Chemically synthesized SLN, palmitoylated or not (pSLN or SLN), and recombinant wild-type rabbit SERCA1a expressed in S. cerevisiae design experimental conditions that provide a deeper understanding of the functional role of SLN on the regulation of SERCA1a. Our data show that chemically synthesized SLN interacts with recombinant SERCA1a, with calcium-deprived E2 state as well as with calcium-bound E1 state. This interaction hampers the binding of calcium in agreement with published data. Unexpectedly, SLN has also an allosteric effect on SERCA1a transport activity by impairing the binding of ATP. Our results reveal that SLN significantly slows down the E2 to Ca2.E1 transition of SERCA1a while it affects neither phosphorylation nor dephosphorylation. Comparison with chemically synthesized SLN deprived of acylation demonstrates that palmitoylation is not necessary for either inhibition or association with SERCA1a. However, it has a small but statistically significant effect on SERCA1a phosphorylation when various ratios of SLN-SERCA1a or pSLN-SERCA1a are tested.Sarcolipin (SLN), a single-spanning membrane protein, is a regulator of the sarco-endoplasmic reticulum Ca SERCA1a uses ATP as an energy source to drive transport of calcium ions into the sarco-endoplasmic reticulum (SR) lumen to promote muscle relaxation. During catalysis, as described by the Post-Albers scheme1, SERCA1a switches between Ca2+-bound E1 states and Ca2+-free E2 states , and of a large cytosolic headpiece comprising three domains: the nucleotide binding domain (N domain), the domain of phosphorylation (P domain) which possesses the conserved aspartate residue that is transiently phosphorylated during the transport cycle, and the actuator domain (domain A) which triggers dephosphorylation of the aspartate for cycling of the enzyme 4. PLB and SLN share the same binding groove within the Ca2+-ATPase, between transmembrane helices M2, M6 and M9. Interestingly, the same groove is occupied by lipids in some structures5. Whereas PLB is 52 amino-acid long, with a transmembrane helix and a long cytosolic extension that interacts with the N domain of SERCA1a6, SLN is only 31 amino-acid long and is composed of a transmembrane helix with very short cytosolic and luminal interfacial stretches7 18.In addition, PLB and SLN undergo post-translational modifications. Residues in their N-terminus are target for kinases and phosphorylation affects association with the pump13 and Supplementary Table 20, heterologous expression of rabbit SLN, an acylated peptide, is more problematic in particular because it is difficult to control the yield of acylation. Additionally, the hydrophobicity of SLN makes its purification from native sources such as SR very tricky7. Experiments performed using purified native SLN gave inconsistent results. While some groups reported an effect of co-reconstitution of native SLN with native SERCA iosforms either on the turn-over rate or the accumulation of calcium21, others did not observe such an effect though they used similar native sample22. As a matter of fact, we demonstrated by combining Size Exclusion Chromatography (SEC) and MALDI-TOF analysis of full-length proteins that mild detergent are unable to totally dissociate native rabbit SLN:SERCA1a complexes15: 20 to 30 percent of endogenous SLN remains bound to SERCA1a after SEC as estimated from SDS-PAGE after modification of tryptophan by haloalkanes. Therefore, preparation of purified SERCA1a from native source contains some endogenous SLN that should be taken into account in the stoichiometry  from the same species, i.e. rabbit, (ii) deprived from any endogenous regulatory peptide as no SERCA isoforms nor PLB nor SLN homologues have been detected in S. cerevisiae31. Moreover, the stoichiometry can be tightly controlled in our experiments. In addition, the role of the palmitoylation of SLN on the regulation of SERCA1a can be investigated. We show that our synthetic peptide is biologically active whether acylated or not, thus providing a deeper understanding of the functional role of SLN. In particular, we reveal that both pSLN and SLN slows down the E2 to Ca2.E1 transition of recombinant wild-type rabbit SERCA1a as demonstrated by using phosphorylation from radiolabelled ATP. We show that SLN has an allosteric effect on SERCA1a by impairing both the binding of calcium and the binding of ATP. Thus, our results demonstrate that even if SLN shares some properties with PLB in its mechanism of regulation of SERCA1a, it also presents important specificities.In the present study, we used chemically synthesized S-palmitoylated and non-palmitoylated rabbit SLN 15. To investigate a possible role of palmitoylation on SERCA1a activity, pSLN and SLN were chemically synthesized by a Ser/Thr ligation (STL) method  in rabbit SR on its Cys9hod Fig. a followi33. Indeed, proteinase K cleavage is relatively unspecific for amino-acids and is, however, dependent on the accessibility of the amino-acyl chain. Proteinase K treatment in the presence of calcium to form a Ca2.E1 state leads to a major cleavage of SERCA1a at position T242-E243 that can be detected on SDS-PAGE as two major bands at 83 kDa and 28 kDa  has been extensively used to discriminate E1-like states from E2-like states, and therefore to evidence overall conformational changes of either the cytoplasmic domain or the transmembrane domain, revealed by the binding of several ligands such as calcium, nucleotide or inhibitors15. To decipher the role of SLN on SERCA1a, we perform the same proteolytic experiment but using recS1a, in the absence or supplemented with chemically synthesized palmitoylated SLN . Since SERCA1a was embedded in its native membrane it was thus co-purified with native acylated SLN2.E1 state and also with the E2.Tg state. This observation is in agreement with the crystal structures of the complex obtained at 3 Å resolution4 and more recently with cross-linking experiments showing an interaction of SLN with SERCA1a at all the major steps of the catalytic cycle11.When palmitoylated SLN was added to recS1a Fig. B, proteo23, it has been admitted that SLN, as well as its cardiac homolog phospholamban (PLB), are moderately reducing apparent affinity for calcium (from 0.35 µM in absence of SLN to 0.51 µM in its presence). Effect on the turnover is less clear as it seems to highly depend on the system used for functional characterization. Whereas some authors observed a significant increase of the Vmax up to 140% of the Vmax measured in absence of SLN, others showed a clear decrease of the maximal rate of hydrolysis to about 70–80% in the presence of SLN , suggesting that pSLN does not affect the Ca2.E1 ~ P phosphoenzyme formation.Since the pioneering work of MacLennan and coll. in 19982+-deprived state. We know that the detergent-solubilized ATPase can be stable for a few minutes in absence of calcium34. Samples were supplemented first with EGTA to reach nanomolar concentration of free calcium, followed by simultaneous addition of [γ32P]ATP and calcium to initiate phosphorylation. Interestingly, when starting from a calcium-deprived state (most probably a E1-like state at pH 7 as previously shown35) the rate of phosphorylation is significantly lower in the presence of SLN or pSLN . Binding of ATP and nucleotide analogues on calcium-deprived SERCA1a can be useful for probing the E2 to E1 equilibrium38. ATP is typically able to bind to the calcium-deprived E1 state and its presence accelerates binding of calcium40. Structures of the SERCA1a-SLN complex were obtained in the presence of AMPPCP3 or TNPAMP4, two non-hydrolysable ATP analogues. As these two molecules induce themselves subtle local reorganization of the nucleotide binding pocket, the authors could not conclude for any specific effect of SLN on the nucleotide binding site in presence of such analogues. To investigate whether SLN impacts the binding of nucleotide, we studied the effect of Mg.ATP on the tryptophan fluorescence emission of the DDM-solubilized and glycerol-stabilized SERCA1a in the absence or presence of pSLN . Such high concentrations are enough to fully saturate magnesium binding sites on the ATPase, either the catalytic site in the cytosolic domain or the calcium binding sites in the transmembrane domain44. Based on these crystal structures, the authors hypothesised that SLN stabilizes an E1.Mg state. However, effect of magnesium on the E2 to Ca2.E1 equilibrium has been extensively studied for 40 years, demonstrating that magnesium itself is enough to stabilize an E1.Mg state, whether SERCA1a is from native SR or from recombinant source35. Additionally, recent in silico studies have shown that the E1.Mg state is likely a non-physiological state that is not favored in the cell, and that SLN, as phospholamban, may populate a metal ion-free intermediate state45. In summary, these results strongly suggest that the E1.Mg state as illustrated by the published SERCA1a/SLN structures is indeed not a physiological intermediate in the catalytic cycle and probably not a SLN-inhibited state, even if SLN remains associated with this conformation despite the presence of metals as magnesium in the published structures11.The Nissen and Toyoshima groups reported simultaneously in 2013 the first two and only crystal structures of a SERCA1a/SLN complex2+ binding sites occlusion at E309, leading to an increase in the distance E771-D800, which produces incompetent Ca2+ binding sites and allows Ca2+ backflux to the cytosol37. These results are in agreement with the increase of cooperativity we observed .Recent molecular dynamics studies have also provided insight in the impact of SLN on SERCA1a, notably on the calcium binding sites. The presence of SLN perturbs the Caved Fig. A. A tougSLN Fig. . Such lo4 whereas this chain is undoubtedly linked to the peptide, the samples being prepared rigorously in the same conditions15. This post-translational modification is also present in pig SLN and presumably in a few other mammals and birds, as judged from the amino-acid sequences15. Among the other hundred sequences aligned covering most of the animal kingdom, other species including human present a phenylalanine instead of a cysteine at position 9  with a palmitate or an oleate. No electronic density could be assigned to a palmitate or oleate chain on the electronic density map of the crystal of SERCA1a-SLN complex15. However, the persistence of native lipids after such a purification could account for such an intermediate rate of phosphorylation or inhibition by vanadate compared to the recombinant enzyme that is certainly deprived of any lipids after purification. Detergent to lipid ratios can affect the equilibrium at several steps of the catalytic cycle, especially during the transition from the E2 to the Ca2.E1 state49. Noteworthy, we demonstrated that addition of chemically synthesized SLN on DDM-solubilized SR led to an increased inhibition of the rate of phosphorylation  and phospholemman (PLM) are palmitoylated17. Acylation of PLB and PLM is functionally relevant by having an impact on the oligomerisation of the peptides, their association with their dedicated pumps  and on their lifetime18. PLB pentamerization is now clearly demonstrated by molecular dynamics simulations, and by solid state or solution NMR53. PLB pentamers act as reservoir and dissociation into monomers leads to the inhibitory function54. Interestingly, palmitoylation of PLB:Cys 36 , a Ser (~ 3%) or a Phe (~ 1%) residue  and the rate of dephosphorylation (Ca2.E1 ~ P to E2) of SERCA1a are not affected by SLN. Surprisingly, we observe an impact of SLN on nucleotide and vanadate binding suggesting that SLN not only prevent calcium binding as early proposed but also disrupts interdomain communication. While palmitoylation appears not to be essential for calcium binding and turn-over, it has a small but statistically significant effect on SERCA1a phosphorylation when various ratios of SLN:SERCA1a or PalmSLN:SERCA1a are tested. If the role of SLN is now better understood, more studies are needed to decipher the role of post-translational modifications of SLN in relation to SERCA1a activities.In summary we demonstrate for the first time the impact of SLN on partial reaction of the enzymatic cycle of SERCA1a: SLN slows down the E2 ground state to E1 state transition impairing the binding of calcium; the rate of phosphorylation . Octaethylene glycol mono-n-dodecyl ether (C12E8) was purchased from Nikkol Chemical , and n-dodecyl β-d-maltopyranoside (DDM), from Anatrace . Streptavidin Sepharose high performance resin was provided by GE Healthcare . Thapsigargin  was from VWR International . [γ32P]ATP was from Perkin-Elmer . All other chemical products were purchased from Sigma . Sequences for SERCA1a heterologous expression and for SLN chemical synthesis were both from rabbit.Sarco-endoplasmic Reticulum (SR) membranes were prepared from rabbit muscles as previously described12E8, an amount that corresponds to the detergent brought with chemically synthesized SLN. After 30 min at 20 °C, samples were supplemented with either 1.1 mM EGTA (Ca2+free ~ 1 mM), or 50 mM EGTA (Ca2+free ~ 150 nM) and 0.01 mg/mL Tg. After an additional incubation for 10 min at 20 °C, proteolysis was achieved by addition of 0.3 mg/mL proteinase K. Proteolysis was stopped by addition of 1 mM PMSF and incubation on ice for 10 min. After proteolysis arrest, samples were diluted twice in a 4 M-urea containing denaturation buffer, boiled for 60 s, and about 0.75 µg protein were loaded on a 9% SDS-PAGE prepared in the presence of 1 mM Ca2+32. After electrophoresis, gels were stained with Coomassie blue.Yeast-expressed and purified SERCA1a (recS1a) was supplemented with SLN at a mole:mole ratio of 1:10 recS1a:SLN. In absence of SLN, recS1a was supplemented with 0.07 mg/mL C27. Briefly, signals were monitored with excitation and emission wavelength set at 295 and 320 nm, with bandwidths of 2 and 10 nm, respectively. Changes in tryptophan fluorescence were recorded at 20 °C after dilution of protein samples to a final concentration of about 6–7.5 µg/mL into a medium containing 50 mM MOPS-Tris pH 7, 100 mM KCl, 5 mM MgCl2, 20% glycerol (v/v) and 10 mg/mL DDM. In presence of SLN, recS1a and SLN were first mixed together and incubate for 1 h on ice before measurement. In absence of SLN, recS1a is supplemented with 0.07 mg/mL C12E8 to compensate the detergent present with SLN. The Maxchelator program was used for estimating [Ca2+]free59 (program available at https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/downloads.htm). The dissociation constant of the Ca.EGTA complex was taken to be 0.4 µM at pH7 and was assumed to be the same in the presence of glycerol.Intrinsic fluorescence of SERCA1a was measured with a SPEX Fluorolog spectrofluorometer  as described before32P]ATP followed by acid quenching, and filtration. For most experiments, 50 µL of enzyme at 10 µg/mL in a buffer containing 50 mM Tes-Tris pH7.5, 1 mM KCl, 5 mM MgCl2, 20% glycerol (w/w) and 0.5 mg/mL DDM, supplemented or not with SLN, was stored on ice. Concentrations of EGTA and calcium are specified in figure legends in order to reach the appropriate [Ca2+]free and therefore the desired starting state for the ATPase. For kinetics of phosphorylation, we initiate reaction by addition of 2 µM [γ32P]ATP (5 mCi/µmole), and calcium at 100 µM when specified. Reaction was quenched by addition of 2 mL of cold quenching medium (500 mM TCA + 30 mM H3P04) under vigorous mixing, and immediately stored on ice for 20–30 min to improve precipitation, a period of time critical for retention of the precipitate on the filter in presence of detergent60. This was followed by filtration on GS filters  and careful rinsing of the filters with diluted quenching medium (50 mM TCA + 3 mM H3P04). The amount of radioactivity bound to the filter was estimated by liquid scintillation counting (Perkin Elmer TriCarb Counter). When starting from a calcium-deprived state , indicating that the number of detergent-SLN-SERCA1a complexes is likely to be less than could be inferred at first view since a number of SLN could be present in the micelle–SLN complexes devoid of SERCA1a. Thus if we assume that the excess SLN gets distributed in empty micelles, for a given micelle, the SERCA1a:SLN ratio is likely to be much lower, maybe close to 1, a ratio close to the actual molar ratio in SR membranes as determined by MacLennan and coll. or others42. Nevertheless limited proteolysis assays demonstrate that interaction between SERCA1a and SLN occurs in such conditions ) and about 0.85 mM assembled in micelles. As the aggregation number of DDM is about 80–120 molecules, the concentration of DDM micelle is about 7–11 µM in the samples. A SERCA1a monomer binds about 100–120 mol of DDM, so an amount of detergent corresponding more or less to a micelleExperiments were conducted at least thrice. The purified protein came from two independent batches of yeast membranes. Two batches of chemically synthetized pSLN and SLN were also compared giving strictly identical results. We analysed the complete set of data by nonlinear regression as mentioned in figure legends, and whenever possible, we submit data to statistical analysis with GraphPad Prism software. Average values are accompanied with standard deviation. Student one-tailed paired test was applied to calculate p-values. We consider a P > 0.05 as not significant.Supplementary Information.


Escherichia coli, coliform and Enterococci) and some accessory parameters  provided for Italian Legislative Decree 31/01. Although 54.5% of samples were compliant, the results highlight a vast contamination of food ice by bacteria and fungi. In particular, 95.8% of samples were contaminated by fungi, stressing no difference between compliant and non-compliant samples. Their presence is generally attributable to the poor sanitation conditions in the production and/or administration phase and to the incorrect sanitization and ordinary maintenance procedures. It seems appropriate to suggest the need to carry out a specific risk assessment with respect to the self-control plans.Food ice is used as an ingredient or as a coolant in drinks and in the storage of food, especially fishery products. Studies show that ice can be polluted both by chemical substances and by bacteria and fungi. In particular, the presence of fungi in these food matrices has acquired an important role in Public Health, as it can represent a risk factor for fungal complications in immunocompromised subjects. In the present study we evaluated the hygiene–sanitary quality of food ice from public and collective catering establishments in a large area of Southern Italy, investigating the mandatory parameters ( Worldwide food ice production has increased in recent years because of the high demand for ice cubes in public places such as bars, pubs and restaurants. Today, commercial ice production is estimated at around 500,000 tonnes per year in Europe and 5,600,000 tonnes per year in the United States . Used inAmong European countries, Spain has the highest ice consumption at over 400,000 tonnes per year, of which 50% is self-produced, whereas the other 50% is produced and packaged in production plants . In ItalAlthough numerous benefits are conferred by the use of food ice, the scientific literature shows how it can be contaminated by different chemical and physical substances ,4. In faClostridium perfringens, Pseudomonas aeruginosa, Yersinia spp. and Salmonella spp. in Greece [Salmonella Saintpaul in Australia [Escherichia coli ESLB (extended-spectrum-beta-lactamase) in Vietnam; and some episodes of cholera related to Vibrio cholerae O1 Ogawa ctxA have been linked with ice melted in tea [Many studies have investigated the microbiological quality of food ice and have confirmed its potential role as a vehicle for foodborne illnesses. In fact, Enterobacteriaceae, Enterococci and fungi have been isolated from ice in Italy ,6,7; totn Greece ,9; Salmoustralia ; multi-ad in tea ,12.The yeast and mould contamination of food ice has also acquired an important role in Public Health, as it is considered among the risk factors for fungal complications in immunocompromised subjects .Finally, ice produced from contaminated water caused an epidemic of a Norwalk-like virus in Puerto Rico with approximately 5000 cases .With this background in mind, the aim of the present study was to investigate the hygiene–sanitary quality of food ice from public and collective catering establishments in a large area of Southern Italy, and to assess whether these establishments complied with Decree No. 31/2001  on food Ninety-nine ice samples were taken directly from the storage compartments of ice machines located in public and collective catering establishments in an urban geographic context. Each sample equal to 1 kg of ice in cubes was placed in sterile food bags and transported to our laboratory in a special isothermal fridge at a controlled temperature of 4 °C to be analysed within 24 h of collection.All ice samples were taken in public and collective catering establishments during the normal operation of the food business activity from active ice machines connected to a public water supply network.E. coli, coliforms and Enterococci, and some accessory parameters relating to Staphylococcus aureus, P. aeruginosa and fungi. Each sample was filtered through a cellulose ester membrane with a diameter of 47 mm and a pore size of 0.45 μm . The samples were considered compliant when E. coli, coliforms and Enterococci were absent in 100 mL of each melted ice sample.After melting at room temperature, the ice samples were evaluated for the mandatory parameters provided for in Decree No. 31/2001 relating to E. coli, and the salmon pink, oxidase negative ones were identified as coliforms [A 100 mL aliquot of melted ice was filtered and the membrane placed on plates containing Chromogenic Coliform Agar . After incubation at 36 ± 2 °C for 24 ± 2 h, the blue-violet colonies that formed were identified as oliforms .A 100 mL aliquot of melted ice was filtered, the membrane was placed on Slanetz and Bartley agar medium  and incubated at 36 ± 1 °C for 48 h. When dark pink-red colonies developed the membrane was transferred to a plate containing Bile Esculin Azide agar medium  and incubated at 44 °C for 2 h. Brown colonies with brown-black halos and positive catalysis were identified as Enterococci .P. aeruginosa [A 250 mL aliquot of melted ice was filtered, the membrane was placed on a plate containing Pseudomonas Selective Agar supplemented with cetrimide (0.20 g) and nalidixic acid (15 mg)  and incubated at 36 ± 2 °C for 48 h, and green colonies were confirmed to be ruginosa .S. aureus [A 250 mL aliquot of melted ice was filtered and the membrane was placed on a selective, differential ground plate containing Mannitol Salt Agar . After 48 h of incubation at 36 ± 1 °C, the yellow colonies that were yellow halo-, catalase- and coagulase-positive were identified as . aureus .A 100 mL aliquot of melted ice was filtered and the membrane placed on Sabouraud dextrose agar containing chloramphenicol (0.5 g/L) . After 8 days of incubation at 28 °C, yeast colonies were identified using a semi-automated sugar assimilation system , while filamentous fungi were identified by evaluating their macroscopic and microscopic morphological characteristics according to the methods described elsewhere .Two 1 mL aliquots of melted ice were separately mixed with yeast extract agar  in Petri dishes, and incubated at 36 ± 2 °C for 48 h and 22 ± 2 °C for 72 h, respectively. The colonies grown in each plate were counted and the results were expressed as the number of colony-forming units per millilitre (cfu/mL).p-values of multiple comparisons were corrected by applying the Bonferroni correction. Bacterial counts are a quantitative variable, but as they were not normally distributed, they are described herein as the median and range. Comparisons between independent groups were conducted with the non-parametric Mann–Whitney U test. p-values <0.05 were considered to be statistically significant. Data were analysed with the MedCalc Statistical Software version 19.1.3 .Because compliance with mandatory or non-mandatory parameters is a qualitative variable, we summarised as a count or a percentage. The chi-square test was employed for comparison between independent groups and, where appropriate, the P. aeruginosa was isolated from 14.8% of them and S. aureus from 11.1% of them. The remaining 45 (45.5%) samples were non-compliant for the mandatory parameters   was not statistically significant  , differently from S. aureus (p = 0.669).According to Decree No. 31/2001, of 99 food ice samples 54 (54.5%) complied with the mandatory parameters. Of these, 52 (96.2%) were positive for additional parameters, in that fungi were isolated from 96.2% of them, i 24.4%) . Of thes 0.5405) . After Bp = 0.8867) (Aspergillus (42.3%), Penicillium (17.4%), Cladosporium (16%), Fusarium (6%), Paecilomyces (6%) and Alternaria (3%). The following yeasts were also identified: Candida humicola (29.4%), Rhodotorula mucilaginosa (20%), Candida lipolytica (17.9%), Candida inconspicua (12.6%), Candida intermedia (8.4%), Saccharomyces cerevisiae (6%) and Candida lusitaniae (5.2%).Fungi were identified in 95.8% (95/99) of the samples and no statistically significant difference between the compliant and non-compliant groups was observed after Bonferroni adjustment ( 0.8867) . Altogetp = 0.3514, p = 0.5597, The median count of mesophilic microbes at 22 °C in the compliant samples was 574 cfu/mL (range 11–3000 cfu/mL), whereas in the non-compliant samples it was 536 cfu/mL (range 11–3000 cfu/mL); a non-statistically significant difference  principles or to the Manual of Good Hygiene Practice for the production, treatment, storage and use of ice .E. coli, Enterococci, Pseudomonas spp., Acinetobacter spp., Stenotrophomonas maltophilia and Yersinia spp. in such ice [Candida parapsilosis, Cryptococcus spp. and Penicillium glabrum were all able to survive in alcoholic and soft drinks in the presence of contaminated ice, and proposed that Enterococci, Pantoea conspicua and Stenotrophomonas maltophilia contamination should be reduced in such ice [Some studies have investigated the microbiological quality of food ice, highlighting the presence of bacteria such as such ice ,21,22. Fsuch ice .In developing countries where hygiene and health conditions are considered to not be optimal and cholera is spread in its endemic form, the consumption of food coming into contact with ice was shown to be an important risk to human health. In these geographical areas, the consumption of beverages produced and purchased by street-food and food businesses not constantly subject to specific controls performed by the health authorities contributes to an increasing transmission risk of foodborne diseases ,23,24.In Italy, the Ministry of Health has validated and disseminated the Manuals of Good Hygiene Practice delineated by the National Food Ice Institute . They hiE. coli and Enterococci (non-compliant samples), although they were derived from potable water. In fact, all ice samples, except four, were positive for P. aeruginosa, S. aureus and fungi . Notably, fungi were isolated from approximately 95% of the samples, with filamentous fungi  being most prevalent. It is important to focus attention on the fact that almost all the samples suitable for the mandatory parameters were positive for other microorganisms that fall within the additional parameters, according to Decree No. 31/2001, probably related to some variables such as water network or unsuitable sanitation of machineries for the production and distribution of ice. Of concern, some microorganisms such as P. aeruginosa and S. aureus are pathogenic bacteria that are able to cause infections in different bodily sites. Man himself could represent a source of contamination for these microorganisms, especially regarding S. aureus; not only its presence among food workers was demonstrated, so too the diffusion of methicillin-resistant S. aureus (MRSA) [In the present study, almost half of the food ice samples we tested were contaminated with s (MRSA) . Thus, cs (MRSA) ,27, and Enterobacter cloacae during cardiac cardioplegia, and food ice remains the subject of current recommendations for pain relief resulting from mucositis and candidiasis of the oral cavity in patients receiving chemotherapy treatment [In this regard, some studies have reported on epidemic levels of infection of surgical wounds with reatment ,29. Therreatment ,31,32,33In light of these considerations, it is important that careful monitoring is undertaken to verify compliance with the parameters set out for drinking water in food-producing companies to ensure that they comply with the principles of the HACCP System, including monitoring the sampling checks performed by such companies, as well as drawing up water safety plans, especially for companies that do not benefit from a public water supply.It is appropriate to remember that under the current legislation, the Services of Prevention Departments in the Local Health Units involved in territorial activities  must carry out annual local planning of the official controls, taking into account the provisions of the Multiannual Control Plan, a national plan aimed at rationalising controls and optimising the use of available resources. Therefore, local planning departments must provide for the use of a “risk-based” method for food ice, taking into account the results of the present investigation, and where no official samples of food ice are normally included it would be appropriate to consider incorporating this matrix into their sampling schemes.Finally, we highlight that our ice samples were collected in public and collective catering establishments from active ice machines connected to a public water supply network, without highlighting failures or reporting anomalies on plants. It is possible to suppose that public water supply was already contaminated. However, with the Guidelines for Drinking Water Quality  of 2004,Therefore, as regards the equipment, it is strictly necessary to follow the instructions provided by the manufacturer for adequate maintenance; to implement the frequency of proper sanitisation procedures for the internal walls of ice machines, promoting abundant rinsing later; to provide for the replacement of any filters and carry out near-term ice sampling, collected by Food Business Operators, in order to detect any non-compliance early and to promptly take corrective actions. Furthermore, a storage tank, if present, should be subjected to daily checks of hygiene conditions, and its cleaning and descaling should take place at least weekly.Our results, although preliminary, have highlighted important sanitation problems in the distribution of ice for use in food and drink. In the face of the evidence shown herein and the desirable planned control actions, which should be adopted by the competent authorities, it seems appropriate to suggest the need for a thorough evaluation of where and when food ice is used, and for the application of the relevant protocols and regulations to ensure proper maintenance and sanitation of ice production plants, as well as timely information and training of staff involved in all aspects of food ice production, storage and use.In conclusion, further studies need to evaluate the presence of other microorganisms  and to verify a possible relationship between the degree of contamination of the ice and the correct use of the various equipment. Furthermore, research could be extended to other regions of Italy, evaluating also geographic position as a variable and comparing public and collective catering establishments. This would define a more complete and detailed scenario.


Background. The most common problem associated with dental implants is the abutment screw loosening. This researchaimed to investigate the effect of the type of connection on screw loosening, using a finite element method (FEM).Methods. Periosave system and different types of the implant–abutment connection were used for modeling. After beingmeasured, CAD files were modeled using CATIA software and imported to the ANSYS analysis software, and the model wasloaded.Results. A force of 100 N was applied at 0.1 second, and no force was applied at 0.42 second. The screw head deformationat 0.1 and 0.42 seconds was 8 and 3.8 μm, and 7.6 and 2.8 μm at morse taper and octagon dental implant connections, respectively. The displacement rate of the internal surface of the abutment at 0.1 and 0.42 seconds was 10.7 and 8.4 μm, and 5.7 and5.6 µm in the octagon and morse taper dental implant connections, respectively. The displacement of the implant suprastructure–abutment interface from the screw head at 0.1 and 0.42 seconds was 9 and 7 μm, and 7 and 6 μm in the morse taper andoctagon dental implant connections, respectively. At intervals of 0 to 0.1 seconds and 0.6 to 0.8 seconds, the octagon connection was separated at the maximum screw head displacement and the internal part of the abutment, but the morse taper connection did not exhibit any separation. In the above time intervals, the results were similar to the maximum state in case ofthe minimum displacement of the screw head and the internal part of the abutment.Conclusion. Screw loosening is less likely to occur in the morse hex connection compared to the octagon connection due tothe lack of separation of the screw from the internal surface of the abutment. A loose screw can lead to crestal bone resorption.3 Screw loosening is costly and time-consuming. Screw copings are usually the weakest connection in the prosthetic chain. Any kind of occlusal, casting, or force inconsistency can cause screw loosening or fracture.4 When the screw is tightened for the first time, an initial tensile preload is created within the screw. Preloading leads to the imposition of pressure on the abutment–implant components and friction between the screw and the threads of the implant, screw head, and abutment, as well as the upper part of the implant and the lower part of the abutment. This pressure causes resistance to the external shearing forces and increases the fatigue strength of the connection.4 Preload is created in the screw by creating cracks.4 The settling effect (embedment relaxation) is another effective factor. When the abutment is placed in the implant, the settling effect occurs by applying torques, which increases due to the micro-roughness between the metal surfaces of the implant and the abutment. Abrasion of the contact surfaces brings the two surfaces closer.5 The settling effect eliminates the initial preload by 2–10%.5 In the past, implants with Branemark's external hexagon connections were used, which resulted in complications, such as screw loosening and rotational misfit, and the resultant microbial penetration led to changes in the external hexagon and internal connection.2 External and internal connections are distinguished based on the presence or absence of the appendage extending out of the implant body. In external connections, an appendage is found that extends out of the implant body, while this appendage is placed in the internal side of the implant body in implants with internal connections.3 There is an anti-rotational feature for the abutment connection in internal connections. The most common designs include hexagonal, octagonal, morse taper, and hex morse taper.4 The internal hexagonal implants are the most commonly available type, with a hexagonal shape. The morse taper implants include a tapered design in the abutment design that is inserted into the indentation of the tapered implant. This type of connection depends on the friction fit to remove the rotation at the abutment–implant interface and prevent screw loosening. This type of connection also causes resistance against bending forces. Internal octagonal implants present as octagonal connections.7 Implant–abutment connections or prosthetic connections with unstable coupling surface exert unwanted stresses on the fastening screw.2 Khraisat et al8 investigated the effect of lateral cyclic loading on the abutment screw loosening in the external hexagon implants. Simon9 investigated the success rate of implant restorations of the single implant-supported molar and premolar crowns, and the results showed that the implant failure rate was 4.6%, of which 7% was related to the abutment screw loosening. Binon10 observed a direct relationship between the rotational misfit of hexagon implants and screw loosening. Since conducting clinical studies on screw loosening is a time-consuming and costly process, studies should be carried out using a systematic approach. This approach is the finite element method (FEM), a powerful and effective method to predict the mechanical behavior of implant restorations.1 Although most of the FEM-based implant-supported dental prosthesis analyses have been performed under static conditions, the dynamic response of dental implants has been studied less frequently. Since the dental loading model is transient in nature, the nonlinear and transient dynamic analysis is widely used in industrial fields; this analysis is also expected to predict the dynamic behavior of dental implants. There are few reports on the FEM components for the evaluation of dental implants.1Titanium endosteal implants, which are osseointegrated, are widely used for their mechanical advantages and excellent bone connection. Long-term follow-up studies have shown many complications after the prosthetic phase of treatment, such as the loss of osseointegration, abutment screw loosening, abutment screw fracture, and other problems.With the help of this analysis (sometimes referred to as transient time history [TTH]), one can calculate the dynamic responses of one structure under the influence of time-dependent loading. In this analysis, we can calculate displacements, strains, and stresses and time-dependent forces in a structure. The main equation of a transient dynamic analysis is as follows:MẌ+CẊ+KX=F(t)(t) is the time-dependent load vector.where M is the matrix of the system mass, C is the damping matrix, K is the stiffness matrix, Ẍ is the acceleration vector, Ẋ is the velocity vector, X is the displacement vector, and FThe Periosave system was used for modeling of the implant and abutment fixture, and the implant and abutment with the usual diameters of 4 mm and 4.5 mm were selected. In order to imitate the clinical conditions, the height of the abutments was adjusted to 5 mm. Implants, abutments, abutment screws, and connections were accurately measured using a profile projector, and their real dimensions were later obtained . The devDeformations were investigated at 0.1 second when a maximum force of 100 N was applied to the suprastructure and at 0.42 ssecond when no force was applied to the suprastructure. An average downward displacement of about 3.8 μm was obtained in an octagon connection at 0.1 second. This deformation is due to the application of a 400-N preload (3 N.cm torque) to the screw, as well as a 100-N force to the suprastructure . An averWhen loading was carried out on the suprastructure from 0 to 0.1 second and from 0.6 to 0.8 second, the displacement of the octagonal abutment increased as compared to the screw head, resulting in the separation of the screw surface from the internal surface of the abutment . At othe11 The present study aimed to determine the effect of the type of implant–abutment connection and its geometry on screw loosening. External forces always lead to transient dynamic deformations in the screw connection. The misfits or deformations already present in the implant components increase the rate of screw loosening. The weak interface between the implant components increases the initial displacement and causes abrasion in the connecting areas, which also increases the distance in the screw connection region. The application of force might lead to the creation of tensile forces in the screw and contribute to screw loosening.11 Only two-piece abutments and cemented restorations were used in the present study. According to the Bickford study, failure in the screw connection region occurs in two stages. In the first stage, the external functional forces gradually reduce the clamping force, and then the remaining clamping force is rapidly reduced.11 In the present study, a different degree of abutment screw loosening occurred inside the implant at 30-N.cm torque in the selected connections, with the lowest rate observed in the morse hex connection. Two types of connecting methods, including screw and interface with fit taper, which is also called morse taper, are commonly used to connect an abutment to an implant. In screw-based systems, the implant–abutment connection depends on the screw preload. However, morse taper interfaces depend on long contact pressure and frictional resistance. In screw-dependent connections, mechanical complications, such as screw loosening or creep at the screw–implant interface, can lead to clinical complications. However, in cases where morse taper connections are used, the abutment loosening is less problematic.12 Limited data is available on the number of cycles needed to loosen the screw, especially in the oral cavity, and the presence of biological tissues, such as bone, periodontal ligaments, and temporomandibular joint with differences in modulus of elasticity, has made the analysis more complicated. In addition, the screw-related factors, such as the screw yield point, the type of screw used, the duration of the use of the screw, and its potential for fatigue, affect screw loosening. Therefore, the potential of screw loosening is variable, and a large number of related factors are still unknown.13 The screw material can also be effective in the amount of the resultant preload. The tensile strength and yield point for the gold-redesigned screws are greater than the former titanium screws. Therefore, a higher preload can be made in gold alloy screws.8 Therefore, in this study, we used one screw type in both types of connection to create similar conditions. Static and dynamic forces are used to analyze micro-gap formation at an implant–abutment interface. Zipprich et al14 analyzed ten different systems of implant and concluded that connections with a clearance fit showed micro-motion under a static force of 200 N, although the same force did not create any micro-motion in the precise conical connections, such as Astra Tech and Ankylos.14 Micro-motion is an important parameter that has received relatively little attention in the implant-supported prosthetic field. It is necessary to consider this parameter as a long-term success factor for implants, which can predict the initial stability of implants within the bone and the stability of the implant components. Occlusal forces, which occur during actions such as clenching and chewing, are also transmitted to the components of the dental implants, leading to movements between the implants and abutments.14 The present study investigated three-dimensional models to evaluate the micro-motion of implant components under simulated occlusal forces. Overall, different connection forms showed different patterns of micro-motion. The results of the present study revealed the highest and lowest micro-motions in the morse hex and octagon connections, respectively. The micro-motion distribution pattern in abutments with morse hex connection was different from that of other connections, attributable to the geometry of this connection, which is of polygonal and cylindrical shapes in the coronal and apical regions, respectively.14 Ameen Khraisat et al assumed that the twist resulting from the lateral exterior central loading could be countered by the clamping force created at screw–hexagon rotational interface, although it was shown that a 32-N.cm clamping torque cannot completely counteract the external torque forces. The size of the rotation created by the external central forces might depend on the rotational abutment freedom, considering the role of the hexagon and the frictional forces produced between the surfaces by the clamping forces. The interaction of these forces produces a reversible hexagonal internal rotation in the abutment against the corresponding region involved in the implant. A larger lateral micro-movement in the abutment leads to a greater loss of the preload. An important result of the rotational misfit in these single implant-supported restorations is the susceptibility to these micro-movements. A better fit between the implant components will result in greater stability of the implant.8 According to the results of the present study, the lowest rate was observed in the morse-hex connection, one of the possible reasons for which could be a higher fit in the morse-hex connection than the other connection, which results in lower preload loss. The relationship between the amount of torque  and the approximate values of the resulting preload force are calculated for the usual screws (with a friction coefficient of 0.05). The 0.2 coefficient might vary depending on the amount of torque and friction.The present study investigated the rate of screw loosening in two different types of implant connection, using the finite element method (FEM). The results showed different rates of screw loosening in the two types of connection during the application of 30-N.cm torque in the different types of connection used. The types of the implant–abutment connections were also effective in the screw loosening rate. Complications of an implant-supported prosthesis can be divided into two categories of mechanical and biological problems, a significant portion of which is attributed to screw loosening.The momentum applied in the assumption of the problem is considered to be15 However, according to available resources, the amount of preload force is considered to be about 400 N, which was also used in the present study to solve this problem according to the formula proposed in the articles. The rate of screw deformation caused under the preload conditions is calculated as follows . This equation is fully correct when there is no material under the screw head.11According to the approximate formula, a 750-N preload is obtained using the above approximate formula.Fi: Preload forceL: Screw lengthE: Screw modulus of elasticity (Young)A: Cross-sectionThe deformations are calculated for these two types of implants.2 was considered in the equations used to calculate the octagon connection.1 Since the screw of the two samples was different due to the internal conical geometry of the abutment, and different rates of screw deformation were thus recorded in the two above-mentioned samples, it should be noted that the same results were also observed in FEM analyses. However, since different materials were used under the screw head in different samples, the values would not be obtained in accordance with the above equation. However, the results obtained by the ANSYS analysis were close to the aforementioned calculated values. Considering the exact and somewhat realistic modeling carried out in ANSYS, the resultant values are more accurate, and the above calculations are only given to show approximate estimates.The morse hex taper connection15Practically, considering the complex geometry of the implant components, it is even impossible to calculate the rate of the screw deformation caused by preload and other components affected by its use of very similar complex formulas. Therefore, analytical softwares, such as ANSYS, are used. However, what is perceived in practice to understand the cause of the implant components’ static deformations is obtained from the general formula below.i = 400 N). So why is the rate of deformations (δ1 = 2δ) not the same? Certainly, according to the formula, this is attributed to the difference in the interface stiffness. Since the material of both interfaces is identical (E1 = E2), the conic inside of the abutment is not of the same screw length, and the length of the implant components is the same (L1= L2); therefore, the remaining difference is related to the cross-section of the implants in the area on which the force was applied. In fact, the different geometries of the implants have created different cross-sections under the screw and created different stiffness levels. As the cross-section increases, the lower the abutment deformation ; and the higher its springiness, the lower the degree of separation and screw loosening.The study was planned by AP and SJA. The literature review was performed by RN, and VG contributed to writing the manuscript and English editing. The statistical analyses and interpretation of data were carried out by RN. VG contributed to the publication of the article and developing the protocol. SJ and AT contributed to the development of the protocol. SJA contributed to the thesis, article writing, and publication. All authors have read and approved the final manuscript.Hereby, Dr. Mahboobkhah is appreciated for his sincere guidance.This study was supported by the Vice Chancellor for Research (VCR), Faculty of Dentistry, Tabriz University of Medical Sciences (TUOMS), Tabriz, Iran.The authors declare no conflict of interests with regards to the authorship and/or publication of this article.Not applicable.


Mycobacterium tuberculosis complex (MTBC) members, which affects various domestic animals, wildlife, and humans. Some wild animals serve as reservoir hosts in the transmission and epidemiology of the disease. Therefore, the monitoring and surveillance of both wild and domestic hosts are critical for prevention and control strategies. For TB diagnosis, the single intradermal tuberculin test or the single comparative intradermal tuberculin test, and the gamma-interferon test, which is regarded as an ancillary test, are used. Postmortem examination can identify granulomatous lesions compatible with a diagnosis of TB. In contrast, smears of the lesions can be stained for acid-fast bacilli, and samples of the affected organs can be subjected to histopathological analyses. Culture is the gold standard test for isolating mycobacterial bacilli because it has high sensitivity and specificity compared with other methods. Serology for antibody detection allows the testing of many samples simply, rapidly, and inexpensively, and the protocol can be standardized in different laboratories. Molecular biological analyses are also applicable to trace the epidemiology of the disease. In conclusion, reviewing the various techniques used in MTBC diagnosis can help establish guidelines for researchers when choosing a particular diagnostic method depending on the situation at hand, be it disease outbreaks in wildlife or for epidemiological studies. This is because a good understanding of various diagnostic techniques will aid in monitoring and managing emerging pandemic threats of infectious diseases from wildlife and also preventing the potential spread of zoonotic TB to livestock and humans. This review aimed to provide up-to-date information on different techniques used for diagnosing TB at the interfaces between wildlife, livestock, and humans.Tuberculosis (TB) is a chronic inflammatory and zoonotic disease caused by  Mycobacterium tuberculosis complex (MTBC); it affects livestock, wildlife, and humans [Mycobacterium tuberculosis, inducing increased morbidity and mortality in developing countries [Mycobacterium bovis occurred globally in the year 2018. However, only 16 countries reported the detection of M. bovis among pulmonary or extrapulmonary TB patients in 2018. Since 2007, TB has been the 10th leading cause of death worldwide from a single infectious agent, ranking above HIV. Most cases in 2018 occurred in South East Asia (44%), followed by Africa (24%), the Western Pacific (18%), the Eastern Mediterranean (8.1%), the Americas (2.9%), and then Europe (2.6%) [Tuberculosis (TB) is a reemerging and chronic infectious disease caused by d humans . The disd humans -4. Studiountries -9. It waThis review is intended to provide an up-to-date information of the antemortem and postmortem diagnostic techniques for TB at the interfaces between wildlife, livestock, and humans, that could help researchers and decision-makers to choose the most appropriate diagnostic protocol and to obtain a better understanding of their performance for effective TB surveillance and control strategies under different epidemiological situations in wild animals.M. tuberculosis, M. bovis, M. bovis BCG , M. africanum, M. microti, M. mungi, M. canetti, M. caprae, and M. pinnipedii [Oryx bacillus [M. orygis [The members of MTBC are nnipedii -18. Othebacillus , known a. orygis , and the. orygis . The MTB. orygis .Over 40 free-ranging wild animal species are known to be infected with MTBC bacterial species, causing the disease. Host species may be “maintenance hosts” (infection persists without input from other sources) or “spillover hosts”  . TubercuM. tuberculosis was isolated from four Asian elephants by bacterial isolation and sequencing [M. tuberculosis and confirmed by molecular methods [Reports of TB in wildlife, mainly in Asian elephants from various countries in Southeast Asia, have been published. For example in Thailand, quencing ,37. The quencing ,39. Repo methods . In the  methods ; in Aust methods . Studies methods ,43.Several factors influence the transmission of TB, including the number of infected animals, susceptible animals, routes of infection, anatomical location and lesions of disease, the structure of tuberculous lesions, pathogen shedding, and the infective dose in each infection route . EnvironFor antemortem diagnosis, single intradermal tuberculin test (SITT), single comparative intradermal tuberculin test (SCITT), and gamma interferon test (IFN-ɣ), which is regarded as a subsidiary test, are used -58. Thesin vitro IFN-γ assay involves two steps. In the first step, fresh blood samples are incubated to initiate the release of IFN-γ by sensitized T lymphocytes. The second stage requires separating the plasma from the blood and observing the IFN-γ in the plasma by an ELISA assay. A benefit of the IFN-γ assay is that the interpretation criteria are adjusted to fit local situations, such as the epidemiological conditions, TB prevalence, and the extent of the local bovine TB control program. The IFN-γ test has higher sensitivity than the skin test because the former detects TB disease in livestock as early as 14 days after infection [The nfection  and 60-1nfection . The mosnfection -73. Morenfection .For the rapid identification of MTBC infections, a direct smear is required for the detection of TB organisms from tissue samples with suspected infection followed by Ziehl–Neelsen (ZN) stain.. The basic principle of the stain is that MTBC available in the smear can take up an aryl methane dye, for example, carbol fuchsin, into the cell, resulting in acid-fast staining reaction, after treatment with a weak acid-alcohol solution. This is attributable to the mycolic acids that are present in the thick bacterial cell wall ,76. The Mycobacterium spp. relies on gross findings and histopathological analyses. One reliable method is histopathological analysis with hematoxylin and eosin staining [M. bovis infection in cattle [In free-ranging wildlife, the diagnosis of staining . This ten cattle .Culture is regarded as the gold standard technique for diagnosing MTBC, as described previously ,80, giveFor the isolation of mycobacteria, three types of media are typically used. The first is egg-based media, such as Stonebrink’s medium and Lowenstein–Jensen, with either glycerol or pyruvate. The second is agar-based media, such as Middlebrook 7H10 and 7H11, and tuberculosis blood agar medium, B83. The third type is liquid media, such as Middlebrook 7H9. The use of solid and liquid media together should maximize the sensitivity, an approach that has been approved for the isolation of mycobacteria in laboratories for human TB diagnosis globally -92. TherM. bovis (bovine PPD) [®kit test (bPPD) [Diagnosis based on an antibody is the most widely used approach for detecting TB in wild boar and pigs because of the maintenance of antibodies during transportation, storage, and handling, and the fact that the protocol can be standardized in different laboratories -97. A wiine PPD) ,98,99. Hine PPD) ,101, MPBine PPD) , or CFP1ine PPD) ,104. Resine PPD) -108. Morine PPD)  and 20 mine PPD) . Hence, ine PPD) . Severalt (bPPD) , INgezimt (bPPD) , and P22t (bPPD) .IS6110 element, especially targeting the 123 bp and 245 bp fragments. Insertion sequences (IS) are mobile genetic elements that constitute a form of repetitive DNA in bacterial genomes. Insertion IS6110 is an IS of 1361 bp that is not present in mycobacteria other than members of the MTBC [M. bovis isolates relies on the amplification of a 500 bp DNA segment, especially targeted for the 123 bp and 245 bp fragments inside the RvD 1Rv2031c genomic sequence [M. bovis from other members of MTBC by amplification of two DNA fragments [Multiplex PCR methods produce a higher rate of the detection of MTBC. These techniques amplify two or more target genes in a reaction simultaneously ,115. A sthe MTBC . Anothersequence ,119. Mulragments ,120.Digital PCR (dPCR) was more accurate  while sorrs (16S rRNA), ITS (16S-23S rRNA), IS6110, groEL2 (hsp65), dnaJ, fbpA (32 kDa protein), MPT64 (MPB64), devR, PPE24 (KS4), and lepA [Some important DNA markers used in differentiating MTBC from non-tuberculous mycobacteria (NTM) in the diagnosis of TB include and lepA .Studies have revealed that the 16S rRNA sequence can be used for the detection of mycobacteria, requiring fewer than 10,000 bacteria . For speMycobacterium, which is applied to distinguish between MTBC and NTM based on the specific primer sequence [23S rRNA genes. Its size ranges from 270 to 360 bp among species, with greater sequence variation than the 16S rRNA [IS6110 gene is lacking, sequence analysis of ITS helps in identifying MTBC members [M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti [PCR amplification of the 16S-23S rRNA internal transcribed spacer (ITS) is used to recognize the genus sequence . This bi16S rRNA . ITS seq16S rRNA . When th members . Slow gr microti .IS6110 is the most available and easily characterized insertion element, which is found mainly in MTBC; it is used to distinguish MTBC from other mycobacteria. Several copies of this element are available at different positions in the genome, which facilitates better strain genotyping [IS6110-restriction fragment length polymorphism (RFLP) has been widely used for epidemiological research on TB and has high discriminatory power, but is not suitable for species with less number of IS6110 copies [IS6110 PCR and real-time PCR have shown high sensitivity (71-87.9%) and specificity (95-98%) compared with culture and acid-fast bacilli [Insertion sequences (ISs) are short mobile genetic DNA elements encoding proteins responsible for the transposition activity, which permit them to spread within the genome. In the bacterial genome, the insertion sequence causes deletions, duplications, and rearrangements due to changes in the genomic plasticity of mycobacterial species . IS6110 notyping . IS6110-0 copies . IS6110  bacilli .hsp65 gene, which encodes a heat shock protein [hsp65-PCR-RFLP can distinguish between mycobacteria and NTM [hsp65 can be applied for specific recognition of NTM and can identify M. avium complex and M. intracellulare [hsp65 sequencing analysis is more accurate (88.9%) for identifying NTM than 16S rRNA (75.9%) [The identification of all mycobacterial strains is achieved using the  protein . hsp65-P and NTM , with a  and NTM . hsp65 cellulare . hsp65 s (75.9%) .dnaj gene encodes a cold-shock protein, which is a genus-specific gene that is amplified from almost all mycobacteria [dnaj sequences, while NTM has nucleotide substitutions at positions 1415 (A-C), 1417b (G-T/A/C), and 1442 (C-A), which are distinguished by the application of dnaj-PCR-RFLP [dnaj gene to detect the presence of MTBC and MAC [dnaj, 16S rRNA, and hsp65 genes, mean sequence similarity levels were 80.4%, 96.6%, and 91.1%, respectively. This proved that dnaj has higher discriminatory power than 16S rRNA and hsp 65 genes and is useful for differentiating NTM [The bacteria . MTBC haPCR-RFLP  GENECUBE and MAC . When 56ting NTM .fbpA is a secreted protein that is present in most MTBC strains but absent in non-MTBC strains [fbpA gene is similar between MTBC groups, but has distinct sequences within MTBC species, such as M. kansasii, M. gastri, M. gardone, and M. malmoense [fbpA gene has better discriminatory power than the hsp65 gene for identifying different NTM species [ strains . Sequencalmoense . This fb species .IS6110 PCR and has the capacity to decrease false-negative results in strains lacking the IS6110 element [The MPB64 gene is generally found in members of the MTBC and is used as an indicator in the diagnosis of paratuberculosis (PTB), TB meningitis, and extrapulmonary TB . PCR usi element . Gene mu element .devRencodes the cytoplasmic response regulator DevR, which works together with the membrane-bound sensor kinase DevS to form DevRS. During latent infection in the host, DevRS is responsible for adaptation and dormancy [devR gene significantly increased the sensitivity of TB diagnosis [devR PCR assay is lower than that of MPB64 and IS6110 [dormancy . Despiteiagnosis , the send IS6110 .M. fortuitum and M. xenopi [Some MTBC genes that have been less used for biomarker include the KS4 fragment of the Rv1753c gene, encoding the protein PPE. It has sensitivity of 98% and specificity of 98% in identifying MTBC, and cross-reacts with . xenopi .The Rv2404c gene encodes an elongation factor that is required for protein synthesis in MTBC members, but not in NTM . It has Molecular typing uses genetic markers to search for sources of outbreaks, to detect the epidemic or pandemic spread of particular strains, or to reconstruct the evolutionary trends of a specific group of bacteria. The majority of the genotyping methods were developed in the 1990s. At this time, efforts were made toward standardizing typing protocols to increase the quality of epidemiological research . GenotypBstEII, PvuII, and BclI, for typing M. bovis isolates [This technique uses three restriction enzymes, namely, isolates  and the isolates ,171. In isolates , but is M. tuberculosis isolates [M. bovis and M. bovis BCG [The technique of pulsed-field gel electrophoresis (PFGE) was initiated to bypassthe problem of analysis of excessive smaller number of DNA segments produced by REA. The PFGE process utilizes restriction enzymes that provide a smaller number of sections, which are very large, to be differentiated by standard agarose gel electrophoresis; but it is easily resolved using a pulsed electrical field. Initially, this approach was developed for isolates , but it ovis BCG ,175.M. tuberculosis, M. bovis and M. bovis BCG [M. bovis infection and its effect on local epidemics.Publication of the whole-genome sequences of ovis BCG -178 is aovis BCG . At presM. bovis strains [The introduction of whole-genome sequence data for different species of the MTBC has made it possible to apply microarray technology to compare strains, which has been particularly used to sequence reference strains . The maj strains . It also strains -186. HowPvuII or AluI, followed by southern blotting and gel electrophoresis of the fragmented isolated DNA onto a nitrocellulose or nylon filter [IS6110 is the genotyping method commonly applied for M. tuberculosis strains, which contain up to 20 copies of this IS [M. bovis; it is not frequently used for typing or routine diagnosis [Restriction fragment length polymorphism (RFLP) analysis utilizes the restriction enzyme n filter . RFLP ba this IS . However this IS . This teiagnosis ,189-191.http://www.Mbovis.org; http://www.pasteur-guadeloupe.fr/tb/bd_myco.html) [Spoligotyping, also known as spacer oligonucleotide typing, is a rapid and robust technique that enables MTBC isolates to be typed at high throughput, with no need for DNA purification . In a prco.html) ,197.M. tuberculosis genome contains about 75 bp tandem repeats situated in the large ORF [Variable number tandem repeat (VNTR) typing has focused on genetic loci that are found in the entire genome; these loci contain an enormous amount of data of repeated sequences. Due to various polymorphisms in the copy number of the repeats, the tandem repeats were successfully used to fingerprint bacterial genomes . VNTR tyarge ORF . It was arge ORF  that somarge ORF , these large ORF ,205.M. tuberculosis, excellent results have been obtained by applying this approach in combination with IS6110, referred to as IS6110-ample printing, which makes use of the gap between IS6110 and copies of MPTR sequences. Because the results of ample printing differ significantly in terms of discrimination and reproducibility, it is not regularly used as a diagnostic technique [Ample printing uses a polymorphic tandem repeat (MPTR) sequence , which iechnique -209.M. tuberculosis isolates, as described previously [M. bovis because of poor results in terms of discrimination ability [Because of its simplicity, RAPD is usually used for genotyping organisms, as a PCR-based fingerprinting method . It was eviously . However ability ,212. ProThe primary purpose of MLST is to categorize organisms in terms of the level of neutral sequence diversity . It has M. tuberculosis from other members of the MTBC and RD4 which is not found in all of M. bovis isolates. As a result, RDs were used as PCR targets as a rapid method of species identification; more recently, additional RDs were discovered, namely, RD2seal [M. pinnipedii, M. microti, M. dassie bacillus, M. mungi, M. orygis, and M. bovis BCG, respectively, from other species [Region of Deletion (RD) typing is used for the differentiation of species that are members of MTBC. RD9is used to differentiate  RD2seal , RD1mic  RD2seal , RD1das  RD2seal , RD1 mun species ,20,219.  species ,221, or  species , to loca species ,223. Bec species , these m species ,186.M. bovis [M. caprae [M. caprae-specific SNP in the M. tuberculosis H37Rv lepA (Rv2404c) gene, which is particular to bovine and cervidae isolates [M. tuberculosis [M. bovis [M. bovis BCG and differentiation of MTBC species [SNP typing is an essential form of genotyping analysis that enables differentiation among members of MTBC, for example, M. bovis  and M. c. caprae . A previ. caprae  describeisolates . Genotyprculosis ,227 and M. bovis ,229. A c species , which m species -36.Tuberculosis remains a global threat at the livestock – wildlife – human interface which require different diagnostic approach in the disease detection, prevention and control strategies. Each of these diagnostic methods has its own merits and demerits in terms of application and efficiency. The antemortem diagnosis detected CMI responses using the SITT, SCITT, and gamma interferon test (IFN-ɣ). The skin test requires animals to be handled twice, which is dangerous, especially for some wildlife, and the test cannot be repeated for about 90 days due to immunological sensitization to the PPD antigens. The higher sensitivity of the IFN-γ test relative to the skin test is because the IFN-γ test detects TB disease in animals as early as 14 days after infection and 60-120 days faster than the SCITT test. For postmortem diagnosis, direct smears are the fastest, cheapest, and simplest way to detect acid-fast MTBC. For this reason, a direct smear of tissue samples can be stained following the Ziehl–Neelsen technique to provide a presumptive identification of MTBC. Histopathological techniques have high specificity and the advantage of producing results within a few days. A correlation was found between the results obatained using histopathology and culture methods. Serology or antibody detection is the most widely used approach for the detection of TB in wild animals because of the maintenance of antibodies during transportation, storage, and handling, the ability to standardize the protocol in different laboratories, and the fact that the results are obtained within a short period of time. One disadvantage of antibody detection is that humoral responses to infection occur at the later stage of the disease. Bacterial culture remains the gold standard diagnostic method for MTBC, but requires several weeks to obtain positive results due to the extremely fastidious growth of tuberculous mycobacteria. It is used for species differentiation and has high sensitivity and specificity. Molecular diagnostic techniques of MTBC have advantages over conventional methods due to their sensitivity and specificity, being less complicated, less time-consuming, but expensive and not cost-effective compared with conventional methods for the early confirmation of the diagnosis of TB in both humans and animals. Molecular genotyping methods are used to differentiate between MTBC and NTM, and for species differentiation, which are not possible by the conventional approach. The combination of antemortem, postmortem, and molecular diagnostic techniques is the best option for controlling and preventing TB in both humans and wild animals.YML and AC conceived and designed the review outlines, collected the data and analyzed the data. PTO, SO, MM, SZR, SJ, and FFAJ analyzed the data and supervised the writing of the manuscripts. All authors have read and approved the final manuscript.


However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with iK* = 0.53 nM. Reversible binding of TFK provides a long residence time, τ = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the “isomerization” step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called “transition state analog”, followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer’s disease and protection of central AChE against organophosphorus compounds.Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with  SolutioDithiobisnitrobenzoic acid (DTNB) was from Sigma-Aldrich . 50 mM solution of DTNB was prepared as described in Ellman . Calbioc−4 M. The working enzyme was diluted 20,000 time in 0.1 M phosphate buffer containing 1 mg/mL BSA ([E]w = 1 × 10−8 M).Recombinant human AChE monomer  was expressed in CHO-K1  cells . AChE pu−10 M.Kinetic studies of human recombinant AChE inhibition by TFK were performed in 0.1 M sodium phosphate buffer pH 8.0 at 25 °C, using the Ellman’s method  with aceiv is the initial velocity, ssv the steady-state velocity and obsk, the first-order rate constant associated with establishment of the steady state. The reciprocal of obsk is the lag time before steady state. This is characteristic of slow binding inhibition  the substrate concentration, mK the Michaelis-Menten constant, [I], the inhibitor concentration, ciK, the competitive inhibition constant, and uiK, the uncompetitive inhibition constant. Rearranging Equation (2) as 1/iv or [S]/iv as a function of [I] provides two complementary plots as decribed by Cornish-Bowden ) and Cornish-Bowden plots ([ATC]/iv vs. [TFK]) at 3 different ATC concentrations [S] = 0.1; 0.5 and 1 mM). The initial reversible inhibition step was analyzed from the tangents of progress curves. The general case for linear reversible inhibition is described by following rate Equation (2):h-Bowden . In the h-Bowden  by buildobsk vs. [TFK] at 3 different ATC concentration   = 0.53 ± 0.07 nM. This value is in agreement with values previously determined by two different methods (0.4 and 0.56 nM) in the presence of 5 mM ATC in 0.1 phosphate buffer pH 7.4 after 70 min incubation at 23 °C [The binding kinetic parameters were determined from non-linear curve fitting of secondary plots s. [TFK]  obtainedat 23 °C .offk rate constant, τ = 1/koff) and the fractional occupancy of AChE (tFO) t can be calculated from Eqation (5). Assuming that the initial concentration of TFK in blood is 4.3 μM at time 0 , it follows that tFO0 = 99.9%. After a certain time (t’) when concentration in blood has dropped to 4.3 nM, tFO0 = 88.7%. If after time t”, the concentration has decreased by another order of magnitude (4.3 pM), then tFO0 = 7.3%. Thus, it is clear that with a residence time on AChE of about 20 min, the potential pharmacological efficacy of TFK could be maintained at a high level even though the drug concentration in blood has decreased to a very low value.Because both the concentration of drug in the target compartment and the residence time on physiological target as a function of time, determine the duration of action of a drug in the body, the pharmacological efficacy of a drug depends on the fractional occupancy of the enzyme as a function of time (FOt) ,44. ThenBinding of a transition state analogue, like acetophenone, to ChEs, the formation of a transient hemiketal conjugate mimicking the acetyl tetrahedral intermediate was observed or hypothesized in some cases ,43. Thus−8 M TFK. Then, the enzyme activity was monitored up to 180 min, far beyond establishment of reversible steady-state equilibrium. During this second step, the recovery of enzyme activity was monitored without dilution or after dilution in order to drop TFK concentration in the medium. As seen in −11 M), enzyme reactivation was completed in 40 min with reactk ≈ 0.05 min−1; t1/2 ≈ 14 min (−8 M TFK (The reaction process was performed under pseudo-first order conditions to reach complete inhibition of enzyme by 2 × 10τ) on AChE calculated from reversible SBI is 20 min, the subsequent acylation and deacylation processes provide an overall residence time of about 60 min.The slow enzyme reactivation process was interpreted as spontaneous slow deacylation of bound TFK from AChE active center. Molecular modeling (see next section) confirmed the occurrence of transient acylation of AChE active site serine after TFK binding and shed light on molecular interactions involved in these acylation and deacylation steps. Then, considering that the residence time (t = 60 min. The slow recovery of enzyme activity depends on the TFK concentration present in the medium. In the presence of remaining 0.02 nM TFK (green curve), it takes less than 40 min to regain an activity similar to that of control enzyme at Molecular modeling was used to depict in terms of molecular events the different steps of TFK interactions with huAChE. Preliminary results about interaction between TFK and AChE were recently reported . 3 group with Trp286 ring  or bottleneck area . The dom286 ring . In anot286 ring  in a way286 ring , showing286 ring . In gene286 ring , orientetert-butyl moiety outside cation-binding site Trp86 and Glu202  profiles for binding of TFK and TMTFA to hAChE calculated using REMD-US method are in agreement with docking results C. For TFIn addition to the two distinctive positions inside the gorge, PMF for TFK allows to assume non-specific interactions on the protein surface  and TorEnergy scan for reactivation process shows that while inhibitor is inside the active site, i.e., carbonyl oxygen atom is within the oxyanion hole, formation of the tetrahedral adduct occurs spontaneously. Stable noncovalent complex was obtained only when the TFK group left the oxyanion hole B. Though reaction itself occurs barrier-less, it has associated energy barrier for incorporation of the TFK group into the oxyanion hole, estimated with QM/MM method as 19.3 kcal/mol for TFK and 13.0 kcal/mol for TMTFA. Energy barrier for TMTFA estimated with QM/MM method corresponds to values obtained with REMD-US , and high energy barrier associated with induced-fit entrance of the inhibitor into the active site. Absence of positive charge additionally slows down this process compared to positively charged analogs. Then, we investigated the possible modulating or protecting effect of TFK on AChE phosphylation by two different OPs. The first OP was cresylsaligenyl phosphate (CSP), the active metabolite of tricresylphosphate. CSP is a strong phosphorylating agent of ChEs ,58. InhiThis type of SBI followed by a covalent step, i.e., phosphylation, is related to enzyme hysteresis . These mInhibition of hAChE by CSP (0.21 µM) performed after different pre-incubation times in the presence of various concentrations of TFK (0.1–1 nM) showed that the biphasic progressive inhibition process of the enzyme is affected by TFK. The biphasic progressive inhibition was analyzed as a function of pre-incubation time and concentration in TFK.Ln  vs time of the fast phosphorylation process and pre-incubation duration the residual activity increased as pre-incubation duration and TFK concentration increased. The deleterious effect of TFK was maximum for 10 nM TFK  vs time and pre-incubation duration the residual activity increased as pre-incubation duration and TFK concentration increased. The protective effect was maximum for 10 nM TFK  on AChE phosphorylation by 50 nM paraoxon. Unlike progressive inhibition of AChE by CSP, first order inhibition of the enzyme by paraoxon is linear as with most OPs. It was found that pre-incubation of AChE with TFK decreased the observed rate of phosphorylation. Thus, the effects of pre-incubation duration and TFK concentration on the rate of inhibition was directly quantified from the slope of 0 nM TFK  and reac0 nM TFK .A recent in vivo work provided evidence that the slow-binding inhibitor C-547 has a long protective action, up to 3 days, on peripheral AChE against inhibition by paraoxon with no side effects . This suKinetic analysis and molecular modeling of hAChE inhibition by TFK showed that this ligand is a SBI of type B. Moreover, after formation of the final complex, a transiently stabilized tetrahedral conjugate is formed, and then slowly dissociates. The existence of covalent and stable acyl tetrahedral intermediates in ChEs ,63,64 isτ = 70 h and iK = 0.26 nM for rat brain AChE -6-methyluracil dibromide) [Other possible pharmacological applications may be considered. In particular, research of new molecules for protection of ChEs against phosphylation by OPs is an active field. At the moment, the currently used molecules for pre-treatment of OP poisoning have limited and short protective actions and may induce behavioral and locomotor side effects . Certainbromide) ,66 has abromide) . Thus, t


Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity. The kinase, in its inactive form, exists as a holoenzyme composed of two catalytic subunits and two regulatory subunits. Activation of the kinase is generally understood to occur through the increase of cAMP, which leads to the dissociation of the catalytically active holoenzyme. Kinase activity can further be spatially regulated through the existence of A-kinase anchoring proteins which anchor the holoenzyme to specific subcellular localizations4.As one of the earlier kinases to be characterized, Protein Kinase A (PKA) has been discovered to play an important role in numerous physiological processes including cardiac physiology5. A more generalizable variant of this design was established by Zhang, et al. which involved creating a chimera that consisted of FRET-compatible fluorescent proteins, a phosphopeptide binding domain, and a short kinase substrate sequence6. Upon phosphorylation, the binding affinity between the phosphopeptide binding domain and the substrate sequence dramatically increase, leading to a conformational change, which is then measured through changes in FRET. This design has been the basis for a number of generations of PKA FRET reporters in the literature12.This well-characterized nature of PKA has naturally led to it being used as a platform for a number of interesting concepts for kinase reporters. One of the earliest such designs was the utilization of Förster resonance energy transfer (FRET) to monitor the dissociation of the catalytic and regulatory subunits of the protein, a basic design that was used to demonstrate the localness of cAMP concentration and PKA activity13, utilizing polarization of light as a readout14, or designing proteins that change subcellular localization in response to phosphorylation15.One of the main advantages of fluorescent kinase reporters is the ability to analyze kinase activity on a single cell level in real time. Despite this advantage, FRET reporters have the inherent downside of requiring two fluorophores, limiting the ability to monitor multiple kinases simultaneously with most experimental setups. Numerous groups have reported various approaches to overcome this limitation in respect to PKA. These approaches have included using multiple FRET pairs16. In summary, arginines in the P-2 and P-3 positions are important residues for substrate recognition18. By placing the amino acid sequence for the substrate in tandem with minor modifications, we designed an amino acid sequence that loosely conformed to known consensus nuclear localization sequence (NLS) motifs19. This peptide responded to PKA activity by shuttling between the nucleus and the cytosol. We furthermore performed basic optimization and demonstrated its use in simultaneously monitoring two different signals within a cell.Our approach here utilizes a similar principle that we reported in a previous study where we designed a membrane-bound phosphorylation switch4 cells/cm2 in a 6-well plate containing a 25 mm coverslip one day before transfection. They were transfected with multiple plasmids at an amount of 50 ng each using FugeneHD (Promega) according to the manufacturer’s protocol. On the following day, culture media was replaced with serum-free DMEM in the morning. Imaging took place in the afternoon of the same day.HeLa cells were cultured in DMEM with 10% FBS and routinely passaged. Cells were seeded at a density of 2.0 × 102 . Images were acquired for either 15 or 25 minutes at 1-minute intervals. Drugs were added at the 5-minute and 15-minute point. PKA activation was achieved through treatment with 50 µM forskolin and 100 µM IBMX, unless otherwise noted. For the experiment demonstrating reversibility, the media was replaced with new media containing 40 µM H89 at the 15-minute point, as residual forskolin and IBMX led to an incomplete inhibition of PKA. Images were acquired with a CMOS camera  in a 60x oil objective  with the appropriate excitation and emission filters driven by a filter wheel controller. Metamorph was used to control the hardware associated with the microscope which includes the motorized stage , filter wheels , and LED light source . To measure response with the FRET reporter, cells were excited in the CFP channel, and images were acquired in the YFP channel.Images were acquired on an inverted microscope  with a heated incubator that maintained the chamber at 37 °C and 5% COAll cells were co-transfected with H2B-mCherry Figs. –3, S1 or3 labeling. All the reporter variants were inserted into the EYFP-C1 vector (Clontech) at the SacII and BamHI restriction sites using annealed oligonucleotides. The forward sequences of the annealed oligonucleotides are tabulated below:The cDNA for H2B (gift from Sergi Regot) was amplified by PCR and inserted into NheI and AgeI sites of the mCherry-C1 and mCerulean3-C1 vectors (Clontech). YFP-PH(Akt) is a standard PH domain construct for PIP1: GGGCctgagaagagccagcctgggcTAGGSub2: GGGCctgagaagagccagcctgggcaagagaagagccagcctgggcTAGGSub3: GGGCctgagaagagccagcctgggcaagagaagagccagcctgggcaagagaagagccagcctgggcTAGGSub4: GGGCctgagaagagccagcctgggcaagagaagagccagcctgggcaagagaagagccagcctgggcaagagaagagccagcctgggcTAGGSub3-NesA: GGGCctgagaagagccagcctgggcaagagaagagccagcctgggcaagagaagagccagcctgggcgtggaccagctgagactggagagactgcagatcgacgagTAGGSub3(AAA)-NesA: GGGCctgagaagagccgccctgggcaagagaagagccgccctgggcaagagaagagccgccctgggcgtggaccagctgagactggagagactgcagatcgacgagTAGGSub3-NesB: GGGCctgagaagagccagcctgggcaagagaagagccagcctgggcaagagaagagccagcctgggcgaccccctgcccgtgctggagaacctgaccctgaagagcTAGGSub3-NesC: GGGCctgagaagagccagcctgggcaagagaagagccagcctgggcaagagaagagccagcctgggcaaggtggccgagaagctggaggccctgagcgtgaaggagTAGGSub3-NesD: GGGCctgagaagagccagcctgggcaagagaagagccagcctgggcaagagaagagccagcctgggcgccctgcagaagaagctggaggagctggagctggacgagTAGGSub17, a well-established PKA substrate, as the working unit of the design, which contains the two arginines in the P-2 and P-3 position considered important for substrate recognition. A monovalent substrate alone does not conform to known NLS. However, placing these sequences in tandem leads to a sequence that conforms loosely to NLS motif . Due to the overlapping nature of the NLS with the phosphate acceptor, phosphorylation of this peptide may result in a shift in localization, thus functioning as a switch. To test this idea, we utilized the Kemptide (LRRASLG)tif Fig. . We trantif Fig. . The pretif Fig. . We also20. Using this as a reference, we fused a 13-amino-acid, leucine-rich fragment from FMR1, TFIIIA, RanBP1, and Map2K1  to the trivalent substrate. A previous study established the relative strengths of the various NES’s2K1 Fig. . We obse2K1 Fig. . This alK1 Fig. 2,D. Among9 , intending to measure PKA activity and Ca2+ simultaneously. As a result, we obtained a distinct temporal response of these two signaling molecules; while Ca2+ indicated a sharp and transient increase  and the PKA phosphorylation switch (EYFP), we simultaneously monitored the synthesis of PIPane Fig. . This waane Fig. . PKA actane Fig. . We alsoEGF Fig. . Second,ase Fig. , PKA actase Fig. . Togethe 2+ Fig. .de novo amino acid sequence based on simple observations of PKA consensus sequence and NLS motif. Using this information, we designed a switch that reproduced the kinetics measured with a well-established FRET reporter. In contrast to FRET reporters, this type of single-fluorophore reporter frees up a fluorescent color, allowing for an additional channel of analysis. We then demonstrated this capability monitoring two different signaling molecules by overexpressing cells with the PH domain from Akt and the PKA reporter.Our PKA phosphorylation switch is a A switchable NLS alone did not reproduce the kinetics of PKA activation observed with a FRET reporter. The distribution of a protein observed in a cell is determined by the net flux of the nuclear import and nuclear export transport processes. With peptides containing only NLS’s, the nuclear import is driven by facilitated diffusion while nuclear export is driven by passive diffusion. As passive diffusion is slower than facilitated diffusion, the time required to reach steady state is longer, reflected by the decrease in response kinetics. The addition of an NES mitigated this issue. An ideal reporter, thus, should be rate-limited by the kinetics of the underlying phosphorylation, not by auxiliary processes.11. A phosphorylation prediction algorithm, NetPhos3.122, strongly suggests PKA as the target kinase for this reporter. Furthermore, based on the current literature of AGC kinases, PKA is the only AGC kinase that requires two arginines at P-2 and P-3 position24. These reports do not rigorously rule out non-specific phosphorylation, as substrates are not necessarily required to conform to consensus sequences. However, given current literature and our data, PKA activity appears to be the driving force behind the shuttling.Another important property of reporters is kinase specificity. In our study, we have used a well-characterized substrate sequence of PKA that has been used in previous FRET reporterset al. developed a similar reporter which shuttles between the cytosol and the nucleus using a different strategy15. The phosphate acceptors in this reporter are placed in a much weaker, proline-rich substrate recognition sequence derived from c-Jun. The affinity towards the substrate is increased through docking sequences, which elevates the effective concentration of the substrate to the kinase26. We were unable to observe a measurable response with this PKA reporter. However, we speculate that this reporter design relies on a much weaker interaction with the kinase than our design, requiring a more careful optimization of experimental conditions.We used the interaction of positively charged residues in the substrate sequence with the catalytic subunit of PKA to design the reporter. However, this is not necessarily the only method of achieving specificity. Regot In summary, we have designed a reporter based on a rearrangement of a PKA substrate to create a NLS. In principle, this is generalizable to all basophilic kinases, as this mechanistically relies on disruption of electrostatic interactions through phosphorylation. Due to the single-fluorophore nature of this reporter and its comparable performance to a FRET reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.Supplementary Figures 1-2.