In order to determine the mode of action, the plants were prepared as methanolic and aqueous extracts and tested for their effects on intestinal glucose and fructose absorption in Caco2 cells. The results of this screening showed significant and reproducible inhibition of glucose uptake between 40 and 80% by methanolic extracts made from the fruits of Aronia melanocarpa, Cornus officinalis, Crataegus pinnatifida, Lycium chinense, and Vaccinium myrtillus; the leaves of Brassica oleracea, Juglans regia, and Peumus boldus; and the roots of Adenophora triphylla. Furthermore, glucose uptake was inhibited between 50 and 70% by aqueous extracts made from the bark of Eucommia ulmoides and the fruit skin of Malus domestica. The methanolic extracts of Juglans regia and Peumus boldus inhibited the fructose transport between 30 and 40% in Caco2 cells as well. These findings can be considered as fundamental work for further research regarding the treatment of obesity-correlated diseases, such as Diabetes mellitus type 2.The traditional use of plants and their preparations in the treatment of diseases as a first medication in the past centuries indicates the presence of active components for specific targets in the natural material. Many of the tested plants in this study have been traditionally used in the treatment of Diabetes mellitus type 2 and associated symptoms in different cultural areas. Additionally, hypoglycemic effects, such as a decrease in blood glucose concentration, have been demonstrated  In the last years, not only did the absolute number of people suffering from Diabetes mellitus increase from 108 Mio in 1980 to 422 Mio in 2014 but also the prevalence of this disease rose from 4.7 to 8.5% among adults during this time period worldwide . Apart fAllium sativum, Ceratonia siliqua, Cuminum cyminum, Juglans regia, Nigella sativa, Olea europaea, Sarcopoterium spinosum have been used in the treatment of Diabetes mellitus . The following herbal drugs were obtained from Alfred Galke GmbH : flowers of Hibiscus sabdariffa and Syzygium aromaticum; fruits of Ceratonia siliqua; fruit skin of Citrus limon; leaves of Brassica oleracea,Camellia sinensis, Ilex paraguariensis, Juglans regia, Melissa officinalis, Origanum creticum, Rosmarinus officinalis, and Salvia officinalis; and seeds of Cuminum cyminum and Vitis vinifera (pomace). The plant materials used in Traditional Chinese Medicine, such as the bark of Eucommia ulmoides; flowers of Rosa rugosa; fruits of Cornus officinalis,Crataegus pinnatifida, and Lycium chinense; and the roots of Adenophora triphylla and Pueraria lobata were acquired from Zieten Apotheke . The fruits of the cucurbitaceae Momordica charantia were kindly given by Dr. Serhat Sezai Çiçek . The green seeds of Coffea arabica were purchased from Ridders Kaffeerosterei . The leaves of Peumus boldus were gained from Heinrich Klenk GmbH & Co. KG , whereas the leaves of Artemisia dracunculus and the fresh fruits of Malus domestica were obtained from a local supermarket. The fresh fruits of Malus domestica were washed with water. The fruit’s skin was cut into small pieces, dried in a drying cabinet at 40°C for 3 days, and freeze-dried for 48 h. The dried plant material was ground with a M20 mill from IKA®-Werke GmbH & Co. KG . 10.0 g of the plant’s powder, adjusted to the drug extract ratio as listed in The methanolic and aqueous extract preparations were performed according to 2-supply during the whole time. Passages 20–35 were used in the experiments. The buffers and solutions for cell culture were prepared with water obtained from the ultrapure water system LaboStar from Siemens AG Wasseraufbereitung .Caco2 cells were purchased from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH  and seeded in 24-well plates, which were acquired from Greiner Bio-One GmbH , for every experiment. As general medium DMEM (Dulbecco’s modified eagle medium) without phenol red containing 1% UltraGlutamine 1, both acquired from Lonza Cologne AG  and 5 or 20% FBS derived from Bio&SELL GmbH  for experiment or mother line, respectively, was used for the handling with Caco2 cells. Cells were washed once with PBS from Lonza Cologne AG  for every medium change and washed twice before Trypsin (TrypLE™ Express without phenol red) from Gibco Life Technologies corporation  was applied to detach the cells for seeding. The cells were incubated at 37°C and 5% COFor uptake studies, cells were seeded in a density of 400,000 cells per well. Confluence appeared 24 h after seeding and was controlled visually. The radiolabeled substrates 1 mCi/ml glucose (specific activity: 30 Ci/mmol) and 1 mCi/ml fructose (specific activity: 5 Ci/mmol) were obtained from Biotrend Chemikalien GmbH . Although fructose absorption into cells was determined by using 54 nM solution of radiolabeled substrate as final well concentration, the 2 nM radiolabeled glucose solution was spiked with 0.001 µM non-radiolabeled glucose, which was purchased from Merck KGaA . For uptake studies with extracts, cells were used at day three and day fifteen after confluence for glucose and fructose, respectively. On the day of the experiment, cells were washed once with 2 ml buffer containing 20 mM Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], which was obtained from Carl Roth GmbH + Co. KG  and 150 mM NaCl purchased from Fisher Scientific GmbH . The pH of the buffer was adjusted with sodium hydroxide from Merck KGaA  using a 766 Laboratory pH meter from Knick Elektronische Messgeraete GmbH & Co. KG  to 7.4. After 1 h starving incubation with Hepes-NaCl buffer at 37°C, substrate or substrate-extract solution were added to the cells and incubated at 37°C. After 1 h, the buffer was discarded, the cells were washed once with 1 ml ice-cold Hepes-NaCl buffer, and 500 µL Hepes-NaCl solution containing 1% Triton X 100 was added to each well. The samples were taken from the well plates, added to a sufficient volume of scintillation cocktail in scintillation tubes, both purchased from Hidex , mixed for 20 s using a vortexer from Heidolph Instruments GmbH & Co. KG , and measured with a β-Counter from Hidex . The plant extracts, which showed the strongest inhibition on intestinal glucose transporters, were repeated and tested for their influence on fructose uptake with a focus on methanolic extracts. Randomly, well plates, which were cultivated and treated under the same conditions as cells used for uptake studies, were tested in Hoechst Assay as described in the via Shapiro–Wilk test manually before using Excel’s tool “Data analysis” for provision of the F-test and the t-tests. If the data was not normally distributed, Mann–Whitney U test was applied for further statistical analysis. IC50 data was calculated with GraphPad Prism 5.0.All statistical tests were performed using Microsoft Excel. Normal distribution was established Aronia melanocarpa (uptake inhibition: 57.1%),Cornus officinalis (uptake inhibition: 73.8%), Crataegus pinnatifida (uptake inhibition: 73.7%), Lycium chinense (uptake inhibition: 75.2%), and Vaccinium myrtillus (uptake inhibition: 79.9%); the leaves of Brassica oleracea (uptake inhibition: 82.8%), Juglans regia (uptake inhibition: 52.4%), and Peumus boldus (uptake inhibition: 47.5%); and the roots of Adenophora triphylla (uptake inhibition: 64.6%) indicated a strong and reproducible inhibitory potential between 40 and 80% on intestinal glucose transporters as visualized in Eucommia ulmoides (uptake inhibition: 68.7%) and the fruits of Malus domestica (uptake inhibition: 57.5%) inhibited glucose uptake significantly and reproducibly. Additionally, for the methanolic extract of Brassica oleracea and the aqueous extract of Eucommia ulmoides, IC50 was calculated as 0.0904 and 0.4285 mg/ml, respectively. These extracts decreased glucose uptake in a dose-dependent manner. The methanolic extracts of Juglans regia (uptake inhibition: 30.2%) and Peumus boldus (uptake inhibition: 32.6%) inhibited the fructose uptake significantly.In order to prevent decreased metabolic activity due to the use of toxic extracts, methanolic and aqueous plant preparations were tested in MTT assay and the concentration, which did not reduce cell viability significantly, was used for uptake studies as shown in Adenophora triphylla,Aronia melanocarpa, Brassica oleracea, Cornus officinalis,Crataegus pinnatifida, Eucommia ulmoides, Juglans regia,Lycium chinense, Malus domestica, Peumus boldus, and Vaccinium myrtillus as listed in Juglans regia and Peumus boldus decreased glucose as well as fructose uptake possibly related to inhibition of GLUT2, which is able to transport both monosaccharides. These findings show that the traditional use of Brassica oleracea, Cornus officinalis, Eucommia ulmoides, Juglans regia, Lycium chinense, Malus domestica, and Vaccinium myrtillus and their preparations as antidiabetic remedy and adjuncts to conventional treatments in the therapy of Diabetes mellitus is reasonable. Extracts of these plants show inhibitory activity on intestinal glucose and fructose transporters resulting in lower blood concentrations of the monosaccharides and reduced concomitant health problems.According to the results of the present study, the observed hypoglycemic effects in animal and/or human studies after treatment with preparations of Adenophora triphylla (uptake inhibition: 13.4%) and Cornus officinalis (uptake inhibition: 16.4%), but other results as for Crataegus pinnatifida (no uptake inhibition) are not consistent with our findings (Eucommia ulmoides (Malus domestica (Aronia melanocarpa, Peumus boldus, and Vaccinium myrtillus showed strong lipase and α-amylase inhibition as well as inhibition of intestinal monosaccharide transporters in our studies. This multi-target effect of the mentioned plant preparations describes a partial, but multiple drug action and is proposed to exceed drugs with single-target effects. Based on synergistic mechanisms, the plants that affect various targets to alleviate monosaccharide uptake show therapeutic advantages concerning higher intended and lower adverse effects (In literature, there is not much comparable data available, because the inhibition of intestinal transporters by crude plant extracts in Caco2 cells has not been investigated very well . Kim et findings . Zhang eulmoides . Manzanoomestica , which somestica . Buchholomestica . The met effects . Since t effects , which a effects  and the  effects . Further


In most individuals, kinetics showed a reduction in the specific IgE at the time about nine months after deployment to Antarctica. Five participants had the highest specific IgE levels after returning to the “normal” world. The examination of the specific IgE relative to house dust mites and storage mites showed different kinetics. Six out of 10 had the highest specific IgE concentrations at the inner Antarctic measurement time point. These data corresponded well to the general situation in the stations. At the stations themselves, there were almost no pollen particle load, especially at Concordia. (4) Conclusions: Antarctic long-term confinement can induce an altered immune function, which is in some individuals pronounced after return to the familiar allergen environment. Future prospective studies in larger cohorts are needed to further specify these first results. Antarctica is the most hostile continent on Earth—it is the coldest, driest, and windiest, but is also the cleanest place on our planet. The average annual temperature ranges from about −10 °C (coast) to −60 °C at the highest parts of inner Antarctica [About a decade ago, our group started immunological human studies in this extreme environment in one year overwintering crews, primarily focusing on innate and adaptive immune alterations ,3 but alDuring the last years, several personal communications have reached us in which returnees from Antarctica reported that they experienced severe or even new allergy typical symptoms such as hay fever, wheezing, or even throat swelling. For example, one overwintering participant described that a new onset of asthmatic symptoms occurred in the presence of their neighbor’s cat after their return. Another one, who had previously had allergic reactions  to cats and dogs complained about newly developed hay fever, wheezing, and throat swelling in the presence of grass and flowers, also after return.These communications brought our attention to retrospectively investigate potential sensitizations and type-1 hypersensitivity  typical symptoms in five previously studied overwintering crews at Neumayer III and Concordia Station.Two different Antarctic research stations served as study locations for the original investigations ,3,5: ConIn contrast to the Northern Hemisphere, the seasons are inversed—the Antarctic summer lasts from November to February and the winter from May to August. Due to the close location to the South Pole, a light/dark cycle is missing during the Antarctic summer, resulting in 24 h of constant sunlight. Summertime average outside temperatures are around −40 °C at Concordia and −3 °C at Neumayer III. During wintertime, no sunlight is present for almost three months. This leads to outside temperatures around −60 °C and sometimes also to −80 °C at Concordia. At Neumayer III, winter darkness lasts some weeks less/fewer with average temperatures of −30 °C. These extreme temperatures are not the only challenges for the expeditioners. Particularly at Concordia, the humidity is very low, which causes a very dry environment, leading to mucosal defects—nose bleeding is hereby common during the first weeks of deployment. Both stations are completely isolated  from the rest of the world from mid-February to mid-October. All telecommunication/internet is only possible via satellite and depends on the weather situation.Confinement and Hypobaric HypOxia on Immunity in the Antarctic Concordia Environment) included sample collection at baseline (about two months before departure (pre)), on a monthly basis, and about 3–4 months after return (post). For this retrospective investigation, an “inner Antarctic” time point was chosen with the longest isolation time from the outer world—for both stations, this was the September collection (Sept). Samples of three overwintering crews from Concordia and samples of two crews from Neumayer III were analyzed. In detail, blood samples (serum tubes) from 23 Concordia crew members  and 16 Neumayer participants were analyzed . Complete sample sets were present in 34 cases, whereas five sets from Concordia were incomplete (2 × September and 3 × post missing).The original study CHOICE  containing more than 280 allergen extracts and molecular allergens.ALEX tests were performed from 200 µL serum plasma at the MacroArray Diagnostics Laboratory at 1230 Vienna, Austria. This test measures the semi-quantitative total IgE concentrations as well as quantitative allergen specific IgE. The ALEX test clusters eight different allergen groups  and further investigated cross-reactive allergen families containing 12 different components .<20 kU/L—Allergy unlikely20–100 kU/L—Allergy possible>100 kU/L—Allergy likelyGeneral classification of total IgE (kU/L) concentrations in the ALEX analyses for adults are as follows:All participants were contacted via email during springtime 2020 and asked to fill in a questionnaire including questions about allergy typical reactions, history of allergy, and changes after return from Antarctica.t-test for normally distributed data. A p-value < 0.05 was regarded as statistically significant.All data were tested for normal distribution using the Shapiro–Wilk test. Between-group comparisons were performed using the Mann–Whitney Rank Sum test for non-parametric data and a The majority of the data are presented in this report as raw data to provide the maximal possible scientific overview and not to “diminish” biological effects after statistical calculations.®  and IBM SPSS Statistics  were used for the statistical analyses and figure design.Data in figures are displayed as single values. SigmaPlotAside from the smaller group size (23 vs. 16), no significant statistical differences in demographics were present between the stations .Thirteen out of the 39 participants had at one of the three time points a total IgE (tIgE) level above 100 kU/L. According to the test definition, an allergy is likely when tIgE is >100 kU/L. When looking at the September tIgE concentrations, 13 participants showed elevated levels compared to the baseline .Alterations of tIgE concentrations were defined here if values were affected by more than ±10 kU/L. Aside from two participants in which tIgE levels increased further, all levels dropped again after return.By further analyzing the ALEX test, we observed in 26 participants elevated specific IgE (sIgE) levels according to the test ranges/classification  and the molecular allergens (MA) of the grass pollen cluster revealed, aside from 2  out of 13, a reduction in specific IgE levels in September. September values were in most cases also lower than the post data. In two individuals , some AE and MA were moderately higher after overwintering when compared to the baseline values .The specific IgE concentrations for grass pollen were classified at any time point very high for three participants, high in three, moderate (three), and low in four individuals .Twelve participants had sensitizations toward tree pollen. In general, concentrations were high in six cases, moderate in four cases, and low in two cases. Interestingly, three overwinterers showed higher sIgE levels in September than at the baseline . Five individuals had the highest levels for some specific IgEs after the Antarctic expedition . Two of them  were within the low range (0.3–1 kUA/L). Con21 had no specific IgE before Antarctica, but low levels afterward .Ten participants showed specific IgEs against AE and MA toward house dust mites and storage mites. Three had very high, four high, and three moderate sIgE concentrations. Six individuals had peak levels for some AE/MA in September . The majority of the post values were lower than the pre data .Three participants were sensitized toward Alternaria alternans. One had very high, one high, and one had moderate levels. In September, the levels showed the lowest values. One participant presented the highest levels after the expedition, whereas the other two had remarkably lower ones compared to the baseline .Specific IgE reactions toward pet AE and MA were observed in seven participants. One with very high levels, one with high, two with moderate, and three with low levels. Interestingly, three showed the highest levels after return. One participant developed a new sensitization toward various pet AE and ME (Con21) .The return rate of the questionnaire was low (11 out of 39 (28.2%)). Five declared that they had no history of allergy or any typical sensitization, whereas six indicated a history of allergy including hay fever, wheezing (asthmatic reactions), and increased swelling after insect bites.Two of the participants with a history of allergy experienced more hay fever after return . Total IgE levels after return were in both cases lower than pre departure. Specific IgEs showed no increase in the post data collection, respectively.Two other expeditioners reported on the newly developed hay fever after their return . In one case (Con21), total IgE concentration was the highest at the post time point (176 vs. 26 (pre) kU/L). A new sensitization after return against the ash extract and molecular allergen could be detected. In the other case (Con23), no alterations in terms of an increase were observed. Total IgE levels dropped from pre 719 to post 521 kU/L.Another subject (Con9) who had hay fever from olive trees explained that this reaction was gone after its return. Total IgE levels in this case were also the lowest after return, but the specific IgE toward ash , olive (Ole e1), and the Ole e1 family were higher post expedition. This is in contrast to the clinical description of an improvement in allergic symptoms  and speaAs initially described, this retrospective investigation was initiated after the incidence of personal communications increased where returnees wintering over for a year in Antarctic stations experienced more allergic reactions than they had at home before deployment to Antarctica. To the best of the authors’ knowledge, this is the second report 44 years after Lund´s et al.  first inDue to the few available sample volumes from five overwintering crews, the study team tried to obtain the maximal possible scientific outcome one could achieve on a retrospective basis. In this light, we have chosen a newer chip based multiplex assay (ALEX Allergy Explorer) as it contained a very broad variety of allergen extracts and molecular allergens. The ALEX test showed comparable results to the Immuno Solid-phase Allergen Chip  test , whereas®, Chicago, IL, USA) with the evaluation of its type IV reaction [In addition to the specific IgEs, the ALEX test also measures the total IgE concentrations. In contrast to previous findings from Lund  on totalreaction . The altreaction . Howeverreaction ,10. In greaction . CertainFrom the specific IgE levels of the pollen cluster, one can assess that in the majority of the participants, sIgEs levels were lower in September and also after return. Interestingly, five individuals had the highest levels after return toward distinct tree pollen allergen extracts and molecular allergens. Certainly, elevated total IgE and specific IgE concentrations should not be stressed as singular markers in the diagnosis of allergies or allergic reactions. However, these findings in the overwintering/returning crew could give a hint that somehow sensitization to pollen could be affected by the year-long isolation from such allergens and lead to the personal communications presented in the introduction.Due to the long distance to the next Antarctic coast (~1000 km) with wildlife possibly transporting pollen, one can assume that there is no significant pollen present at Concordia Station. Though Neumayer III is situated at the coast on shelf-ice, the closest island (Bouvet Island) with some spare vegetation is more than 1600 km away. Significant amounts of pollen are not expected to be on this station. Overall, the here presented data on pollen sIgEs are in good accordance with the general known sIgE kinetics that specific IgE tends to decrease when not exposed and go up in response to exposure .Elevated sIgEs after return were also present in two other clusters—microorganisms (one out of three) and pets (three out of seven). The kinetic over time was otherwise similar to the pollen cluster.In terms of pet allergens, one has to acknowledge that there is also no animal life at Concordia Station. At Neumayer III, winter breeding places of the Emperor penguin can be found not far from the station. The ALEX test does not investigate bird allergens, which could have detected possible new sensitizations. Most sensitizations were in context with cat allergens. After several months, specific IgE levels such as the Fel d1  are, in general, shed ,13. At tSomehow different was the cluster “house dust mites and storage mites”. Six out of 10 individuals presented the highest peak sIgE levels in September. One explanation therefore can be that these stations are not at all allergen free areas and trigger specific IgE generation. Though the humidity is low at these stations and additionally, a low oxygen pressure is present at Concordia, van Houdt et al.  describeIn contrast to Neumayer Station, wildlife does not exist at the inner-Antarctic Concordia Station, possibly leading to a lower overall environmental antigen load and altered immune stimulation normally occurring in a regular environment ,16,17. TAs Hamilton et al.  stated tThis investigation and the generated data do have several shortcomings as to the retrospective data acquisition and the divergent questionnaire/sampling time points. This must be carefully differentiated between the presence of allergen specific IgE, a single lab parameter, from a clinical allergic sensitization. Since up to 30% of individuals can have allergen-specific IgE via serum test and/or skin test and have no clinical symptoms, most allergists would be unwilling to call someone “allergic” because their total IgE was elevated. The ALEX test cluster of total IgE uses this common definition allergy likely/unlikely depending on total IgE. In this report, this separation was not used. Instead, the authors tried to show new findings on sensitizations in an extraordinary environment.Further limitations have to be addressed when looking into the data of specific IgE levels. Potential sensitizations with pollen (after unpacking stored food packages during winter over) or after sweeping distinct areas could have influenced the measurements in September as the supplies were not packed in an allergen-free environment. In future studies, one certainly has to measure the allergen load ). Food sensitizations detected by the ALEX test were not further analyzed and discussed as food sensitizations/allergies are considered as different entities.Taken together, the here presented data provide additional information on a possible altered immune function in the sense of a type 1 reaction during the Antarctic deployment, which is in some individuals pronounced after return to the familiar allergen environment.A further study in a prospective designed model is warranted—if such a risk of new sensitization and “overshooting” reaction exists—as such findings could have an impact on future long-term expeditions or space missions  when an allergic sensitization during mission could have potentially more serious consequences when maximal medical treatment is not guaranteed. That the here presented results are not random display other observations made in extreme conditions of living environments such as on the International Space Station published by NASA from one of the authors’ team (B. Crucian) ,20,21. T


Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with emerging multiresistant isolates causing a significant burden to public health systems. We identified 2-sulfonylpyrimidines as a new class of potent inhibitors against S. aureus sortase A acting by covalent modification of the active site cysteine 184. Series of derivatives were synthesized to derive structure-activity relationship (SAR) with the most potent compounds displaying low micromolar KI values. Studies on the inhibition selectivity of homologous cysteine proteases showed that 2-sulfonylpyrimidines reacted efficiently with protonated cysteine residues as found in sortase A, though surprisingly, no reaction occurred with the more nucleophilic cysteine residue from imidazolinium-thiolate dyads of cathepsin-like proteases. By means of enzymatic and chemical kinetics as well as quantum chemical calculations, it could be rationalized that the SNAr reaction between protonated cysteine residues and 2-sulfonylpyrimidines proceeds in a concerted fashion, and the mechanism involves a ternary transition state with a conjugated base. Molecular docking and enzyme inhibition at variable pH values allowed us to hypothesize that in sortase A this base is represented by the catalytic histidine 120, which could be substantiated by QM model calculation with 4-methylimidazole as histidine analog. S. aureus ΔSrtA knock-out mutant is attenuated in mouse infection models compared to the wild type  are cleaved between threonine and glycine by the membrane-anchored SrtA and subsequently ligated to the pentaglycine tail of the peptidoglycan layer to yield covalent attachment of these surface proteins . The face agents .S. aureus SrtA is an eight-stranded β-barrel protein with three conserved catalytic residues: His120, Cys184, and Arg197, each of which cannot be mutated without disrupting enzymatic functionality  and is therefore significantly less nucleophilic than structurally related cysteine proteases such as enzymes of the papain family (184 is inefficient with cysteine protease-specific warheads such as Michael acceptors and similar electrophiles (1) The catalytic Cysn family . Hence, rophiles .M value of 5.5 mM (2) The active site is predominantly defined by the intrinsic flexibility of the β6/7- and β7/8-loops . Bindingf 5.5 mM .in cellulo. Hence, most active compounds were found to be irreversible covalent inhibitors containing an electrophilic warhead that reacts with the active-site Cys184 of SrtA, such as disulfides, benzisothiazolinones, thiadiazolidine-3,5-diones, and 1,3,4-thiadiazoles (3) To compensate for the low activity and the high KM value, both the LPXTG- and the pentaglycine-substrate are co-localized with the SrtA enzyme at the bacterial outer membrane . Spatialdiazoles .However, the privileged position of SrtA is protected from pharmacological manipulation by several structural and biochemical properties of this enzyme. ionality . Three S4  is lower by a factor of 10–1,000 and thus 2-sulfonylpyrimidines may have the potential to be mild enough to achieve favorable target vs. off-target selectivity  to form the respective pyrimidyl thioether and release the corresponding sulfinic acid (1–R3) increase the polarization of the electrophilic C-2 atom and generally lead to higher reaction rates  were coupled to various commercially available or synthesized amines (1a–o) in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) to provide the inhibitor scaffold precursors (3a–z). The potential sulfonylpyrimidine inhibitors  were subsequently synthesized by oxidation with potassium peroxymonosulfate  showed slightly higher overall inhibition potency  than parent compound 4 (5c) showed no SrtA inhibition even at a final inhibitor concentration of 300 µM. From the inhibition results, we concluded that the binding geometry of the previously optimized disulfanylbenzamide recognition sequence .The chimeric 3-phenoxyphenyl glycine derivative . Subsequently, molecular docking to the binding pocket of SrtA was carried out using the Glide docking algorithm .As a second attempt, we tried an approach : Using vlgorithm . Potenti4) for the 3-phenoxyaniline fragment (5a) led to a significant improvement of the overall inhibition potency by a factor of 10 . The docking pose of 5a with putative interactions in the SrtA binding pocket can be seen in 120, Cys184, Arg197) and is thus reasonably positioned for a nucleophilic attack of the Cys184 thiol to the pyrimidine ring. The 3-phenoxyaniline moiety fits well to the L-shaped binding pocket of SrtA and shows interactions with the hydrophobic residues  in the S3/S4 substrate binding pockets.The exchange of the 2-benzylaniline fragment (kobs) in the presence of inhibitor 5a varied hyperbolically with the concentration of the inhibitor. A limiting value was approached asymptotically at higher inhibitor concentrations indicating two-step mechanism inactivation kinetics.Exemplarily, the substrate conversion plot with time-dependent inhibition is shown in 5d–k) were also assayed using the fluorometric enzyme assay at an inhibitor concentration of 100 µM but showed no measurable inhibition of SrtA  to 3-phenoxyaniline derivate (5a) resulted in a tenfold increase of the inhibitory potency , hence, we hypothesized that the inhibitor’s recognition sequence can be further systematically optimized. Therefore, we conducted a SAR study around virtual screening hit 5a. The inhibition results of the synthesized compounds 6a–n are shown in The subtle structural transformation from 2-benzylaniline derivate (6a–f) did inhibit SrtA in the fluorometric enzyme assay, but with varying potency. Strikingly, we observed a drop in SrtA inhibition for most modifications on the 3-phenoxyaniline motif. These structural “activity cliffs” highlight the importance of the accurate positioning of the warhead towards the catalytic Cys184 for a successful covalent reaction  or the methylene derivate (6d). However, the exchange to a ketone connecting unit (6a) resulted in a significant increase of inhibition potency which might be due to beneficial interactions with Arg197. Based on the k2nd value, compound 6a  appeared to be 2.5-fold more potent than virtual screening hit 5a. Alteration of the meta-substitution pattern resulted in the complete abolition of inhibition for the ortho (6g) and para (6h) derivatives. After truncation of the connecting group to the biphenyl derivative (6f), only minor inhibition potency remained (k2nd = 588 M−1min−1). On the other hand, with connector elongation to the benzyl ether (6k) or an amide derivate (6m), the inhibition was completely abolished.Only small changes in inhibition potency were observed upon changes of the aryl ether connecting group of compound 6c, k2nd = 6,593 M−1min−1) but resulted in a severe loss of inhibition when downsized to a methoxy group . Other transformations such as amide inversion at the sulfonylpyrimidine ring (6i), rigidization to a benzofuran derivative (6l), or decyclization of the central phenyl ring (6n) resulted in complete loss of inhibition. For derivatives 6e,f, two-step kinetics could not be confirmed because of a lacking kobs saturation at higher inhibitor concentrations. The KI values of these compounds were estimated to be significantly higher than 100 µM. In these cases, the inhibition potency  was determined by Lineweaver-Burk linearization.Modifications of the terminal phenyl ring were well tolerated in the case of an exchange to an aliphatic cyclohexyl ring  > Cl (5a) > H (7c) > CH3 (7d) >> NH2 (7e). The increase in inhibition was mainly caused by an increase in the kinact value and was not due to affinity changes for the target . The electronic effects of the substituents on the pyrimidine ring predominantly influenced the warhead reactivity, which can be quantified and is discussed by the analysis of the Hammett constants . The optimization strategy is summarized in A positive correlation was found between the intensity of the electron-withdrawing properties of the substituents and the inhibition potency: Br (onstants . In summSNAr reactions often occur at the aromatic carbon atom with the substituent representing the best leaving group (7a and cysteine in phosphate buffered solution at pH 7.5. The reaction between compound 7a (100 µM) and cysteine (100 µM) occurred quantitatively in less than 10 min to form the 2-pyrimidyl cysteine adduct (m/z = 501.03) as described by the mechanism in 5c, the analogous reaction adduct could be detected by LC/MS . Furthermore, the serine proteases NS2B-NS3 from Zika virus (ZIKV), the homologous protease from Dengue virus (DENV), and the human protease urokinase plasminogen activator (uPA) were tested for their susceptibility. The results of the inhibition data are summarized in The sulfonylpyrimidines studied in this work were characterized as mild covalent inactivators of the SrtA enzyme  and exhi6g. A time-dependent inhibition, as with SrtA, could not be observed over the measurement duration of 30 min, which leads to the conclusion that sulfonylpyrimidines probably do not react covalently with cathepsin-related proteases.Cathepsin B/L, rhodesain, and SrtA are all structurally related to papain-like proteases, thus, we used their relatedness to study the selectivity of our inhibitors . The selA significant difference between cathepsin B/L, rhodesain, and SrtA is that in contrast to SrtA, the catalytically active cysteine is present in thiolate form in the first three proteases . This ob6g, however, showed 67% inhibition, but this inhibition was observed to be not time-dependent and thus presumably due to reversible binding.The ZIKV NS2B/NS3 protease is a serine protease and contains two non-catalytic cysteine residues, thus as expected, only <30% inhibition at 100 µM inhibitor concentration was found. The related DENV NS2B/NS3 protease has no cysteine residues in its protein sequence and was inhibited by a maximum of only 15% . The uro184 is responsible for the reaction with sulfonylpyrimidines, enzyme assays were performed at different pH values and in different enzyme buffers. It is known that the catalytical Cys184 of SrtA is predominantly (99.94%) in the protonated thiol form under physiological conditions at pH 7.5  of the DMSO control and the inhibition potency k2nd (M−1min−1) of compound 5a were determined at each pH value (To investigate which thiol protomer (Cys-SH or Cys-S¯) of SrtA Cyst pH 7.5 . The pKaitration . Thus, iitration . Enzyme pH value .kcat) at pH values above pH 7.5 is in agreement with the literature . Hence, the inhibitory potency at pH 7.5 did not differ significantly between all three investigated buffers indicating no involvement of a buffer molecule for the reaction in an enzymatic context.The observed activity increase of SrtA . However, the enzymatic activity (kcat) did not decrease as much with this pH change, which is in agreement with the literature reports  sulfonylpyrimidines are added at a much higher concentration than cysteine (5 µM) and the probe (5 µM), the kobs values depend substantially on the kchem value. The compound-specific second-order inactivation parameter kchem can be determined by linear regression from the kobs values of the dilution series . Our determined kchem for the reaction of the identical compound with cysteine is 63 M−1min−1 and thus, as expected, in the same order of magnitude. The determined kchem values ranged from 6 to 104 M−1min−1, i e., they are 10–10,000 times lower than for the reactions of the reported Michael acceptors 1-penten-3-one, methyl propiolate, and methyl acrylate with GSH (The observed with GSH . This cowith GSH .kchem (cysteine assay) and k2nd (SrtA inhibition), selectivity indices (S.I.) were calculated that compare how efficient the inactivation of SrtA was in comparison with the in-solution reactivity towards free cysteine (6a and 7a were superior to 3-phenoxyaniline derivatives 5a and 7b in terms of the S.I. by about a factor of two. This is in agreement with findings from the enzymatic context that the recognition sequence mainly influences the affinity of binding (KI), with the halogen substituent influencing the reaction rate .From the kinetic constants cysteine . It was 5a or 5c in water/DMSO mixtures. Likewise, no reactivity was detected between 5c and 2-phenylethanethiol in chloroform. These observations are in agreement with the literature. In organic synthesis, such reactions required a base and elevated temperature  was observed in phosphate-buffered solution during the preparation of ESI-MS experiments  also in bicarbonate buffered solution at an identical pH 7.5. The reaction rate in bicarbonate buffer appeared to be significantly reduced (kchem = 21.9 ± 1.27 M−1min−1). Mechanistic participation of the buffer molecule is discussed during the quantum chemical investigations  and Hammett’s substituent constants (σ), which allow qualitative mechanistic conclusions to be drawn  was investigated  allowed us to generate Hammett plots for the reaction of sulfonylpyrimidines with SrtA  aqueous solution was selected as a model system. The relative orientation of the reactants together with the main reaction coordinate is depicted in thiol–C-2inhibitor)). To obtain single points of the corresponding reaction paths this distance was frozen while all other degrees of freedom were optimized. In the course of the calculations, only the R (Sthiol–C-2inhibitor) distance was frozen but not the relative orientation of the two fragments to each other. Hence, the corresponding reaction path represents the minimum energy path (MEP) of the reaction. More details about the calculations are described in the experimental section.To gain insights into the mechanism of 2-sulfonylpyrimidine inhibitors in an enzymatic environment, QM/MM calculations would be desirable. However, the available X-ray structures of 5c with methanethiol as well as methanethiolate were investigated. As shown in 5c with a neutral thiol is not expected to take place. This is in line with the experimental findings that a reaction is not observed in organic solvents or neutral water/DMSO mixtures . The proton transfer to the buffer occurs at R (Sthiol–C-2inhibitor) = 2.3 Å. This point represents the top of the barrier. For the thiol, the top of the barrier is at about R (Sthiol–C-2inhibitor) = 2.0 Å. Neutral buffer molecules of H2CO3 are not able to bind the additional proton, hence no catalysis would be expected. Indeed, our computation predicted a reaction barrier of 124 kJ/mol, which lies only 20 kJ/mol below the barrier computed for unbuffered thiol (142 kJ/mol). CO32– as buffer molecule facilitates a fast reaction with a barrier height of merely 24 kJ/mol. For this species, the proton transfer occurs earlier in the reaction at R (Sthiol–C-2inhibitor) = 2.5 Å. For the reaction catalyzed by HCO3¯ structures resembling the Meisenheimer complex are found to be about 50 kJ/mol higher in energy than the fragments, showing that no Meisenheimer complex is formed. These predictions that Meisenheimer complexes are energetically unfavorable supports the conclusion drawn by Hammett’s analysis  = 7.8 Å. A similar result is found for the docking pose of inhibitor 5a in the zwitterionic cathepsin-like protease rhodesain , a reaction was observed.In summary, our investigations show that 2-sulfonylpyrimidines act as irreversible inhibitors for 3¯, CO32¯, act as base-catalysts. The barriers drop because these bases induce a proton transfer from the thiol, thus increasing the nucleophilicity of the attacking agent. As expected, the decrease of the barrier heights correlates with the basicity of the catalyst. Additional computations show that imidazole can also act as a catalyst. It reduces the reaction barrier from 142 kJ/mol to about 64 kJ/mol.While the enzyme inactivation measurements indicate that 2-sulfonylpyrimidines react preferentially with protonated thiols, the investigations of in-solution cysteine reactivity indicate that this reaction is hampered. To get a better understanding of these conflicting data, we performed quantum chemical model calculations in water/DMSO, bicarbonate, and phosphate buffers. Our computations predict that thiolates react with 2-sulfonylpyrimidines without any barrier. This is not the case for a protonated thiol for which a very high barrier of 142 kJ/mol is computed. The barrier is considerably lowered for bicarbonate or phosphate buffer solutions because the conjugated bases, e.g., HCO+ dyad in their reactive center are less affected. This contradiction is resolved by docking studies, which predict that 2-sulfonylpyrimidines only enter the active sites of enzymes with neutral Cys/His dyads. If the dyad of SrtA is artificially switched to a Cys¯/His+ dyad, the 2-sulfonylpyrimidine does not fit the active site. For rhodesain, which possesses a zwitterionic Cys¯/His+ dyad, the same results are found. Intermolecular interactions prevent the formation of an enzyme-inhibitor complex whose geometrical arrangements allow the reaction. As a catalyst for the reaction of 2-sulfonylpyrimidines with SrtA, the His120 moiety can act as a base catalyst. In line with the Hammett plots, our computations indicate that Meisenheimer complexes are energetically unfavorable for the energy surface of the SNAr substitution reaction.The computations predict that a deprotonated cysteine should react considerably more efficiently with 2-sulfonylpyrimidines than protonated cysteine moieties. In contrast, our enzyme inhibition measurements only show an efficient inhibition of SrtA with neutral Cys/His dyads while enzymes possessing a zwitterionic Cys¯/HisSynthesis protocols of all compounds with their analytical data and the spectral appendix are provided in the supporting information.S. aureus SrtA was performed as described previously (E. coli strain BL21 Gold (DE3) cells  and grown in LB medium with 100 µM ampicillin at 37 °C to an OD600 of ∼0.7. Expression was then induced with 1 mM isopropyl-d-thiogalactoside (IPTG) for 16 h at 20 °C. Harvested cells were resuspended in lysis buffer  and lysed by multiple cycles of sonication . After centrifugation (45 min at 15 krpm), the cleared lysate was subjected to IMAC  to isolate crude SrtA. For further purification, SrtA containing fractions were subsequently subjected to a gel-filtration step  and eluted in the storage buffer . For storage at –80°C, the pure SrtA was concentrated, aliquoted and shock frozen in liquid nitrogen. Throughout all steps, protein concentrations were determined via absorbance at 280 nm and sample purity was assessed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Expression of the eviously . The pETS. aureus SrtA transpeptidation reactions were performed as described previously  with 25 µM of the FRET-pair substrate Abz-LPETG-Dap (Dnp)-OH  and 0.5 mM tetraglycine . Inhibitors were added from DMSO stocks. Reactions were initiated by addition of SrtA and monitored for 30 min at 30°C in an Infinite M200 Pro plate reader with λex 320 nm/λem 430 nm . Three technical replicates were carried out for each inhibitor in black flat-bottom 96-well plates . The enzyme kinetics of irreversible SrtA inactivation were analyzed as described previously  was added and incubated for 10 min at room temperature. The negative labeling control was performed by mock treatment with DMSO. Samples were analyzed by LC/MS using an Agilent 1100 series HPLC system with an Agilent Poroshell 120 EC-C18 column  and electron spray ionization with the Agilent 1100 series LC/MSD Trap in positive ionization mode.To a solution of 100 µM cysteine in phosphate-buffered saline , compound S. aureus SrtA stock solution was diluted in 100 μL assay buffer  to a final concentration of 10 µM. Compound 7a was dissolved in DMSO and added to SrtA at a final concentration of 100 μM. The protein samples were allowed to react for 1 h at room temperature and subsequently, these were desalted using ZebaTM Spin desalting columns  according to the manufacturer’s instructions. For MALDI sample preparation, the desalted solution was mixed 1:1 with a matrix combination of equal amounts α-cyano-4-hydroxycinnamic acid (20 mg/ml dissolved in 70% acetonitrile and 30% of formic acid in water at a concentration of 5%) and 2,5-dihydroxybenzoic acid (20 mg/ml dissolved in 70% acetonitrile and 30% of trifluoroacetic acid in water at a concentration of 0.1%). The resulting mixture was allowed to dry slowly on the MALDI target prior to introduction into the mass spectrometer. The measurements were carried out on a rapiflexTM MALDI-TOF/TOF mass spectrometer . The instrument is equipped with a scanning smartbeam 10 kHz Nd:YAG laser at a wavelength of 355 nm and a 10 bit 5 GHz digitizer. The acceleration voltage was set to 20 kV and the mass spectra were recorded in positive ion linear mode. Calibration was done with the Bruker protein calibration standard II in a mass range from 10 to 70 kDa. Samples were measured at a laser power of 60% with random walk ionization across the sample spot.An 3, 100 mM NaCl, pH 7.5). Three technical replicates were carried out for each sulfonylpyrimidine in black flat-bottom 96-well plates. For this purpose, 50 µL buffered solution (including 5% (v/v) DMSO) of the respective sulfonylpyrimidine derivative  and the cysteine reactive probe 8  were premixed. Reactions were initiated by the addition of 50 µL cysteine , dissolved in the respective buffer, and monitored for 30 min at 30°C in an Infinite M200 Pro plate reader with λex 380 nm/λem 460 nm. To calculate kobs and kchem values, fluorescence curves were fitted using the same non-linear regression as for in vitro inhibition of SrtA  or individual cases in bicarbonate buffer (20 mM NaHCO of SrtA .http://www.chemaxon.com)  or Cys25 (rhodesain) were untethered and reprotonated. Receptor preparation was performed using the automated binding site, protonation, and energy minimization routine within Maestro “Protein preparation” and “Receptor grid generation.” Ligands were energy minimized using the “LigPrep” routine. The docking protocol was performed under default parameters with extra precision (XP) settings. Subsequently, predicted binding modes were analyzed to select the most promising candidate compounds for wet-lab synthesis. Ranking of the virtual screening output was performed according to the total docking score and in a second step according to the predicted binding mode .JChem for Office (Excel) was used for the virtual synthesis of an in-house docking library. JChem for Office 20.18.0, 2020, ChemAxon (xon.com) . For thiorksuite . The prothiolate-C-2inhibitor) = 1.8/1.9 Å the restraint was removed to ensure the correct product geometry. Reactants and products were confirmed by calculations without restraints and frequency calculations.All calculations were carried out with the Gaussian16 program . The DFT


PTSD symptom severity was assessed with the Clinician-Administered PTSD Scale, before undergoing nine sessions of TF-CBT. They were then reassessed post-treatment to estimate reduction in fear and dysphoric symptoms of PTSD. Although neural responses during the inhibitory task did not predict overall symptom change, reduced activation in the left precuneus and the right superior parietal cortex predicted greater improvement in dysphoric symptoms. ERP responses during response inhibition indicated that lower P3 peak latency predicted greater reduction of dysphoric symptoms. There were no significant predictors of changes of fear symptoms. These findings indicate that neural activity associated with response inhibition can act as a predictive biomarker of TF-CBT response for PTSD symptoms. This pattern of findings underscores the importance of delineating the role of biomarkers to predict remission of subtypes of PTSD.Although trauma-focused cognitive behavioural therapy (TF-CBT) is the frontline treatment for posttraumatic stress disorder (PTSD), up to one half of patients do not respond optimally to this treatment. Inhibitory functions are important for successful management of PTSD, yet there is a dearth of knowledge regarding the extent to which neural mechanisms unpinning response inhibition are associated with TF-CBT response. Treatment-seeking PTSD patients ( This situation has led to many attempts to understand predictors of treatment response, including neural markers that can predict who will respond to TF-CBT. The majority of these studies have focused on functional magnetic resonance imaging (fMRI) during emotional processing or regulation tasks6. Other studies have employed resting state MRI to identify neural profiles characteristic of TF-CBT responders7. These studies have led to variable findings that have not provided consistent regions that predict TF-CBT outcome; nonetheless, one review indicates that there is convergence that better treatment response is associated with increased pre-treatment dorsal anterior cingulate activation and decreased amygdala and insula activation8.Trauma-focused cognitive behavior therapy (TF-CBT) is the primary frontline treatment for posttraumatic stress disorder (PTSD). Despite the demonstrated success of this treatment for many patients, 30–50% of those with PTSD do not respond to this therapy9. There is convergent evidence that PTSD is associated with impaired executive functions10, and this includes deficits in inhibitory functions14. Deficits in inhibitory function are associated with severity of PTSD symptoms15, and may be particularly associated with level of re-experiencing symptoms16. Consistent with these findings, people with PTSD display less activation of the prefrontal cortex during inhibition tasks relative to controls19. There is evidence that the deficit in inhibitory functioning in PTSD may be associated with deficits in fear inhibition in that fear-potentiated startle and extinction to a conditioned stimulus are associated with reduced ventromedial PFC activation during a Go/No-Go paradigm19. The observation that fear inhibition may involve generic capacity of inhibitory functions in PTSD raises the possibility that propensity to activate inhibitory neural networks may be influential in how PTSD patients respond to TF-CBT. Several studies have investigated how response inhibition in PTSD patients may be associated with TF-CBT response. One small pilot study showed that reduced activation of frontal and left dorsal striatal networks during a Go/No-Go task predicted poorer response to TF-CBT20. Another study employed a response inhibition task that allowed delineation of contextual processing and inhibition, and found that treatment responders had increased activation of the inferior parietal lobe during contextual processing that non-responders21.Inherent in most theories of TF-CBT is that successful treatment response involves the capacity to recruit inhibitory functions because one needs to manage trauma memories and strong negative emotional states22 and a shorter N2 latency23 during inhibition trials. One study also found that people with PTSD had larger P3 amplitude on both Go and No/Go trials24. Whereas the N2 is a negative component elicited approximately 200 ms after a Go/No-Go stimulus , the P3 is a positive component elicited approximately 300 ms after the stimulus . Despite the evidence regarding distinct ERP patterns in PTSD patients during Go/No-Go tasks, this important temporal index of response inhibition has not been investigated in relation to predicting response to TF-CBT.Reflecting temporal patterns during response inhibition, PTSD patients display aberrant evoked response potentials (ERPs) during Go/No-Go tasks. PTSD has been associated with longer latency of the P3 componentTo date there have been no studies that have investigated the relationship between TF-CBT response and comprehensive assessment of inhibitory functions, including both fMRI and ERP measures. Using both indices is important because it allows assessment of both spatial and temporal measures of neural mechanisms of response inhibition. To this end, this study administered treatment-seeking PTSD patients  a Go/No-Go task during separate fMRI and ERP recordings. PTSD patients then underwent a course of TF-CBT. On the basis that TF-CBT response involves intact inhibitory functions, we hypothesized that better treatment response would be predicted by stronger activation of prefrontal cortical networks and shorter P3 latencies during the Go/No-Go task.27, with evidence of two latent factors that comprise fear  and dysphoric  symptoms of PTSD29. These latent symptom constructs potentially represent a neurobiologically meaningful approach to understand capacity to predict symptom remission because evidence indicates that depression may contribute distinctly to executive function deficits relative to PTSD31. Moreover, there is evidence of different neural profiles in PTSD patients with and without comborbid depression36, which may be attributed in part to the greater reward deficits in depression36. Accordingly, it may be an over-simplification to relate neural process to a global reduction of PTSD symptoms, and it may be more meaningful to consider how response inhibition may predict treatment response on these two dimensions. On the basis of evidence that both depression and PTSD are associated with greater parietal network recruitment during response inhibition37, we predict that reduced parietal activation may be associated with greater reduction of dysphoric symptoms, but not necessarily of fear symptoms.We were also interested in how neural responses could predict remission of different subtypes of PTSD. Increasing evidence points to the heterogeneity of PTSD38) to diagnose PTSD. The ‘2/1’ method was used to detect symptoms, indicating that a given symptom was experienced at least twice a month and caused moderate levels of distress. Participants were excluded if they reported substance dependence, bipolar disorder, psychosis, neurological disorders or moderate to severe brain injury . The Depression, Anxiety, and Stress Scale (DASS40) was used to measure levels of depression and anxiety in all patients. Table Forty treatment-seeking PTSD patients  participated in this study. Participants had experienced assault, childhood abuse, motor vehicle accidents, or police-related trauma leading to PTSD. Masters or doctoral-level clinical psychologists diagnosed DSM-IV PTSD used the Clinician-Administered PTSD Scale , generalized anxiety disorder, social phobia, panic disorder, agoraphobia, obsessive compulsive disorder, and substance use disorder. Participants underwent clinical and lab (EEG and MRI) assessments at baseline, underwent a 9-week TF-CBT treatment course, and were assessed for PTSD severity following treatment.The Go/No-Go task assesses response inhibition. Participants were instructed to respond to ‘Go’ trials by pressing a button as quickly as possible and withhold a response on ‘No-Go’ trials. The ‘Go’ stimuli was the word “PRESS” in green writing, and the ‘No-Go’ stimuli was the word “PRESS” in red writing. Each stimulus was presented for 500 ms, with a 750 ms interstimulus interval. In total, 180 Go stimuli and 60 No-Go stimuli were presented in a pseudorandom order to ensure that the No-Go stimulus did not occur more than three times in a row. Because the Go/No-Go task requires an all-or-nothing decision to either act or refrain from acting, it reflects participants’ abilities in inhibitory control. The Go/No-Go task was repeated three times in the session for each participant. We measured performance on the test, including commission errors (failing to withhold a response), omission errors (failing to correctly respond), and reaction time. The first task was conducted without neural recordings. The task was then repeated while continuous electroencephalogram (EEG) data was being recorded and subsequently during magnetic resonance imaging machine.41. The TF-CBT comprised an initial session of psychoeducation about psychological responses to trauma, followed by six sessions of 40-min imaginal exposure to the trauma memory, implementation of in vivo exposure to avoided situations, and cognitive reframing of maladaptive thoughts related to the traumatic event. Two further sessions reinforced cognitive restructuring exercises, and a final session focused on relapse prevention42. This trial was prospectively registered at Australian and New Zealand Clinical Trials Registry, ACTRN12612000185864.Approximately 2 weeks after completing the Go/No-Go testing, participants began a 9-week TF-CBT treatment administered by experienced doctoral-level or masters-level clinical psychologist. Sessions occurred weekly and were 60–90 min in length. The therapy is consistent with prescribed TF-CBT protocols29.The change in PTSD symptom severity across treatment was calculated by subtracting post-treatment CAPS scores from pre-treatment scores. This score then was divided by the pre-treatment CAPS score, to produce a CAPS-change score independent of initial symptom severity. This change in PTSD severity score was used for correlation with task measures. To examine change in PTSD severity, while considering the heterogeneity of the disorder, change in CAPS factor scores (Fear factor scores and Dysphoria factor scores) were also calculated in the same way as the overall CAPS-change scores, and were correlated with neural measures. Fear factor was defined as total score of re-experiencing, active avoidance, hypervigilance, and elevated startle symptoms and dysphoric factor as a total score of passive avoidance, sleep disturbance, concentration difficulties, and irritability symptomsBaseline clinical data , and behavioral Go/No-Go data  were tested for association with PTSD symptom improvement using Pearson correlations. Regressions analyses were conducted to examine the strength of each clinical or behavioural measure as a predictor of symptom improvement.43.Electrophysiological data was continuously recorded from 32 EEG channels at 500 Hz with a skin resistance of <5 kOhms, using a Quick Cap and NuAmps DC system (Neuroscan). Twenty six cephalic sites, 4 electro-oculogram (EOG) sites, an orbicularis oculus site, and a masseter site comprised the 32 channels. To record horizontal eye movement, electrodes were placed 1.5 cm lateral to the outer canthus of each eye. To record vertical eye movement, electrodes were placed 3 mm above the left eyebrow and 1.5 cm below the left lower eyelid. The threshold for artefact rejection was set at 100 µV. Each event-related potential epoch was filtered using a low-pass Tukey filter. No-Go trials were averaged together to form ERP waves, which were then hand-scored to determine the peak-value and latency of the N2 and P3 peaks. The peak-value and latency of the N2 wave (defined as 180–220 ms) was examined for the Fz, FCz, and Cz electrodes. In addition to these, the Pz electrode was also included for analysis of the P3 wave (defined as 250–550 ms). These waveforms and electrodes were selected based on previous studies in response inhibitionTo examine whether pre-treatment EEG data was associated with a reduction in PTSD symptoms, we conducted repeated-measures ANOVAs using the set of electrodes as a within-subjects variable and CAPS-change values as a between-subjects covariate. A 3 × 1 repeated-measures ANOVA was conducted on both the N2-amplitude and N2-latency data. Similar analyses were conducted on the P3-amplitude and P3-latency data using a 4 × 1 repeated-measures ANOVA. Further posthoc correlations between individual electrodes and CAPS-change values were conducted on significant electrode sets.2 and the matrix size was 64 × 64. We also acquired a T1-weighted anatomical image with 1 mm3 isotropic voxel resolution to normalise the fMRI data to standard space. This was produced using a 3D spoiled gradient echo sequence in the sagittal plane with the following parameters: TR = 8.3 ms, TE = 3.2 ms, flip angle=11°, TI = 500 ms, NEX = 1, ASSET = 1.5, S/I frequency direction, 256 × 256 matrix size, and 180 contiguous 1 mm slices.Functional MRIs were conducted on a 3.0 T GE Signa HDx scanner and an eight-channel head coil, using an echo planar imaging protocol. We collected 120 T2*-weighted functional volumes in each task run, with three dummy scans collected prior to the sequence to ensure magnetisation had stabilised to a steady state. Each volume consisted of 40 axial slices parallel to the intercommissural line, with 3.5 mm thickness, 2.5 s TR, 27.5 TE, and 90° flip angle. The field of view was 24 × 24 cmStatistical Parametric Mapping  software was used to realign, normalise (into standardised MNI space), and smooth neuroimaging data. fMRI images were realigned and unwarped to the initial image for the task run to correct for participant motion within the scanner. To normalise data into stereotactic MNI space, we used the FMRIB linear registration tool to co-register the functional data to the T1 anatomical scan, as well as using the FMRIB nonlinear registration tool to normalise the weighted 3D spoiled gradient echo sequence. A mask covering the ventricles and white matter was used to estimate their corresponding signal and correct for any physiological noise. Smoothing was conducted on all fMRI data using an 8 mm Gaussian kernel. For first-level analysis, the BOLD response was estimated by convolving the hemodynamic response with a boxcar function in a general linear model framework. Contrast images for response inhibition were determined by comparing the No-Go versus the implicit baseline.45. These regions of interest (ROIs) were defined using 10 mm radius spheres combined into a single network specific mask. The regions of the DMN consisted of the medial prefrontal cortex (mPFC), the posterior cingulate cortex (PCC) and precuneus. The regions tested as part of the CCN were the dorsolateral prefrontal cortex (DLPFC), dorsal anterior cingulate cortex (dACC), inferior parietal, and superior parietal cortices. We also conducted exploratory analyses at the whole-brain level. All analyses were conducted voxel-wise and judged significant at a family-wise-error-corrected peak p-value of 0.05.We examined brain areas making up the default mode network (DMN) and the cognitive control network (CCN) based on previous meta-analyses and studies, which have outlined the role of these networks in response inhibition processest-test on their respective contrast images. Next, the change in PTSD symptom severity score was regressed (voxel-wise) onto the pre-treatment fMRI signal to determine whether neuroimaging data was associated with reduction in PTSD symptoms; we also repeated analysis with reduction in fear and dysphoria symptoms. Finally, we conducted generalised psychophysiological interaction (gPPI) analyses46 to determine whether functional connectivity across DMN and CCN brain regions were associated with reductions in PTSD symptoms. Significant clusters from activation analyses were used as seeds to evaluate connectivity to the rest of the network regions. As done for the activation analyses, a family-wise error (FWE) corrected p level of 0.05 was used for statistical inferences.We first evaluated group differences between the PTSD and healthy control groups using a voxel-wise two-sample We conducted a few additional analyses. First, we evaluated group comparisons between the PTSD and healthy control on the neurocognitive, EEG and fMRI measures, particularly to test if the identified prognostic measures also characterize PTSD diagnosis. Next, we performed analyses to build predictive models of treatment outcome using all data. For this using hierarchical stepwise linear regression models, we evaluated the additive predictive utility of each of our measures. Specifically, our goal was to determine whether the neural data (EEG and MRI) provided additional useful predictive information above that of the clinical and cognitive behavioral measures. For this we created a regression model in which the clinical and cognitive data were entered into a first block, and the neural measures (i.e. the significant EEG measures and fMRI clusters) were entered using a stepwise method in the second and third blocks, respectively. As an exploratory analysis to find the best overall predictive model, we used all the measures which were significantly correlated with change in CAPS  using a stepwise regression and cross-validation analyses. For this analysis, we used a binary response variable based on 50% decrease in symptoms (i.e. responders and non-responders) to quantify treatment outcome. We used this analysis only to identify the most predictive features from the identified significant measures. We also ran cross-validation analyses on both these analyses to test generalizability of these models beyond our tested cohort. This linear cross-validation was conducted using bootstrapping in R. The data were randomly split into equal-sized training and test datasets (20 participants in each dataset). The training dataset was bootstrapped 200 times, with each bootstrap fitted with a linear regression. The averaged coefficients obtained from the bootstrapped training data were then applied to the test data to determine the generalizability of the model. To test whether our results were affected by SSRI use within the patient group, we repeated all analyses with a smaller group excluding participants who were currently taking SSRI medication.r = −.409, p = .013), as well as change in dysphoria subscale scores  and fear subscale scores , with lower pre-treatment levels of anxiety predicting better treatment response.All participants met the DSM-IV PTSD criteria and completed 7–9 sessions of TF-CBT. Mean CAPS scores prior to treatment was 71.8 ± 17.6, which reduced to 28.13 ± 20.0 at the post-treatment assessment. Twenty-seven (67.5%) participants were classified as treatment responders (50% or greater reduction in PTSD symptoms) and 13 (32.5%) were non-responders. Table Behavioral data for the Go/No-Go task are detailed in the Supplementary Results Table . These mF = 4.43, p = 0.045; shorter P3 latency associated with better improvement). Posthoc correlation analyses found that the P3 peak latency of the Fz electrode significantly correlated with CAPS Dysphoria change  . However, when evaluated in the unmedicated PTSD cohort, this finding was significant at the Bonferroni corrected threshold  Fig. . This coAs observed with the EEG data, fMRI neural activation for the contrast reflecting response inhibition (No-Go vs Baseline) was significantly correlated with only change in dysphoria symptoms. Lower pre-treatment activation in the left precuneus of the DMN and right parietal cortex of the CCN were significantly associated with better improvement of dysphoric symptoms , we performed a backward stepwise regression analysis using all the significant EEG and fMRI measures described above. The EEG indices associated with symptom change, specifically the latency of the P3 peak on the Fz electrode significantly improved the model relative to only using the DASS anxiety scores. Similarly, the fMRI activation in the right parietal cortex further improved the EEG-clinical model  shorter P3 peak latency, and (b) lower activation in the left precuneus and right parietal cortex when participants inhibited response to No-Go stimuli. Further, the inclusion of these fMRI and ERP variables significantly improved prediction relative to clinical data alone; this model provided a cross-validated accuracy of 73% in predicting treatment response.47. Inhibitory performance has been shown to be deficient in PTSD14 and depression48, which is associated with longer latency of the P3 component22; these findings suggest that individuals with dysphoric symptoms of PTSD are less efficient in engaging inhibitory processes. The observation of shorter P3 latency predicting symptom remission suggests that those PTSD patients who have greater efficiency in inhibitory functions prior to treatment may be better able to utilize the strategies of TF-CBT and thereby achieve greater symptom remission.The finding that better treatment response was associated with shorter latency of the P3 component during inhibition needs to be understood in the context that this component represents a later phase of the inhibition process, possibly involving decision response49. PTSD individuals demonstrate a reduced recruitment of this network during response inhibition17. Also, greater PTSD severity is found associated with greater parietal activation during this task17. There is also evidence of engagement of parietal regions during inhibition in patients with depressive states37. On the premise that the dysphoric phenotype of PTSD overlaps with depressive symptoms, it is possible that patients who do not experience remission of dysphoria symptoms may be deficient in inhibition processes and therefore engage inhibitory networks to a greater extent to compensate for this deficit. This interpretation accords with the finding of slower P3 predicting non-remission in more dysphoric patients because this pattern may reflect less efficient inhibitory processes.Greater remission of dysphoria symptoms was associated with less activation in the parietal cortex and precuneus during response inhibition. Many studies have shown that a fronto-parietal network is strongly involved in response inhibition50. This pattern highlights that the role of inhibition in predicting treatment outcome appears to be specific to the treatment modality, and the current finding indicates that TF-CBT may implicate distinct inhibitory networks relative to antidepressants in predicting reduction of depressive symptoms of PTSD.It is interesting that both fMRI and ERP indices of response inhibition predicted remission of dysphoria, but not fear, symptoms of PTSD. This pattern underscores the heterogeneity of PTSD and points to the need for more nuanced and mechanistic approaches to identifying predictors of TF-CBT response instead of the prevailing focus on PTSD as a unitary construct. In this context it is worth noting that one large treatment study found that greater inferior parietal activation predicted remission of depression symptoms for SSRIs, but reduced activation predicted remission after serotonin and norepinephrine reuptake inhibitors (SNRIs)51, behavioral indicators of inhibition did not predict treatment response in the current study. This accords with other studies that have not reported an association between baseline neuropsychological measures of inhibition and treatment outcome52. Further, a meta-analysis of the relationship between neuropsychological measures of executive function found that response inhibition was not related to antidepressant response53. This pattern underscores the conclusion that biological markers may be predictive of treatment outcome even if behavioral performance is not.Although some models may suggest that response to TF-CBT should be associated with better inhibitory functions prior to treatment23, and the evidence points to no robust finding regarding any one ERP component’s latency or amplitude being distinctive to PTSD during this task. The current observation underscores the need for further study of EEG profiles in PTSD during response inhibition.In contrast to previous findings, the biological markers that predicted treatment outcome were not found to be distinct between PTSD and controls in our study. It is likely that the treatment response markers of specific PTSD symptoms could be different from those that characterize PTSD as an overall diagnosis. Interestingly, there were no EEG differences between PTSD and control participants. It should be noted that the body of evidence regarding distinct ERP patterns in PTSD during response inhibition is limitedWe recognize a number of limitations. First, this study lacked a no-treatment wait-list comparison condition, which would allow delineation between the predictive capacity of response inhibition for TF-CBT relative to spontaneous remission. Second, we only tested the predictive capacity of inhibition at post-treatment, and future studies should attempt to replicate this paradigm with longer-term follow-up assessments. Third, we note the modest sample size and acknowledge that a larger sample would allow for closer examination of PTD subtypes. Fourth, the predictive model yielded only modest accuracy, and so the current findings need to be replicated with larger and independent samples before any robust predictive model could be accepted.54. The current findings extend this goal by indicating that neural markers, such as those involving inhibitory processes, differentially predict remission of dysphoria, and fear symptoms of PTSD. Moreover, it shows that better prediction is achieved using multimodal assessments that integrate temporal and spatial indices of response inhibition. Future studies could usefully build on these findings by using complementary measures to assess different functions prior to treatment, and evaluate their predictive capacities for remission of distinct symptom phenotypes across different treatments. This approach is likely to yield more promising results than limiting the focus on diagnostic constructs.In the context of precision psychiatry, there is currently much attention being given to the need for an evidence base that integrates neural indices that promotes matching of particular patients with specific treatmentsSupplementary Results


Infectious bronchitis virus (IBV) initially establishes the infection in the respiratory tract and then spreads to other tissues depending on its virulence. During 2011–2018, the 4/91 IBV strain was isolated from poultry flocks affected by decreased egg production and quality in Eastern Canada. One of the Canadian 4/91 IBV isolates, IBV/Ck/Can/17-038913, was propagated in embryonated chicken eggs and molecularly characterized using whole genome sequencing. An in vivo study in laying hens was conducted to observe if IBV/Ck/Can/17-038913 isolate affects the egg production and quality. Hens were infected with IBV/Ck/Can/17-038913 isolate during the peak of egg lay, using a standard dose and routes maintaining uninfected controls. Oropharyngeal and cloacal swabs were collected at predetermined time points for the quantification of IBV genome loads. At 6 and 10 days post-infection, hens were euthanized to observe the lesions in various organs and collect blood and tissue samples for the quantification of antibody response and IBV genome loads, respectively. Egg production was not impacted during the first 10 days following infection. No gross lesions were observed in the tissues of the infected birds. The IBV genome was quantified in swabs, trachea, lung, proventriculus, cecal tonsils, kidney, and reproductive tissues. The serum antibody response against IBV was quantified in infected hens. In addition, histological changes, and recruitment of immune cells, such as macrophages and T cell subsets in kidney tissues, were measured. Overall, data show that IBV/Ck/Can/17-038913 isolate is not associated with egg production issues in laying hens infected at the peak of lay, while it demonstrates various tissue tropism, including kidney, where histopathological lesions and immune cell recruitments were evident.  Gammacoronavirus that belongs to the family Coronaviridae and order Nidovirales , wa, waS1 geThe complete genome sequence of the Canadian 4/91 IBV isolate, IBV/Ck/Can/17-038913 and 23 reference IBV sequences mentioned in the previous section were aligned using multiple alignments with fast Fourier transformation (MAFFT)  and usedTwenty-four 25-weeks-old SPF hens (at the peak of egg lay) were purchased from CFIA, Ottawa, and allowed to acclimatize for 19 days. Hens were placed in Animal Resource Center (ARC), Foothill campus, University of Calgary. Feed and water were provided ad libitum. The laying hens were kept under a daily light cycle of 16-h light: 8-h dark.6 EID50 per chicken under isoflurane anesthesia. We used the standard dose of IBV used in adult chickens as was performed previously [At the end of the acclimatization period, 12 hens were infected with IBV/Ck/Can/17-038913 isolate at a dose of 1× 10eviously ,59. A to®UniTranz-RT® Media Transport Systems, VWR, Edmonton, AB, Canada). Tissue samples of trachea, lung, cecal tonsils, kidney, oviduct , and proventriculus were collected individually in RNA Save , and preserved at −20 °C, for virus genome load quantification by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In addition, kidney samples were snap frozen in Optimum Cutting Temperature (OCT) medium  for immunofluorescence studies and fixed in 10% neutral buffered formalin  for examining histological changes. Blood samples were collected into plain tubes on the day of euthanasia (6 and 10 dpi) to monitor the anti-IBV Ab response. The blood samples were kept at room temperature for 30 min, centrifuged at 2000× g for 10 min at 4 °C, the serum was separated, and aliquots were stored at −20 °C.OP and CL swabs were collected at 3, 6 and 10 dpi into tubes containing viral transport medium  according to manufacturer’s guidelines. Total RNA was also extracted from the trachea, lung, kidney, reproductive tract , cecal tonsils, and proventriculus using Trizol reagent  following the manufacturer’s protocol. The amount of the extracted RNA was quantified using Nanodrop 1000 spectrophotometer  at 260 nm wavelength. The quality of the RNA was determined based on the 260/280 absorbance ratio. Approximately 1000 ng from swab samples and 2000 ng of tissue samples were used to synthesize cDNA using RT random primers  according to manufacturer’s guidelines.The tubes containing OP and CL swabs were vortexed and 250 μL of the medium was used to extract the RNA using Trizol LS® Green Master Mix  maintaining the final reaction volume at 20 µL. Each reaction volume consisted of 10 µL of SYBR Green master mix, 100 ng of respective samples, 0.5 µL of forward Fw-5′GACGGAGGACCTGATGGTAA-3′ and reverse Re-5′CCCTTCTTCTGCTGATCCTG-3′ specific primers targeting the conserved IBV N gene [The qRT-PCR assay was carried out using a CFX 96-c1000 Thermocycler  to quantify the IBV genome loads in cDNA samples. The assay was performed using Fast SYBRV N gene . ThermalThe kidney samples fixed in 10% neutral buffered formalin were sent to the Histopathology Diagnostic Services Unit at the University of Calgary, Faculty of Veterinary Medicine for further processing, sectioning, and hematoxylin-eosin (H&E) staining.Anti-IBV Abs in serum samples (1:500 dilution) were quantified using IDEXX ELISA kit  according to manufacturer’s guidelines. This ELISA kit does not differentiate different Ab isotypes. The absorbance values were taken at 650 nm using the SpectraMax M2 microplate reader  and the titers were calculated according to the manufacturer’s instruction. Titers greater than 396 were considered positive for anti-IBV Abs.+ T cells, CD8+ T cells and macrophages. On the day of immunostaining, the sections were air-dried for 20 min (min) at room temperature and fixed with cold acetone for 5 min. The tissues were blocked with 5% goat serum diluted in Trizma buffered saline (TBS) buffer  and the mouse monoclonal Ab specific for chicken macrophages/monocytes  or CD8α  were used in a 1:200 dilution in 5% goat serum. The secondary Ab, goat anti-mouse IgG (H + L) conjugated with Dylight® 550  was used in 1:500 dilution in 5% goat serum. The sections were incubated with primary Ab for 30 min and with secondary Ab for 60 min.OCT preserved frozen tissues were cut into sections of 5 µm thickness, adhered to positively charged slides , and preserved at −20 °C until stained for CD4+ T cells consists of an additional 15 min of blocking step with avidin and biotin  before blocking with 5% goat serum in TBS for 30 min. Each step was followed by 3 min of washing twice with Tris-buffered saline containing 0.1% Tween 20 (TBST) and once with PBS. Following blocking, the sections were incubated with 1:200 dilution of mouse monoclonal Ab specific to chicken CD4+ T cells  in blocking buffer and incubated for 30 min. Next, the tissues were incubated for 30 min with 1:250 dilution of biotinylated goat anti-mouse IgG  and 15:1000 dilution of DyLight® 488 (green fluorescence) streptavidin , respectively. Each incubation step was followed by 5 min of washing with TBST buffer twice and once with PBS. Finally, the slides were mounted with 4′,6-diamidino-2-phenylindole dihydrochloride  and cover slipped.Immunostaining of CD4The number of IBV genome copies in 100 ng of RNA was calculated based on a standard curve obtained by serial dilution of plasmids. For quantification of immune cells, 5 areas (x20 magnification) of each tissue with the highest signals were captured using an epifluorescent microscope  along with applicable nuclear stained (DAPI) areas. The density of the cells in each area was quantified using Image-J software .+ T cells, CD8+ T cells, and macrophages at 6 and 10 dpi were detected using the Mann–Whitney U test. All the statistical analysis was performed in GraphPad Prism Software .The difference in egg production between the infected and control groups was analyzed using a Mann–Whitney U test. The Mann–Whitney U test was also used to compare the anti-IBV Ab responses in serum of the infected group at two time points, 6 and 10 dpi. IBV genome load in OP, CL swabs and tissues were compared using the Kruskal–Wallis test followed by Dunn’s multiple comparison. Differences of CD4


Site-specific conjugation of proteins is currently required to produce homogenous derivatives for medicine applications. Proteins derivatized at specific positions of the polypeptide chain can actually show higher stability, superior pharmacokinetics, and activity in vivo, as compared with conjugates modified at heterogeneous sites. Moreover, they can be better characterized regarding the composition of the derivatization sites as well as the conformational and activity properties. To this aim, several site-specific derivatization approaches have been developed. Among these, enzymes are powerful tools that efficiently allow the generation of homogenous protein–drug conjugates under physiological conditions, thus preserving their native structure and activity. This review will summarize the progress made over the last decade on the use of enzymatic-based methodologies for the production of site-specific labeled immunoconjugates of interest for nuclear medicine. Enzymes used in this field, including microbial transglutaminase, sortase, galactosyltransferase, and lipoic acid ligase, will be overviewed and their recent applications in the radiopharmaceutical field will be described. Since nuclear medicine can benefit greatly from the production of homogenous derivatives, we hope that this review will aid the use of enzymes for the development of better radio-conjugates for diagnostic and therapeutic purposes. Bioconjugation techniques that exploit enzymes are a growing field of protein chemistry due to the interest to produce conjugates of proteins with small molecules or macromolecules for applications in research as well as in the biotechnological and pharmaceutical industries . TraditiProteins have shown unique potential to improve human health both as therapeutics and as drug vectors. As therapeutics, the diversity and biospecificity in protein function impart the ability to treat different pathologies including cancer, autoimmune, metabolic, and infectious diseases . In addiWith this aim, different enzymatic approaches have been developed to produce homogenous immuno-conjugates that maintain the affinity for the antigen ,4,5,6,7.Molecular imaging is one of the most fast-developing areas of research. It aims to visualize, characterize, and quantify, in a non-invasive way, processes on molecular or cellular levels in living systems, giving clinicians important information both in the diagnosis and for monitoring the treatment of diseases . Nuclear2, Fab, scFv) as well as engineered mAb fragments , and small protein scaffolds  . T. T28]. Ter costs ,38. For Microbial TGase is a monomeric protein with a molecular mass of 37.9 kDa and a measured isoelectric point of pH 8.9 . The keyWith the aim of protein derivatization, mTG can modify a protein if it contains reactive Gln or Lys residues. This possibility constitutes an advantage of mTG in respect to other enzymes used for protein conjugation, which generally require the introduction of a specific recognition sequence by recombinant methods . Indeed,Regarding the derivatization of reactive Gln and Lys already present in the sequence of proteins, especially in the case of Gln residues, several cases of site-specific protein modification by mTG have been reported in which the sites of conjugation have been identified  46]. Th. Th46]. In the absence of protein reactivity to mTG, Q- or K-tags can be inserted in the sequence of the protein and several different tags have been reported and recently reviewed ,35. FromFor the purposes of protein conjugation, the substrate used for Gln modification has to contain a primary amine without steric hindrance and separated by a spacer of at least four carbon atoms from the payload , especially if this is negatively charged B 74]. I. I74]. Iv/v) ethanol or 60% (v/v) methanol, human growth hormone is derivatized only at Gln141 level, while in the absence of solvent addition also Gln40 is derivatized  ivatized . When thStaphylococcus aureus, which has been developed as a tool for protein conjugation and semisynthesis 5 peptide it is favored [Tc(N)(PNP)]2+strategy (PNP = water-soluble diphosphino-amine) to the labeling of temperature-sensitive biomolecules such as proteins and derivatives (unpublished data) [99mTc][Tc(N)(PNP)]-synthon (w/w) were used. Notably, apoMb contains two Gln residues (Gln91 and Gln152) that can be derivatized by mTG, thus leading to the generation of a bis-derivative adduct. Upon conjugation (37 °C for 4 h), analyses revealed the formation of a main product identified as the mono-conjugated apoMb modified site specifically at the level of the Gln91 residue, and of only a minimal amount of the bis-derivative one modified at both Gln91 and Gln152 residues as result of the high substrate specificity of mTG (99mTc][Tc≡N]2+int precursor with the wsPNP ligand and the Cys~apoMb adduct (10−5 M) at RT for 30 min. The radiochemical yield of the 99mTc-Cys-apoMb is 95%. The radiolabeled construct is highly stable under all investigated conditions, suggesting a role of the [99mTc][Tc(N)(PNP)]-technology in the labeling of (temperature)-sensitive biomolecules for SPECT imaging.Most recently, we have been investigating the possibility of extending the usage of the . Ra. Ra99mTc89Zr-radiolabeled DFO-mAb conjugates with enhanced homogeneity, revealing unappreciated limits of Q-tag site selectivity at positions likely to directly impair function -fluorodeoxy-D-glucose (FDG) and 64Cu sdAb fragments to explore the possibility of truck immune responses [18F]-FDG-tagged Tz to generate, according to the prelabeling method, the final RIC (RCY = 25%). This technique is further applicable to install a selected functionality (such as NOTA) to any other suitably modified biomolecule (18F) for different imaging modalities, and the second for further modification of the fragment with a PEG moiety or a second antibody fragment to tune its circulatory half-life or its avidity -Fluorooctanoic acid ([18F]-FA) and 7-(4-[18F] fluorophenyl)-7-oxyheptanoic acid ([18F] FPOA) as prosthetic groups since octanoic acid is a known substrate of LplA, and the recombinant human Fab antibody fragment 2G10 for proof of concept  for the site-specific coupling of olecules . Site-sp concept . Enzyme-The use of enzymes as highly efficient and site-specific alternatives to the classical random chemical approaches has been gaining increasing interest in biotechnology and it is also finding its role in radiopharmaceutical applications for developing site-specific radiolabeling and highly homogeneous RIC. A precise control of the number and location of payloads on the protein should really result in better-defined and more efficient immunoconjugates and more easily reproducible procedures and may facilitate the approval process. In general, the enzymatic derivatization of proteins offers important advantages such as the use of aqueous mild reaction conditions that are required to preserve the integrity of the biomolecules as well as of BFCA and prosthetic groups. They can be efficiently used to selectively modify proteins and derivatives through one step protocols or stepwise procedures combining mild enzymatic methods with high-yielding bioorthogonal click reactions (vide supra). The inclusion of groups suitable for bioorthogonal reactions enables a modular approach with the potential for adding multiple functional moieties without any apparent important effect on the mAb function. From the studies conducted so far on different enzymatic approaches, it is not obvious to define the best methodology for the design of homogeneous RICs, also because they are “biomolecule and radionuclide dependent” and the optimization of the procedure is in some cases necessary. The four enzymatic approaches considered in this review all proved useful for the radiolabeling of mAb and their truncated derivatives. However, each approach possesses intrinsic pros and cons that are summarized in Site-specific conjugations are also expected to improve the stability and pharmacological profiles of RICs in respect to biomolecules modified using traditional chemical approaches. Indeed, heterogeneous conjugation may result in a high cargo loading that can be responsible for their impaired immunoreactivity, low stability, and suboptimal pharmacokinetic profiles. Nevertheless, if on the one hand, animal studies have shown that site-specifically labeled RICs may feature superior in vivo behavior compared to their randomly constructed counterparts, on the other there is no irrefutable evidence of the added value of site-specific conjugation in comparison to random labeling. In most of the studies comparing both approaches, the site-specifically modified biomolecules seem to not outperform the conjugates prepared by conventional routes. This behavior may be due to the specific biomolecules and/or mouse model used, thus additional studies are urgent needed to support, confirm, or refute that site-specific modification in general will improve nuclear MI with mAb and related.Although the benefits of site-specific labeling seem to be antibody-dependent, a systematic evaluation of the in vivo data of site-specifically derivatized mAbs for which the partial or complete removal of heavy chain glycans is required before enzymatic conjugation reactions, such as in transglutaminase and galactosyltransferase mediate methodologies, show a clear and significant improvement of the in vivo performance of RICs ,144,145.The examples presented here demonstrate the versatility and utility of site-specific enzyme-mediated bioconjugation to improve the efficiency and potential of protein-based radiolabeled products. Most of them are already tested in preclinical studies. However, further efforts concerning scale-up and cost minimization are necessary to realize the entire potential of site-specific protein modification in the clinic. The precise control provided by these bioconjugation methods can be used to identify criteria for selecting modification sites on mAbs and relates as well as determining generalizable design principles to maximize stability and circulation properties, biological activity, and targeted cellular uptake. Future developments in this area will also depend on the identification of new protein scaffolds that have appropriate characteristics in terms of stability, high target affinity, selectivity, rapid clearance, and ease of labeling, etc., as cancer targeting imaging agents. Moreover, by considering the current increased attention to the imaging of inflammatory responses by the direct targeting of T-cells, it is also expected that RICs and relates will be used to specifically target antigens of pathogenic bacteria and viruses, and thus permit the development of new pathogen-specific tracers to discriminate between infectious and sterile sites of inflammation.