ISAjo2 transposase, TnpAjo2, modelled on TnsB of Tn7 revealed two N-terminal HTH domains followed by an RNaseH fold (DDE domain), a β barrel and a C-terminal domain. Similar to Tn7, the outer IS ends are 5′-TGT and ACA-3′, and an additional Tnp binding site, corresponding to the internal portion of the IR, is found near each end. However, the Acinetobacter IS do not encode further proteins related to those required by Tn7 for targeted transposition, and the transposase may interact directly with XerC bound to a dif-like site. We propose that these IS, currently in the IS1202 group in the not characterized yet (NCY) category in ISFinder, are part of a distinct IS1202 family. Other IS listed as in the IS1202 group encode transposases related to TnpAjo2 (25–56 % amino acid identity) and have similar TIRs but fall into three groups based on the TSD length . Those with 3–5 bp TSDs may also target dif-like sites but targets were not found for the other groups.Several insertion sequences (IS) found in various  Her. Herdif dif site .dif sites (dif site in the Acinetobacter baumannii plasmid pD36-4 (GenBank accession number CP012956). Recently, two additional Acinetobacter IS in this group (ISApi2 and ISAso2) have also been reported to be associated with pdif sites [ISAba71 shares 94.1 % DNA identity with ISAjo2 and the two transposases share 97.0 % amino acid identity and, when the sequence of the plasmid containing ISAjo2 (GenBank accession number CP010351 ) was re-if sites . Subsequif sites .not characterized yet) category under the IS1202 group. Here, we first examined the properties of the Acinetobacter-associated IS in the IS1202 group and then compared them to other group members. We propose that these IS form a novel IS family, the IS1202 family, that includes two main sub-groups, the IS1202 sub-group and the ISAjo2 sub-group, distinguished by the length of the TSD they generate.In the ISFinder database (www-is.biotoul.fr), ISAba71, ISAjo2 and ISAba32 and further relatives are currently assigned to the ISNCY (1202 group (last accessed 18 October 2022). The nucleotide and protein sequences for the ISFinder entries were downloaded and compiled into a custom blast database using Geneious version 7.1.9 (Biomatters).To facilitate an accurate analysis of IS related to ISAjo2, a stand-alone database was built by first querying the ISFinder database (www-is.biotoul.fr) with the nucleotide and amino acid sequences of ISAjo2 and ISAba32 to identify all related IS in the ISdif module database was constructed by searching the literature and collating previously identified and named dif modules. The nucleotide sequences of the modules were compiled into a custom blast database using Geneious version 7.1.9.A custom A ClustalW alignment  was performed to align the predicted protein sequences, and the pairwise identity matrix output was used to check for any pairs of IS that fell within the current ISFinder rules for defining isoforms, namely sharing >98 % amino acid similarity and/or >95 % nucleotide identity with any other IS in the database. Only one representative of isoforms was included in the analysis. Alignments were exported to Adobe Illustrator (Adobe).A phylogenetic analysis of the transposases of all IS in the curated data set was performed by building an un-rooted neighbour-joining tree from the ClustalW alignment. A consensus tree was built by resampling the analysis 10 000 times.Acinetobacter-associated IS, the GenBank non-redundant database was queried (last accessed 18 October 2022) with the nucleotide sequence of each IS as given in To determine the prevalence of each of the blast database. The flanking sequences were queried for the XerD pdif consensus sequence (TTATGTTAATT or TTATGTTAAAT). The sequence was then examined manually for an XerC site at the right distance from the XerD sequence. If a potential dif-like site or related sequence was found, the orientation and distance of the IS from the dif-like site was recorded.To examine targeting, the surrounds of three independent examples of each IS were examined and 50 bp of sequence on the left side of the IS (adjacent to the left TIR) was compiled into a custom http://www.compbio.dundee.ac.uk/jpred) using default parameters [Transposase secondary structures were predicted by submitting the amino acid sequences to the Jpred4 server  were modelled with >90 % confidence. TnpAjo2 modelled closely to the TnsB transposition protein encoded by TnB: 7PIK; ) and theB: 7PIK; ). TnpAjodif site hunters may play in the evolution of Acinetobacter plasmids, we sought to understand how prevalent each IS is and whether they have been found outside the genus Acinetobacter. For each of the IS in The IS in ISFinder are named after the species in which they were first observed, and though this can give the impression that they are confined to a particular bacterial species, that is not always so. To understand the importance that these ISAjo2 group pAcinetobacter species, namely A. baumannii, A. bereziniae, A. chinensis, A. defluvii, A. haemolyticus, A. indicus, A. johnsonnii, A. junii. A. lwoffii, A. nosocomialis, A. towneri and A. ursingii. The 70 GenBank entries include 42 entries identified as complete plasmids, and 28 entries identified as circular chromosomes. The latter group are examined further below.The original ISAjo2 is not common. It does not match at >98 % identity to any other sequence in the GenBank non-redundant database, and only 14 GenBank entries (nine plasmids and five chromosomes) contain isoforms sharing >95 % nucleotide identity with it . ISAba71Acinetobacter species  found in six different  species . ISAba54Acinetobacter. In addition, the number of instances of IS sharing >98 % nucleotide identity (often over 99 % identity) in multiple different Acinetobacter species is indicative of relatively recent spread.Hence, the IS examined were never found outside of the genus dif site in an Acinetobacter plasmid, the location of three independent examples of each IS in a plasmid were examined. As the pdif site is adjacent to the left TIR, as shown in dif site. In each instance, the IS was found to be 5 bp away from the XerC side of a pdif site, always in the same orientation relative to the pdif site, and surrounded by a 5 bp TSD  is located between locus tags ABA1_01921 and ABA_01922 in the A1 reference sequence , in addition to a copy adjacent to the primary dif site . In A. tes vary , but thedif site . Findingdif sites incorporated into the chromosome. In two instances (GenBank accession numbers CP046392 and CP024620), ISAba50 (an IS256 family member) had incorporated a fragment of an Acinetobacter plasmid into the chromosome of an A. indicus strain. These plasmid fragments each contained a single pdif site, and this pdif site had been targeted by ISAba71  in A. baumannii, A. junii and A. haemolyticus. ISAlw22 was next to a secondary chromosomal dif site (in four examples) in A. lwoffii. ISAba54 was found in the A. baumannii chromosome in four GenBank entries in an IS1008-bounded pseudo-compound transposon (PCT) and this PCT is the location where ISAba54 was originally found [dif sites, only one of which has been targeted by ISAba54.ISAba54, ISApi2 and ISAlw22 also produced chromosomal hits sharing >99 % identity with the respective IS nucleotide sequence. Examination of those GenBank entries revealed evidence for ISApi2 next to the chromosomal ly found . It incldif modules have been reported, including those containing antibiotic resistance genes, toxin–antitoxin genes and other genes of unknown function [dif sites presents a unique pathway to identifying novel dif modules within Acinetobacter species and potentially more broadly.Over the past few years, several different function . Howeverdif site that had acted as the target was identified and the sequence on the other side of the IS was examined for a second pdif site in the opposite orientation, giving the configuration of a C-type dif module. In total, ISAba71 was found associated with 12 different dif modules  that each share approximately 94 % nucleotide identity with the higAB module described previously, and five modules containing genes of unknown function. In pS30-1, one copy of ISAba71 is in the plasmid backbone .Each IS encodes a predicted transposase ranging in size from 394 to 507 aa. However, the shortest, found in ISTde1, lacks the C-terminal domain  and ISTde1 is >60 bp shorter than the remaining IS. A pairwise analysis of the transposases revealed that they share 25–60 % amino acid identity with the ISAjo2 transposase. Identity to the ISThe TIR lengths recorded in ISFinder for the 21 IS range from 14 to 42 bp, but here a manual examination of each IS found that all TIRs were in the expected range .1202 group members  revealed that most of the amino acids completely or highly conserved lie within the catalytic domain . The transposases fall broadly into three sub-groups, corresponding to the clusters determined by TSD length. One sub-group contained the transposases of ISAjo2 and the other IS found in Acinetobacter species as well as 13 close relatives with short TSDs, including those found in Klebsiella pneumoniae, Salmonella enterica and Burkholderia cenocepacia, amongst others. IS1202 and the nine other IS with longer TSDs formed a distant sub-group, while two IS with no detectable TSD (ISEsa1 and ISCARN112) made up the third sub-group. The only exception to this grouping was ISRor8 which had no detectable TSD but clustered with the short TSD group in the transposase phylogeny and will require further examination.A phylogenetic analysis of the transposases of all 28 IS in the curated data set, performed by building an un-rooted neighbour-joining tree , was lardif targeting in the Acinetobacter ISAjo2 group IS was employed to examine three independent examples of each of the IS with 3–5 bp TSD . The sequence adjacent to the left end of the IS was manually examined for the XerD consensus to determine if the IS was associated with a dif-like site. In each of the three examples examined, ISKpn21 and ISSen13 were found 5 or 3 bp away, respectively, from the C side of a conserved dif/dif-like site (dif module containing a gene encoding a GNAT acetyltransferase in an HI1B plasmid (GenBank accession number CP079726). ISSen13 was found adjacent to a single dif-like site  that is found in multiple sequences belonging to a widespread lineage (type 2) of HI1 plasmids [The strategy used to detect pike site . In one plasmids  that incdif-like site showing moderate conservation in the XerD site sequence but significant divergence in the XerC site sequence. The other four IS in this group  were found 5 bp away from sequences that only very loosely resembled pdif sites (dif-like sites that have degenerated, or they may be related to dif sites with a different consensus found in these species. In all instances, the targeted sites also appeared to be a single dif-like site, rather than a pair of inversely oriented pdif sites in a dif module configuration.ISLad2 was similar, found 4 bp away from a if sites . Interesdif site or any other conserved sequence. In several instances only single examples of the IS were found, or the same structure that been sequenced multiple times. The lack of independent data makes it difficult to determine if they are targeting a different conserved sequence. As more genomes are sequenced additional evidence may emerge.When the IS with either longer TSD or no detectable TSD were examined in the same way, we found no evidence of the IS targeting any sequence resembling a p1202 family all encode a large DDE transposase related to the Tn7 transposase and share strong similarity in the regions around the conserved acidic residues. The similarities extend to the features of the TIRs and to the presence of additional transposase binding sites near the ends. To date, no other IS family whose members encode a DDE transposase has features related to those of Tn7 and its closest relatives, making this family unique.Here, we have proposed the formation of a unique new IS family made up of a group of related IS currently found in the NCY category in ISFinder. Members of the ISAcinetobacter species appeared to be specifically associated with pdif sites [Acinetobacter species were always found in a specific orientation 5 bp from the XerC binding site of the dif site in the terminus region of the chromosome or a dif-like site elsewhere in the chromosome or a pdif site in a plasmid that includes dif modules. Hence, these IS are dif site hunters. Other more distantly related IS listed in ISFinder in the IS1202 group that also generate a short TSD of 3–5 bp may also target dif-like sites, though in some cases the site has clearly diverged or degraded. An IS or Tn targeting the same specific site is relative uncommon. Transposon Tn7 is the best studied example [7 transposition requires three proteins, the DDE transposase TnsB plus TnsA and TnsC, and a fourth protein TnsD is required for targeted transposition [dif or dif-like site.The analysis presented here arose from our earlier observation that a particular group of IS found in plasmids isolated from if sites , suggest example . Howeverposition . Here, n1202 family includes a second large group of IS, the IS1202 group, that generate an unusually long TSD and, in the phylogeny of the transposases, form a clearly distinguishable group (1202 family and the ISAjo2 family, or even three families.The ISle group . A thirdClick here for additional data file.


Ecosystems are often resilient enough to fully recover following a natural disturbance, or to transform into a new equilibrium favourable to the surrounding flora and fauna. However, at a local level, whether this transformation will be beneficial or not depends strongly on the level of disturbance and the available mechanisms for recovery. The Arctic, however, provides a potentially extreme environment for microbial growth and this is reflected in the microbial biodiversity, the in-situ growth rates, the biogeochemical cycling and its sensitivity to environmental change. In this study, we evaluated the current microbial biodiversity and environmental conditions around the landfill site in Adventdalen, Svalbard to identify differences across bacterial communities that might promote or accelerate naturally occurring environmental recovery. Landfill sites can induce changes in the local environment through the input of exogenous chemicals (both organic and inorganic) and microorganisms. Leachate can flow with run-off from the primary location of the landfill site due to rain, snow or ice melt and spread material into soils surrounding the site. In this study we found a strong effect of the landfill site on the bacterial diversity in the local landscape. Intervention is highly desirable to enhance the environment and improve the restoration by subtly altering the conditions at the site (such as the pH or drainage courses) and by encouraging specific groups of naturally occurring indigenous microorganisms to bioremediate the site. Soils he change ,4,5,6. Te change . In thisArctic and boreal environments cover ~22% of the terrestrial surface of the planet and are very sensitive to environmental change—both natural perturbations and those which result from human activity. The Arctic provides a potentially extreme environment for microbial growth, and this is reflected in the microbial biodiversity, the in-situ growth rates, the biogeochemical cycling and its sensitivity to environmental change. A number of local factors are responsible for this, including the arid climate, extreme low temperatures and varying light and UV exposure. In Svalbard 78° North), the temperatures in winter are on average −20 °C, in the summer they average +6 °C [° North, Research interest in the Arctic environment is increasing due to the extreme environmental conditions and the rate of climate change . Global The biodiversity of tundra ecosystems is generally low due to the harsh climate with a bacterial population density that is relatively low for Arctic soils compared to temperate soils . OrganisThe bacterial community composition in soil can change significantly in response to environmental change , althougLandfill sites have been a common method for disposing of domestic waste for centuries and are the most widely employed methods across the globe ,21,22. TAnother contributory factor to the pH story is acid mine drainage (AMD). The Adventdalen landfill site is located on top of an area of an acid mine drainage impact. Indeed, the site was originally chosen due to the effects and impact already seen at that location. In general, acidic sulphur-rich wastewater is produced by industrial operations. However, the most common cause is from the mining industry. The water draining from both active and abandoned mines is often extremely acidic. The low pH increases the solubility of the transition metals causing the drainage to contain elevated metal concentrations, such as iron, manganese and aluminium, with the potential for other harmful heavy metals such as arsenic ,35. AcidRhodococcus sp. [Studying microbial processes in the Arctic is still in its relative infancy but it is crucial as this region responds rapidly to environmental change and is susceptible to climatic control. There is also considerable potential in directly using the microorganisms from the polar regions in bioremediation studies to neutralise or eliminate the pollutants from a contaminated site, resulting in non-toxic or less toxic products ,38, for ccus sp. . Nocardia sp. and Mycobacterium sp., and Gram-negative bacteria such as Pseudomonas sp. and Spirosoma sp. [Bacterial diversity in the polar regions has been found to be dominated by few bacterial phyla. In general, these are the alpha-, beta- and gamma-proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group and high GC Gram-positive phyla. Other dominant groups in polar regions include Actinobacteria such as the soma sp. ,40,41. Csoma sp. . 9 bacteria per gram of soil [The bacterial community composition is also linked to the vegetation type, geographical region, quality of soil organic matter and environmental factors that include but are not limited to the temperature, soil pH, water and nutrient availability . The war of soil . The pro of soil ,45,46,47 of soil .Microbial activity can help curb the impact and rate of environmental pollution as a result of the metabolic processes of the Arctic soil bacteria breaking down hydrocarbon build-up and releasing elements such as methane and nitrogen. However, these organisms have systems that are highly temperature dependent or regulated. The lower temperatures that exist in the Arctic areas, therefore, can slow the rate at which these bacteria can clean up potential contaminants . The polThe aim of this study was to analyse both the organic and inorganic compounds in soil samples taken from the Adventdalen landfill site to determine any change in the environment that could have resulted from leachate from the site and to investigate its potential influence on the attendant microbial community. 2 [2. The mean air temperatures in the Svalbard region within the warmest month  range from 1 °C to 6 °C [The Arctic includes a large area of land, sea and ice covering approximately 40 million km2 ,52. This to 6 °C . In wint to 6 °C . The Arc to 6 °C ,56. In L to 6 °C . However to 6 °C . Most ofThe main landfill site has three streams originating from different melt sources Initially, nine samples were taken of the surface sediment (SS) 100 m and 200 m upstream of the landfill site for SS1, SS2 and SS3 and SS4, SS5 and SS6, respectively, and 50 m downstream for SS7, SS8 and SS9 to investigate whether differences in the microbial biodiversity could be detected upstream vs. downstream of the site . To increase the spatial resolution and investigate patterns in the chemical composition, a further 18 soil sediment samples were then collected the following year on 9 July 2015 . These samples were identified as upstream (U) 1–7 and downstream (D) 1–11 of the landfill site. The upstream sites were sampled at different points on the surrounding streams that appeared to enter the landfill from various sources. The downstream samples were also taken for comparison to the upstream samples to see what influence the landfill site had on the pH, temperature and both organic and inorganic chemical composition. Samples D1–D4 were taken from the floodplain downstream of the bypass stream with influence from the water current from the north-west bend (Zone E). The samples D5–D11 were taken downstream of a stream that flowed directly from the landfill itself (Zone F). The samples were placed into sterile 250 mL wide neck plastic bottles and sealed. At the time of sampling, the pH and temperature of both the soil and water were taken and recorded. The sample map can be found in −1) for 5 min and rinsed twice with sterile Milli-Q water and PBS (5 mL). The bacterial cells were counted using an epifluorescence microscope equipped with a DAPI filter and recorded. Due to the movement of water across the site, additional samples were taken at D13 and D14 in the stagnant ponds next to the stream where there was no water movement. Samples D16 and D17 were also taken at the end of the stream flowing from the landfill at the road. For this study, 5 g of sediment sample was diluted 1:10 in 0.2 µm filtered sterile Milli-Q water and phosphate buffered saline PBS , and then homogenised briefly using a vortex mixer. The cells were harvested using vacuum filtration (~30 kPa) through a 25 mm, 0.22 µm, black, polycarbonate filter membrane. The filters were then stained with DAPI , following the manufacturer’s instructions. The extracted DNA was purified and concentrated directly using Illustra GFX PCR DNA and Gel Band Purification kits , and following the manufacturers’ instructions. These tubes were then sent for 16S rRNA gene sequencing at the MrDNA laboratory in Shallowater, Texas. The sequence data were analysed using QIIME and Mothur software and a principal component analysis was conducted on the data to determine differences in community profiles for each sample site.Samples were taken from the top of the soil using clean trowels or scoops, placed in sterile, non-cytotoxic, non-pyrogenic Falcon™ tubes and stored in a refrigerator or on ice at −20 °C. Approx. 1–2 g of the soil/sediment samples were taken and placed in separate 2 mL microcentrifuge tubes and sealed. The extraction was carried out using the PowerSoilThe pH was directly determined using a hand-held portable pH meter  in the field, due to its influence on the solubility of metals.The inorganic elements were determined using X-ray fluorescence (XRF). Sediment samples were weighed in 50 mL falcon tubes for drying, allowing initial calculations of the percentage water loss. The samples were then placed in a drying cupboard until completely dry. Dry samples were then milled into a fine powder using a Frich Pulverisette 6 Ball Mill. Approx. 4 g of this fine soil was mixed with 0.7 g of a Cereox binder, with both weights being measured to three decimal places. Tubes containing the soil-binder mix were then placed on an electronic shaker for 3 min until fully mixed. The mixture was fashioned into an XRF pellet using a manual desktop hydraulic press at 10,000 kg. This procedure was repeated until there were duplicate pellets from each sampling site. The pellets were then labelled and analysed using a Spectro X-Lab 2000. The duplicate measurements were averaged. 2Cr2O7) was added using an automatic burette. Following this step, 20 mL of sulphuric acid (H2SO4) was added and the flasks were left to cool for 45 min atop the heat-proof surface. The amount of dichromate added to each sample was noted for the final calculation. Then, 170 mL of distilled water and 10 mL phosphoric acid was added to each flask. Two to three drops of diphenylamine indicator were then added to each flask before titration. Each sample was titrated against 0.5 M ammonium ferrous sulphate. Total organic carbon was determined using the Walkley-Black method. Approx. 400 µg of replicate dry sediment samples were added separately to 1 L Erlenmeyer flasks. The flasks were then moved into a fume cupboard with heat mats, where 10 mL of potassium dichromate  of the normalised relative abundance of all the preliminary samples is given in A hierarchical clustering of the samples based on the genus-level classification is given in The total DAPI counts are presented in Duplicate recordings of the pH and temperature of both sediment and water were taken at the time of sampling and the average is presented in In common with other studies, the sequence analysis identified bacteria belonging mainly to the phyla proteobacteria, Actinobacteria and Acidobacteria and to a lesser extent Bacteroidetes and Firmicutes ,41. In tThe XRF data for the 23 elements are presented in The total organic carbon concentrations are given in In total, 14 major organic compounds were detected . The dowFrom the nine initial samples taken upstream of the landfill site at 100 m  and 200 m  and downstream at 50 m , it was very clear that differences in microbial biodiversity could be detected across the landfill site. The total cell counts in landfill leachate presented in The temperature was found to vary considerably over the site which may have had some differential effect on bacterial growth. The pH levels both up and downstream of the landfill site were also highly variable, although they could broadly be classified as acidic (under the influence of AMD) or neutral. Samples U6 and U7 were apparently affected byrun-off from the mining site further up the mountain that joined the bypass stream. This stream continued down and resulted in low pH in sample sites D1–4 (Zone E). Compared to this, the sample sites following the landfill stream (Zone F) were all quite close to neutral in both sediment and water (apart from site D8). The neutrality of the downstream samples appeared to be directly caused by the presence of the landfill site itself, likely due to a neutralizing effect of relatively alkaline run-off from the discarded waste. This difference in pH could be an important driver of biodiversity. As discussed, the prime importance of soil pH as a control of soil bacterial community structure has been known for some time ,58,59, aBrevundimonas, Devosia, Flavobacterium and Rhodoferax  were dominated by the acidophilic genera iphilium . Polaromonas sp., which are ubiquitous in low temperature environments, and Thiobacillus sp., which are involved in sulphur cycling, whilst the species belonging to the genus Ramlibacter degraded isoprene and/or were methanotrophic  were dominated by otrophic .Algoriphagus, Alkalibacterium and Hydrogenophaga  were dominated by the more diverse genera that tended to be more mesophilic, such as enophaga .Flavobacterium sp., Bradyrhizobium sp. and Polaromonas sp. The AMD influenced low pH sites were dominated by Terracidiphilus sp., Acidophilum sp., Acidibrevibacterium sp. and Hydrogenophaga sp., while the landfill leachate sites (Zone F) were dominated by Rhodoferax sp., Bradyrhizobium sp. and Polaribacter sp. Rhodoferax sp. are purple non-sulfur bacteria that are frequently found in stagnant aquatic systems exposed to light [, Bradyrhizobium sp. are common soil-dwelling microorganisms, some of which can fix nitrogen and Polarimonas sp. are psychrophiles. This distribution of species strongly suggests that AMD had a more profound affect on biodiversity than the landfill site.Comparing the top four sequence matches across the sites , U2 was to light   that is soluble in water over time and typically comes from construction materials, such as drywall. When exposed to water, gypsum dissolves, releasing calcium and sulphates into solution. The sulphates may be reduced to produce hydrogen sulphide gas (H2S) via sulphate reducing bacteria [One of the non-biodegradable wastes that was mentioned specifically ,56 was gbacteria . Additiobacteria . This wo2+ can outcompete the other K+ and Mg2+ cations, resulting in leaching of these cations, and therefore, a reduction in the soil concentration [In the soils with low magnesium levels, due to the addition of gypsum and excess calcium, the Cantration . There wIt has been estimated that around 90–98% of soil potassium is unavailable with only 2% ‘free’ potassium . This woThe iron levels in and around the landfill site were high. There was clear evidence of the influence of iron moving from the mine with a sudden increase in the iron concentrations from sample site U2 (this increased after sample point U6 where the acid mine drainage met the stream). Iron could also be seen on the soil directly as an orange streaks. The pH of the site was also quite acidic in places, which could increase the solubility of iron oxide .The patterns of ion concentration vs. the bacterial diversity differed for different ions. The calcium, zinc and strontium showed the same trend upstream and downstream. However, the iron  and bariSample site D11 had higher concentrations of some elements, such as manganese, iron and zinc . This maThe levels of organic carbon in the soils showed wide variation, with the highest (at 12.3%) at site U2 (Zone B) and lowest at sites U6-D4 (Zone D and E). This could either reflect higher bacterial consumption rates or a lower overall biomass. Importantly, it suggests that if significant levels of organic carbon were leached from the landfill site, they were being broken down and not accumulated.In total, 14 major organic compounds were detected . Site U4The landfill site biology and chemistry differed markedly across the site. This suggested that the landfill site (and the underlying presence of AMD) had a noticeable impact on the environment in Svalbard. The bacterial biodiversity was high both up and downstream, with a lower diversity at the site of the AMD ingress. Along with the fact that similar groups dominated the upstream and downstream communities (away from the direct AMD impact), this suggested that intervention through bioremediation could be effective, since the leachate appeared to allow microbial growth and organic material degradation. Upward and downward spikes in the concentrations of the key groups of inorganic and organic chemicals across the landfill site were observed. The variation in the total organic carbon, observation of biofilms and DAPI counts, taken together, suggested that active growth within the ecosystem was occurring. According to these observations, a number of active intervention options would be possible at the site . IntervA number of active management plan options are also available: (i) do nothing and allow the environment to recover by itself , (ii) minor intervention , (iii) medium-scale intervention (an active in-situ bioremediation programme), (iv) full-scale intervention  and (v) further research to determine the likely timescale and outcomes.Hence, our overall recommendations would be to (i) develop a strategy for intervention to help the environment recover and (ii) conduct specific experiments to determine the most effective form of bioremediation for this site . However, if the site is found to be stable, given the unique combination of chemical and physical gradients in close proximity following significant human intervention in an otherwise pristine and rapidly changing environment, another option might be to protect it as a site of special scientific interest or a microbial biodiversity reserve.Intervention is highly desirable to restore the original environmental conditions due to the extreme nature of the changes in the biodiversity of the microbial community caused by both the leachate from the site and from AMD, as well as the likelihood of significant improvement for low investment and the overall cost-effectiveness of such an intervention. However, the area of impact appears to be relatively small and stable, and the underlying AMD appears to have a more significant impact than the landfill leachate . Changes can be made to enhance the environment and improve restoration by subtly altering the conditions at the site (such as the pH or drainage courses) and by encouraging specific groups of naturally occurring indigenous microorganisms to remove key toxic compounds (particularly the organics). Monitoring of the induced changes is desirable to determine whether the area of influence of the landfill site is stable, receding or increasing over longer time scales and to determine the wider applicability of these observations to acid mine drainage elsewhere in Svalbard.


Pseudomonas aeruginosa, toxin production in Vibrio cholerae, resistance to lysozyme in enterococci and antimicrobials in several Bacillus spp, and cell-envelope lipid composition in Mycobacterium tuberculosis. The prominent role of site-2-proteases in bacterial pathogenicity highlights the potential of site-2-proteases as novel targets for therapeutic intervention. In this review, we summarize the role of site-2-proteases in bacterial physiology and virulence, as well as evaluate the therapeutic potential of site-2-proteases.Site-2-proteases are a class of intramembrane proteases involved in regulated intramembrane proteolysis. Regulated intramembrane proteolysis is a highly conserved signaling mechanism that commonly involves sequential digestion of an anti-sigma factor by a site-1- and site-2-protease in response to external stimuli, resulting in an adaptive transcriptional response. Variation of this signaling cascade continues to emerge as the role of site-2-proteases in bacteria continues to be explored. Site-2-proteases are highly conserved among bacteria and play a key role in multiple processes, including iron uptake, stress response, and pheromone production. Additionally, an increasing number of site-2-proteases have been found to play a pivotal role in the virulence properties of multiple human pathogens, such as alginate production in  While the catalytic mechanism varies between the different IMP families, structural determination of prototypic members has confirmed the membrane-embedded location of the catalytic active site for each family  is a signaling mechanism known to mediate transmembrane signaling in Gram-positive and Gram-negative bacteria Urban . RIP is Caulobacter crescentus, iron acquisition in various Gram-negative bacteria and sporulation in Bacillus subtilis  family in response to environmental cues. As reviewed in detail here and by others, S2P-mediated RIP signaling contributes to a vast array of cellular processes, including cell polarity in B. subtilis and Methanocaldococcus jannaschii, all S2P discussed in this review belongs to subgroup I. The two remaining subgroups (Group II and IV), lacks any additional domains; however, none of these putative proteases have been extensively characterized  has been associated with reduced protease activity of S2P in multiple species E. coli aEscherichia coli RseP , previously known as Yael, is one of the most extensively studied members of the S2P family and was first identified as a key regulator of the σE-dependent stress response , the S2P of ily Fig.   and N-acetylglucosamide (NAG) in peptidoglycan in the bacterial cell wall, resulting in increased cell wall permeability and ultimately cell death  cleaves RisV, thereby releasing the cytoplasmic region of RisV containing σV  and RseP  the S2Ps of C. difficile and E. faecalis, respectively, results in decreased σV activity and lysozyme resistance  is essential for heme utilization through the expression of the outer membrane receptor BhuR  , also known as AlgW, is suggested to recognize C-terminal residues of the envelope protein MucE and possibly other OMP, which are accumulated under cell wall stress  in the majority of the mice  has not been investigated in vivo.Although the severe attenuation of rip1 with various stressors, Rip1 was found to be essential for protection against metal and nitrosative stress. Most importantly, the observed attenuated growth of Δrip1 in acute infection in mice was reversed in the absence of nitric oxide production, suggesting that Rip1 directly contributes to pathways defending M. tuberculosis against host-produced nitric oxide , increases the production of multiple sex pheromones, including cCF10, cAD1 and cPD1  is involved in maturation of multiple small linear peptides, including the sex pheromone cAM373    , an FDA-approved protease inhibitor used in HIV therapy, is suggested to inhibit human S2P. The drug results in increased accumulation of the human S2P substrates AFT6 and SREBP1  strain, and batimastat have no inhibitory effect on the wild-type efflux-intact strains  are revolutionizing the field of drug discovery by providing new ways to predict protein structures and identify potential inhibitors. The use of these technologies is expected to grow in the future as they will help researchers to understand the structure of S2P better and screen large libraries of potential inhibitors for those that effectively bind and inhibit S2P.S2P-mediated RIP is a well conserved signaling mechanism common to all forms of life. Notably, S2P and RIP play a key role in physiology and virulence in multiple human pathogens, including enterococci, staphylococci, and