These peptides contain an optimal balance of positive charge and hydrophobicity, which is required for interactions with the phospholipid bilayer to facilitate their application as a drug delivery system. To further optimize them, we synthesized and evaluated a multivalent tricyclic peptide as an efficient molecular transporter. The monomeric cyclic peptide building blocks were synthesized using Fmoc/tBu solid-phase chemistry and cyclization in the solution and conjugated with each other through an amide bond to afford the tricyclic peptide, which demonstrated modest antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli (E. coli) with a minimum inhibitory concentration (MIC) of 64–128 µg/mL. The tricyclic peptide was found to be nontoxic up to 30 µM in the breast cancer cell lines (MDA-MB-231). The presence of tricyclic peptide enhanced cellular uptakes of fluorescently-labeled phosphopeptide , anti-HIV drugs (lamivudine (F’-3TC), emtricitabine (F’-FTC), and stavudine (F’-d4T), 1.7–12-fold), and siRNA (3.3-fold) in the MDA-MB-231 cell lines.The cellular delivery of cell-impermeable and water-insoluble molecules remains an ongoing challenge to overcome. Previously, we reported amphipathic cyclic peptides  Multivalency is a pervasive phenomenon that has attracted attention for therapeutic development with the examples of multivalent aptamers, a multivalent effect in glycosidase inhibition, and many more ,2,3,4. MPeptide-based multivalent interactions at interfaces of a biological system are of great importance. For example, the multivalency of carefully designed supramolecular peptide ligands was utilized in enhancing binding interaction with β-cyclodextrin (β-CD) to study host–guest relationships . In anotc[WR]4 and c[WR]5  of 4 µg/mL against methicillin-resistant Staphylococcus aureus (MRSA) and 16 µg/mL against E. coli [We also discovered that a block of four subsequent tryptophan and positive-charged arginine residues generated amphipathic antibacterial peptide  E. coli . Further E. coli .c[WR]5 series of peptide for their molecular transporter property, we developed a series of bicyclic peptides (c[W5G]-(triazole)-c[KR5] and c[W5E]-(β-Ala)-c[KR5]) containing two cyclic peptides (c[W5G] and c[KR5]) joined through a flexible amide or nonflexible triazole linker (c[W5E]-(β-Ala)-c[KR5] showed higher cellular uptake of a phosphopeptide as compared to monocyclic peptides (c[KR5] or c[WR]4). These data indicated that for better cellular uptake of these cyclic peptides, an optimal balance of positive charge and hydrophobicity is essential for interactions with the cell membrane and deep penetration into the lipid bilayer [c[WR]5 consists of a balance of hydrophobicity and positive charges. We hypothesized that this peptide template could be extended into a tricyclic peptide platform as a multivalent structure for application in drug delivery.Furthermore, to gain a deep understanding of e linker  and eval bilayer . c[WR]5 c[W5E]-(β-Ala)-c[KR5] than bicyclic peptide (c[W5G]-(triazole)-c[KR5]  , presumaThus, to unravel a new multivalent tricyclic peptide as a molecular transporter, herein, we synthesized a tricyclic peptide based on naturally occurring amino acids using a simpler biocompatible and biodegradable amidic linkage instead of a triazole formed through click chemistry. The tricyclic peptide was a trimer of monocyclic peptides containing alternating hydrophobic residues (W) and charged residues (R) in addition to lysine (K) residues that assist in the generation of an amide linkage through their side chain amino group. Herein, we report antibacterial activity and molecular transporter properties of the tricyclic peptide.Staphylococcus aureus (MRSA) (Los Angeles County (LAC) clone) and E. coli (ATCC 25922) were acquired from the Loss Angeles Public Health Department . Pseudomonas aeruginosa (ATCC 27883) and Klebsiella pneumoniae (ATCC BAA 1705) were obtained from ATCC. The media for bacterial experiments were purchased from Hardy diagnostic .Fmoc amino acids, coupling reagents, and amino acid-loaded chlorotrityl resin were obtained from AAPPTec . The solvents and chemical reagents were purchased from MilliporeSigma  and used without further purification. Crude peptides were purified using reverse phase high performance liquid chromatography (RP-HPLC) using Hitachi L-2455 system  with a C18 Phenomenex column , at a flow rate of 10 mL/min with the detection at 214 nm using a gradient of 0–100% acetonitrile (0.1% trifluoracetic acid (TFA)) and water (0.1% TFA) over 60 min. Peptides were characterized for confirmation of their exact mass using Bruker GT 0264  matrix-assisted laser desorption/ionization (MALDI) mass spectrometer with α-cyano-4-hydroxycinnamic acid as a matrix under positive mode. Human breast cancer  cell line was obtained from American Type Culture Collection . Cell culture media were obtained from Fisher Scientific. Bacterial strains: Methicillin-resistant 1)  was swelled in N,N-dimethylformamide (DMF) (50 mL) under nitrogen in a 500 mL peptide synthesis vessel. After 2 h, DMF was drained out, and a solution of Fmoc-Trp(Boc)-OH , 1-[bis(dimethylamino)methylene]-1H-benzotriazolium hexafluorophosphate 3-oxide (HBTU) , and N,N-diisopropylethylamine (DIPEA)  in DMF (20 mL) was added to the resin. The resin was agitated under nitrogen for 30 min for coupling the first amino acid. After 30 min, the reaction mixture was drained and washed with DMF (10 mL × 3 times). A solution (20 mL) of 20% piperidine in DMF (v/v) was added to remove the N-terminal Fmoc group with the agitation of peptidyl resin for 20 min two times followed by washing with DMF (10 mL × 3 times). In a similar way, the subsequent Fmoc-protected amino acids were coupled based on the required sequence of the linear peptides. Once the linear protected peptide was assembled on the peptidyl resin, the side-chain-protected peptide was cleaved from the resin for N to C terminal cyclization. The peptidyl resin was stirred with freshly prepared cleavage cocktail containing trifluoroethanol (TFE)/acetic acid/dichloromethane (DCM)  for 2 h. The resin was filtered off, and the supernatant was evaporated to dryness to get side chain-protected linear peptides . The side-chain-protected peptide (300 mg) was taken in 2 L round bottom flask under nitrogen. DMF and DCM  were added to the mixture. A solution of 1-hydroxyazabenzotriazole (HOAt) (236.8 mg) in DMF and N,N’-diisopropylcarbodiimide (DIC) (269.4 µL) were added. The reaction was stirred for 24 h under nitrogen. The solvents were evaporated under vacuum, and side chains were deprotected by agitation with freshly prepared cleavage cocktail containing TFA/thioanisole/anisole/1,2-ethanedithiol (EDT)  for 2 h. The crude peptides were precipitated by the addition of cold diethyl ether (Et2O) and centrifugation. Crude peptides were purified with RP-HPLC. The peptides were separated by eluting the crude peptides at 10.0 mL/min using a gradient of 0–100% acetonitrile (0.1% TFA) and water (0.1% TFA) over 60 min and then characterized by MALDI TOF mass spectroscopy. The desired fractions were pooled and lyophilized to yield cyclic peptides. MALDI spectra of intermediate and synthesized compounds and selected analytical HPLC have been provided in the In general, all cyclic peptides were synthesized by the Fmoc solid-phase synthesis strategy followed by cyclization in the solution. H-Arg(Pbf)-2-chlorotrityl resin (4) containing free carboxylic acid.[D-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K(Dde)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)] (2-D(OAll)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K(Dde)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-tritryl resin) was assembled on H-Arg(Pbf)-2-chlorotrityl resin  using building blocks of Fmoc-protected amino acids, such as Fmoc-Trp(Boc)-OH (916.3 mg), Fmoc-Arg(Pbf)-OH (1128 mg), Fmoc-Lys(Dde)-OH (926.7 mg), and Fmoc-Asp-(OAll)-OH (687.9 mg), followed by cleavage from the solid support to afford 460 mg (24%) of the linear side-chain-protected peptide (NH2-D(OAll)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K(Dde)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-COOH) (2). MALDI-TOF (m/z) for 2, [C91H124N27O15]: calcd, 1834.9765; found, 1835.1590 [M + H]+.The side-chain-protected linear peptide (NH2 underwent cyclization to obtain side-chain-protected peptide [D(OAll)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K(Dde)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)] (3) using general peptide synthesis method explained in 3, 418 mg (91%) was dried for 24 h before using for the subsequent reaction. MALDI-TOF (m/z) for 3, [C91H121N27O14]: calcd, 1815.9586; found, 1817.9611 [M + 2]+.Compound 4, the protected cyclic peptide 3 (200 mg) was taken in a dry round bottle flask fitted with a rubber septum and under the inert condition with N2. A solution containing Pd(PPh3)4  and CHCl3:AcOH:N-methyl morpholine  was added, and the mixture was stirred for 2 h followed by evaporation of solution under rotatory evaporator. A solution of 0.5% DIPEA and sodium diethyldithiocarbamate (0.5% w/w) in DMF was added to the evaporated residue to remove the catalyst. The solvent was evaporated to remove all the solvents. The crude mixture was washed with water (8–10 mL) to get rid of all side products following with centrifugation . After centrifuging the crude peptide, it was dried overnight to obtain the protected cyclic peptide 4 . MALDI-TOF (m/z) for 4, [C88H117N27O14]: calcd, 1775.9273; found, 1777.8700 [M + 2]+.To obtain compound 5) containing free amino group.[D(OAll)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)]  with 7.8 mL of allyl alcohol was added to the peptide 3 (200 mg) and stirred at room temperature for 2 h. The reactant mixture was evaporated under vacuum to dryness and washed with cold water. The obtained solid was dried to yield side-chain-protected cyclic peptide 5 . MALDI-TOF (m/z) for 5, [C81H111N27O12]: calcd, 1653.8895; found, 1654.5044 [M + H]+.To obtain the Dde-deprotected cyclic peptide 7) containing free amino and free carboxylic acid.Deprotected cyclic peptide [DWRWRKWRWR] (4 was deprotected using 2% hydrazine monohydrate in DMF to afford protected cyclic peptide [DW(Boc)R(Pbf)W(Boc)R(Pbf)KW(Boc)R(Pbf)W(Boc)R(Pbf)] (6). Alternatively, peptide 5 was deprotected using Pd(PPh3)4 and CHCl3:AcOH:N-methylmorpholine as mentioned above to obtain 6 . Finally, peptide 6 was completely deprotected to remove Pbf and Boc groups by stirring with freshly prepared cleavage cocktail containing TFA/thioanisole/anisole/1,2-ethanedithiol (EDT)  for 2 h to yield fully deprotected cyclic peptide [DWRWRKWRWR] 7. MALDI-TOF (m/z) for 7, [C78H105N27O12]: calcd, 1611.8436; found, 1613.2794 [M + H]+.Peptide 1, 1 g, 0.58 mmol) using building blocks of Fmoc-protected amino acids, such as Fmoc-Trp(Boc)-OH (916.3 mg), Fmoc-Arg(Pbf)-OH (1128 mg), and Fmoc-Lys(Dde)-OH (926.7 mg), based on the sequence of amino acids in the diamine cyclic peptide 10 followed by cleavage from solid support to afford side-chain-protected linear peptide NH2-K(Dde)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K(Dde)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-COOH . MALDI-TOF (m/z) for 8, [C100H138N28O15]: calcd, 1971.0896; found, 1972.3850 [M + H]+.Using the general protocol mentioned in 8 was cyclized and deprotected for Dde group using HOAt/DIC and 2%hydrazine in DMF, respectively, to provide side-chain-protected cyclic peptide [K-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)] . Cyclic peptide 9 was divided into two parts (120 mg and 200 mg). Compound v/v/v/v, 10 mL) was added. The mixture was stirred at room temperature for 2 h to fully deprotect side chain protecting groups and afford 10. MALDI-TOF (m/z) for 10, [C80H112N28O10]: calcd, 1624.9116; found, 1626.4005 [M + H]+.To the first part, a freshly prepared cleavage cocktail containing TFA/thioanisole/anisole/1,2-ethanedithiol (EDT)  taken in DCM (25 mL), triethylamine (242.5 µL) was added at 0 °C followed by the addition of succinic anhydride (174.1 mg). The mixture was stirred at room temperature for 2 h. DCM was evaporated under vacuum, and the solid residue was washed with water and dried to afford crude product [K(COCH2CH2COOH)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-K(COCH2CH2COOH)W(Boc)R(Pbf)W(Boc)R(Pbf)] . After drying the crude product, the side chain-protecting groups were deprotected by stirring peptide 11 with a freshly prepared cleavage cocktail containing TFA/thioanisole/anisole/1,2-ethanedithiol (EDT)  at room temperature for 2 h. The peptides were precipitated and centrifuged to obtain a fully deprotected diacid of cyclic peptide [K(COCH2CH2COOH)-WRWR-K(COCH2CH2COOH)WRWR] (12). MALDI-TOF (m/z) for [K(COCH2CH2COOH)-WRWR-K(COCH2CH2COOH)WRWR] (12), [C88H120N28O16]: calcd, 1824.9437; found, 1826.3790 [M + H]+.To the second part of peptide 1, 1 g, 0.58 mmol) using building blocks of amino acids, Fmoc-Trp(Boc)-OH (916.3 mg), Fmoc-Arg(Pbf)-OH (1128 mg), and Fmoc-Lys(Dde)-OH (926.7 mg) based on the sequence of peptide 16. The linear assembled peptide  was cleaved from the solid support and cyclized as mentioned in the general protocol to obtain crude cyclized peptide [K(Dde)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)] .Using the general protocol mentioned in 14 (300 mg), a solution of 2% hydrazine monohydrate in DMF (50 mL) was added to deprotect Dde group. The solution was stirred for 2 h, and the reaction mixture was evaporated under vacuum to dryness and washed with cold water. The solid obtained was dried to obtain side-chain-protected cyclic peptide [K-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)] . Peptide 15 was divided into two parts (154 mg and 200 mg).To peptide 15 (154 mg), a freshly prepared cleavage cocktail containing TFA/thioanisole/anisole/1,2-ethanedithiol (EDT)  was added and stirred at room temperature for 2 h to deprotect side chain-protecting group. The second part of peptide 15 (200 mg) was dissolved in DCM (25 mL) and kept at 0 °C. Triethylamine (121.3 µL) was added to the solution of succinic anhydride (87.1 mg) in DCM was added slowly to the reaction mixture at 0 °C. The reaction mixture was stirred at room temperature for 2 h. DCM was evaporated under vacuum and the residue was washed with water and dried to obtain crude product [K(COCH2CH2COOH)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)-W(Boc)-R(Pbf)] . The side chain protecting group of peptide 17 was deprotected by reacting with freshly prepared cleavage cocktail containing TFA/thioanisole/anisole/1,2-ethanedithiol (EDT)  at room temperature for 2 h.To the first part of peptide 16) and monocarboxylic acid [K(COCH2CH2COOH)-WRWRWRWR] (18). MALDI-TOF (m/z) for [KWRWRWRWR] (16), [C74H101N26O9]: calcd, 1496.8167; found, 1497.7751 [M + 1]+. MALDI-TOF (m/z) for [K(COCH2CH2COOH)-WRWRWRWR] (18), [C78H106N26O12]: calcd, 1596.8237; found, 1598.7387 [M + 2]+.The cleaved peptides were precipitated and centrifuged to obtain fully deprotected cyclic peptides containing monoamine [KWRWRWRWR] (9)  and monocarboxylic acid peptide (17)  were added in 10 mL anhydrous DMF under stirring and inert condition using septum and purge of N2. The reaction mixture was kept at 0 °C followed by slow addition of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl)  and 4-dimethyl aminopyridine (DMAP)  with stirring for 1 h. The reaction mixture was allowed to reach room temperature and stirred for 24 h. The completion of the reaction was monitored by mass analysis of aliquot after complete deprotection of the reaction mixture using MALDI. Once the reaction was completed, the reaction mixture was evaporated to the dryness to yield peptide 19, 24 mg, 80%, and the residue was treated with the freshly prepared cleavage cocktail containing TFA/thioanisole/anisole/1,2-ethanedithiol (EDT)  at room temperature for 2 h. The peptide was precipitated and centrifuged to obtain fully deprotected tricyclic peptide 20, crude 10 mg, 78% that was purified by HPLC as described above to get pure compound 6 mg. MALDI-TOF (m/z) for 20, [C236H316N80O32]: calcd, 4782.5559; found, 4782.1199 [M]+.To a round bottom flask, the side-chain-protected cyclic peptides, diamine peptide (E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27883), and Klebsiella pneumoniae (ATCC BAA 1705) were cultured according to the guidelines of the Clinical Laboratory Standards Institute (CLSI) guidelines. MIC values were determined using microbroth dilution assay. Tricyclic peptide 20 was used to evaluate antibacterial activity in comparison to positive controls, such as Meropenem for Gram-negative bacteria and c[R4W4] for Gram-positive bacteria. Bacterial strains were inoculated into LB (5 mL) and shaken in an orbital shaker at 175 rpm overnight at 37 °C. The cultured suspension was immediately diluted to get turbidity of 0.5 McFarland  using normal saline. The test compounds  were dissolved in 5% dimethyl sulfoxide (DMSO) to generate 256 µg/mL solutions. 200 µL of tricyclic peptide 20, and controls were added to the first column of the 96 well plates, and a twofold serial dilution was performed with the addition of 100 µL of bacterial culture media to the other wells. The corresponding control DMSO 5% alone without the compound in similar concentration exhibited no antibacterial activity when diluted in the final concentration. Bacterial solution (100 µL) was added to all the wells and incubated statically at 37 °C overnight. The minimum concentration at which bacterial growth was not visible to eyes were reported as MICs and experiments were performed in triplicate.The selected strains of bacteria, MRSA (Los Angeles County (LAC) clone), 2 cell culture flasks with Dulbecco’s modified eagle’s medium (DMEM) medium, supplemented with 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution  in a humidified atmosphere of 5% CO2, 95% air at 37 °C. CellTiter 96® Aqueous  one solution cell proliferation assay (MTS) was utilized to evaluate MDA-MB-231 cell viability. Cells were seeded into 96-well plate (104 cells/100 μL) in complete media and incubated overnight at 37 °C with 5% CO2. Different concentrations (0−300 μM) of the peptide solution (10 μL) were added to wells to make the final concentrations of the peptide (0−30 μM). The cells were incubated again  for 24 h, then MTS reagent (20 μL) was added to each well followed by incubation for 2 h under the same condition. The absorbance of the resulting solution was evaluated at 490 nm using a microplate reader to detect the live cells. Cells without peptide were considered as a control. DMSO (30% v/v) was used as a positive control. The compound was used in aqueous solution. All the tests were performed in triplicate. Human breast cancer (MDA-MB-231) cell line was grown on 75 cm5 cells/well) in DMEM overnight. Then different fluorescence-labeled molecules  at 5 μM or F’-GpYEEI (5 μM) in the presence or absence of the tricyclic peptide 20 (25 μM) in opti-MEM were added, where F’ represents carboxyfluorescein and pY represents the phosphotyrosine amino acid). F’-GpYEEI, F’-3TC, F’-FTC, or F’-dT4 and cyclic peptide 20 were premixed in opti-MEM at room temperature for 30 min. The cells were incubated with the premixed solution for 3 h at 37 °C. 5-(6) carboxyfluorescein (FAM or F’) was used as a negative control. After 3 h incubation, the media containing peptide was removed. The cells were washed twice with PBS, followed by digestion with 0.25% trypsin/0.53 mM EDTA for 5 min to remove any false surface binding. Then PBS was added to wash the cells. The cells were centrifuged  to collect the pellet. Finally, the obtained pellet was resuspended in buffer designated for flow cytometry and analyzed by flow cytometry ) using FITC channel and CellQuest software. The data depicted in the results are determined by using the mean fluorescence signal for 10,000 cells collected. Flow cytometry analysis was performed in triplicate.Human breast cancer cells (MDA-MB-231) were grown in 6-well plates  and peptides in different N/P ratios. First, FAM-siRNA was added to 150 mM NaCl solution. Tricyclic peptide N/P = (Number of moles for peptide × Number of Nitrogens)/(Number of moles for siRNA × 48) where 48 represents the average number of phosphates in each siRNA molecule.Cells were seeded in 24-well plates at ~200,000 cells/mL. After overnight seeding, peptide/siRNA complexes were added. Peptide/siRNA complexes were made with peptide: siRNA N/P ratios of 10, 20, and 40, respectively. After 24 h of incubation at 37 °C, the cells were washed with hanks balanced salt solutions (HBBS), trypsinized, and fixed with 3.7% formaldehyde solution. Quantification of siRNA uptake was performed by Flow cytometer  using the FITC channel to evaluate intracellular fluorescence signal. The mean fluorescence of cells in the selected cell population was determined. The results were calibrated by gating with the control cells  group in such a way that the auto-fluorescent cells became ~1% of the total cell population.p-value less than 0.05 was considered as statistically significant and included in the Figures.To evaluate the statistical significance of the results, ANOVA test was carried out using a single factor in MS Excel 2016. In case of cytotoxicity assay, cells without treatment were considered as the control and compared with the treated groups. For cellular uptake studies, only drug treated cells were considered as the control and compared with drug/carrier mixture. N to C terminal cyclization; however, parentheses  represent linear peptides.For the synthesis of click-free tricyclic peptide, we designed and followed various strategies. In the first approach, we started with the synthesis of two cyclic peptides that contained one free carboxylic acid and one free amino group to perform intermolecular conjugation between them or to oligomerize a cyclic peptide containing a free amino group and a free carboxylic acid . Scheme 2) was assembled on 2-chlorotrityl resin starting from H-Arg(Pbf)-2-chlorotrityl resin (1) using Fmoc/tBu solid-phase peptide synthesis strategy followed by cleavage of the protected peptide from the resin using cleavage cocktail (TFE:DCM:AcOH) at the room temperature to afford 2. Linear protected peptide 2 was cyclized under dilute condition using HOAt and DIC to yield protected peptide 3 according to the previously reported procedure  (7) containing both free amino and carboxylic acid groups as new bifunctional cyclic peptide. The conjugation of the cyclic peptide with itself failed to generate any dicyclic peptide.Alternatively, the cyclic peptides 10 and a diacid functional group-containing cyclic peptide 12 using Novazyme-435, a lipase usually used for polyamide or polyester synthesis  10. The second part of cyclic peptide 9 was reacted with succinic anhydride in anhydrous condition to obtain a diacid functionalized side-chain-protected peptide 11. The complete deprotection afforded diacid containing two free carboxylic acids in the cyclic peptide (12).The synthesis was started with the synthesis of side-chain-protected linear peptide 9 with peptide 11 and peptide 10 with peptide 12 were attempted using Novazyme-435 as a catalyst at 50 °C, 60 °C, and 70 °C without success, presumably due to lack of access of the cyclic peptides to the catalytic triad of the enzyme because of the bulky size.The enzymatic synthesis of multivalent cyclic peptide or polymerization of peptide 16) and free monocarboxylic acid functional group (18) as shown in 13) was synthesized, followed by cyclization to afford cyclic peptide 14. The Dde group was removed using hydrazine to afford protected peptide 15, which was divided into two parts. The first part underwent complete deprotection to yield mono amine-containing cyclic peptide [KWRWRWRWR] (16). The second part was functionalized by reacting with succinic anhydride to afford mono acid-containing cyclic peptide 17. The complete deprotection of peptide 17 afforded mono carboxylic acid-containing cyclic peptide 18.In the third approach, we designed and synthesized cyclic peptides with a free monoamine functional group  was not successful due to the steric hindrance associated with the side chains of tryptophan residues in the cyclic peptide. Moreover, steric hindrance may be due to the use of carboxylic acid for conjugation from the aspartic acid side chain, which was in the proximity of the backbone of the cyclic peptide. Therefore, we tried to use a lipase family enzyme named Novazyme-435 to facilitate conjugation and or polymerization of diamine or diacids containing protected or unprotected side chains in the cyclic peptides. However, the MALDI analysis did not show any conjugation. Novazyme-435 was reported to be used in the synthesis of polyamide or polyester from small molecules but did not produce any success with the case of cyclic peptides during our synthesis. Finally, the use of diamine cyclic peptide from the second approach and monofunctionalized carboxylic acid with a succinate linker was examined (third approach) using EDC and DMAP as classical peptide coupling reagents. The third approach afforded the tricyclic peptide, possibly due to the availability of four extra carbon atoms as a linker in the lysine side chain, which enhanced activation of the COOH group followed by conjugation to the diamine of the cyclic peptide.20 was determined by the MTS assay against breast cancer cell lines (MDA-MB-231). Tricyclic peptide 20 showed more than 97% cell viability in MDA-MB-231 cell lines up to a concentration of 25 μM, which slightly decreased to 92% at 30 μM -Gly-(pTyr)-Glu-Glu-Ile (F’-GpYEEI) was analyzed with flow cytometry. The percentage of fluorescently positive cells exceeded 98%, while non-treated cells or phosphopeptide alone were less than 0.6%. In terms of mean fluorescence, the cellular uptake was enhanced at least by 18 times in the presence of tricyclic peptide 20, suggesting the role of the latter as a successful delivery tool , emtricitabine (FTC), stavudine (d4T)  was examined in the presence and absence of the tricyclic peptide 20 using MDA-MB-231 cell line  and E. coli (MIC 16 µg/mL) and showed improved antibacterial activity when co-administered with tetracycline [c[R4W4] peptides revealed the requirement of R and W amino acids   was enhanced in the presence of tricyclic peptide 20 by 1.9–12-fold as compared to the drug alone. Tricyclic peptide 20 also internalized a fluorescent-labeled siRNA into the MDA-MB-231 cells by 3.3-fold at N/P ratio of 10 as compared to siRNA alone. Further optimization in the design and application of tricyclic peptide will be required to access the full potential of these multivalent compounds in the biological system. Our data also reflect the potential of the tricyclic or polycyclic peptides in the area of drug delivery.We designed and synthesized tricyclic [(WR)


Scientific Reports 10.1038/s41598-020-74418-w, published online 16 October 2020Correction to: This Article contains an error in Figure 2 where Greek letters are replaced with Helvetica font. The correct Figure 2 appears below as Figure


However, the diversity of tet(X4)-bearing plasmids and bacteria remains largely uninvestigated. To cover this knowledge gap, we comprehensively identified and characterized the tet(X)-bearing plasmidome in different sources using advanced sequencing technologies for the first time. The huge diversity of tet(X4)-bearing mobile elements demonstrates the high level of transmissibility of the tet(X4) gene among bacteria. It is crucial to enhance stringent surveillance of tet(X) genes in animal and human pathogens globally.Tigecycline is an expanded-spectrum tetracycline used as a last-resort antimicrobial for treating infections caused by superbugs such as carbapenemase-producing or colistin-resistant pathogens. Emergence of the plasmid-mediated mobile tigecycline resistance gene  tet(X) variants were reported in diverse pathogens from different sources. However, the diversity of tet(X)-bearing plasmids remains largely unknown. In this study, the phenotypes and genotypes of all the tet(X)-positive tigecycline-resistant strains isolated from a slaughterhouse in China were characterized by antimicrobial susceptibility testing, conjugation, pulsed-field gel electrophoresis with S1 nuclease (S1-PFGE), and PCR. The diversity and polymorphism of tet(X)-harboring strains and plasmidomes were investigated by whole-genome sequencing (WGS) and single-plasmid-molecule analysis. Seventy-four tet(X4)-harboring Escherichia coli strains and one tet(X6)-bearing Providencia rettgeri strain were identified. The tet(X4)-bearing elements in 27 strains could be transferred to the recipient strain via plasmids. All tet(X4)-bearing plasmids isolated in this study and 15 tet(X4)-bearing plasmids reported online were analyzed. tet(X4)-bearing plasmids ranged from 9 to 294 kb and were categorized as ColE2-like, IncQ, IncX1, IncA/C2, IncFII, IncFIB, and hybrid plasmids with different replicons. The core tet(X4)-bearing genetic contexts were divided into four major groups: ISCR2-tet(X4)-abh, △ISCR2-abh-tet(X4)-ISCR2, ISCR2-abh-tet(X4)-ISCR2-virD2-floR, and abh-tet(X4)-ISCR2-yheS-cat-zitR-ISCR2-virD2-floR. Tandem repeats of tet(X4) were universally mediated by ISCR2. Different tet(X)-bearing strains existed in the same microbiota. Reorganization of tet(X4)-bearing multidrug resistance plasmids was found to be mediated by IS26 and other homologous regions. Finally, single-plasmid-molecule analysis captured the heterogenous state of tet(X4)-bearing plasmids. These findings significantly expand our knowledge of the tet(X)-bearing plasmidome among microbiotas, which establishes a baseline for investigating the structure and diversity of human, animal, and environmental tigecycline resistomes. Characterization of tet(X) genes among different microbiotas should be performed systematically to understand the evolution and ecology.The emergence of novel plasmid-mediated resistance genes constitutes a great public concern. Recently, mobile IMPORTANCE Tigecycline is an expanded-spectrum tetracycline used as a last-resort antimicrobial for treating infections caused by superbugs such as carbapenemase-producing or colistin-resistant pathogens. Emergence of the plasmid-mediated mobile tigecycline resistance gene tet(X4) created a great public health concern. However, the diversity of tet(X4)-bearing plasmids and bacteria remains largely uninvestigated. To cover this knowledge gap, we comprehensively identified and characterized the tet(X)-bearing plasmidome in different sources using advanced sequencing technologies for the first time. The huge diversity of tet(X4)-bearing mobile elements demonstrates the high level of transmissibility of the tet(X4) gene among bacteria. It is crucial to enhance stringent surveillance of tet(X) genes in animal and human pathogens globally. Antibiotics, considered a major breakthrough of modern medicine, are drugs utilized to treat bacterial infections in humans and animals , 2. HoweEnterobacteriaceae (CRE) and colistin-resistant Enterobacteriaceae (–tet(X) was first discovered in the obligate anaerobe Bacteroides fragilis (tet(X) and its variants in clinical pathogenic microbiotas constitutes a potential public health risk (–tet(X) existed in environmental bacteria such as Flavobacterium and Bacteroides (–tet(X). However, the horizontal gene transfer (HGT) of tet(X) among microbiotas, especially those most closely related to human health, was not investigated in detail.Tetracycline antibiotics have been a fundamental antibacterial agent for more than 6 decades and are widely used in clinical settings and animal sectors because of (i) their broad-spectrum activity against Gram-positive, Gram-negative, and atypical bacteria, (ii) their low cost, (iii) and their ability to be administered orally and intravenously –10. Tetr–eriaceae , 14, tigeriaceae , resistaeriaceae –20. Anotriaceae –, 22. Altfragilis , the emelth risk –26. Enviteroides –29, whictet(X3) and tet(X4) conferring high-level tigecycline resistance in bacteria of animal, food, and human origins (tet(X) genes among pathogens is becoming a real threat, which is further demonstrated by the emergence of tet(X) genes from different sources, novel tet(X5) in Acinetobacter baumannii and tet(X6) in Proteus spp. (–tet(X4)-bearing plasmids have been characterized (tet(X4)-bearing genetic structures, especially in the tigecycline resistome of animal fecal microbiotas and surroundings, was not investigated systematically. In this study, we probed the diversity and polymorphism of tet(X4)-bearing plasmids genomically and phenotypically, with the perspective of the resistance plasmidome. The findings imply that the mobile tigecycline resistance gene tet(X4) is present extensively in fecal commensals and environments.Recently, two reports highlighted the emergence of plasmid-mediated  origins , 31. Thieus spp. –38. Varicterized , 39, 40,tet(X)-positive strains, consisting of 74 Escherichia coli strains harboring tet(X4) and 1 Providencia rettgeri strain harboring tet(X6) (RF14-2), confirmed by 16S rRNA gene sequencing and Sanger sequencing of PCR products. The rates of isolation from wastewater and soil samples were the highest (40%), followed by swine feces and ground blood samples (see tet(X)-bearing samples was 28.33%, higher than that in two previous reports (tet(X)-positive strains, especially tet(X4)-bearing E. coli strains, existed in slaughterhouse environments at high prevalence and probably resulted from contamination from imported tet(X4)-bearing fecal microbiotas. Previously, E. coli strains from farm pigs and pork samples in markets were found positive for tet(X4), implying that slaughterhouse processing of pigs was a vital procedure for controlling tet(X4) contamination (tet(X)-positive strains were recovered from the sample among seven samples (see tet(X) genes could be transmitted within the same microbiota. All the tet(X4)-positive strains and one tet(X6)-positive strain exhibited resistance to tigecycline, with MICs from 8 to 32 mg/liter. In addition, all the strains were resistant to tetracycline and florfenicol, with a high rate of resistance to amoxicillin, doxycycline, and streptomycin, and most of them were multidrug-resistant strains. However, all strains were still susceptible to colistin and meropenem, except Providencia rettgeri RF14-2, which had intrinsic resistance to colistin  yielded 75  reports , 31. Thimination , 41. Notples see , demonststin see .10.1128/mSystems.00134-20.4TABLE S1tet(X4)-positive strains and their prevalence among different sources in the slaughterhouse. Download Table S1, DOCX file, 0.01 MB.Numbers of Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00134-20.5TABLE S2E. coli strains harboring tet(X4) and 1 Providencia rettgeri strain harboring tet(X6). Download Table S2, DOCX file, 0.03 MB.Antibiotic susceptibility testing (in milligrams per liter) of 74 Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli isolates and one Providencia rettgeri isolate from different sources, were randomly selected for whole-genome sequencing (WGS) with the Illumina HiSeq 2500 platform. These strains were isolated from different sources, including feces, wastewater, blood, soil, and carcasses. Draft genomes of these isolates were generated via de novo assembly for subsequent analysis. To investigate the evolutionary relatedness of these 26 E. coli strains, a phylogenetic tree based on single nucleotide polymorphisms (SNPs) of core genomes was constructed (E. coli strains were grouped into three clusters. Multilocus sequence typing (MLST) analysis revealed that the 26 E. coli strains were assigned to nine known MLST types, with ST1196 being the most prevalent type, and strain RS3-1 belonged to a novel ST, designated ST10671 by Enterobase (tet(X4)-carrying E. coli strains isolated from swine feces and environmental samples in this slaughterhouse were diverse and had no obvious clonal spread. Other STs of E. coli, such as ST8302, ST101, and ST542, were positive for tet(X4) (tet(X4)-bearing E. coli strains indicated that horizontal gene transfer was the major transmission route for tet(X4) among E. coli strains, although tet(X4) was occasionally found on the chromosome (tet(X4)-bearing strains were positive for at least three categories of resistance genes, with the most prevalent being floR, aadA1, blaTEM, and qnrS1 (tet(X4) has a risk of cotransmission with other resistance genes.Twenty-seven strains, including twenty-six structed . The phyterobase . The phy tet(X4) , 35. Theromosome , 43. Thend qnrS1 , which itet(X4) and tet(X6) genes and their genetic contexts, all 75 strains were subjected to conjugation assay. The tet(X4) gene from 27 isolates and the corresponding resistance phenotype were successfully transferred to E. coli C600, suggesting that the 27 isolates harbored the tet(X4)-positive genetic structures in conjugative plasmids or other mobilizable genetic elements. Plasmid fingerprints of tet(X4)-positive strains and corresponding transconjugants resolved by pulsed-field gel electrophoresis with S1 nuclease (S1-PFGE) were utilized to probe the plasmid profiles. All the donor strains harbored one to three plasmids, and at least one plasmid existed in transconjugants, which indicated that the tet(X4) gene was in self-conjugative or mobilizable plasmids, and other plasmids could be cotransferred to the recipient strain. Notably, the plasmids of six transconjugants were larger than the plasmids of their donor strains, implying that plasmid reorganizations may occur. The genetic basis of the tet(X4)-bearing plasmids and plasmid reorganizations was investigated further.To investigate the transmissibility of the tet(X4)-harboring plasmids with different sizes were selected and sequenced with the long-read Nanopore MinION platform, and these plasmids ranged from 12 to 294 kb in length (see tet(X4)-harboring plasmids were de novo assembled completely by combining short-read and long-read data, and a few of them were de novo assembled based on available long-read data. Sixteen plasmids could be successfully transferred into the recipient E. coli C600. These 27 tet(X4)-harboring plasmids were categorized into nine different replicon types. Eight plasmids, ranging from 97 to 129 kb, belonged to IncFIB(K)/IncFIA(HI1)/IncX1 group of hybrid plasmids. Four plasmids, ranging from 112 to 136 kb, were classified as IncFII-type plasmids. Three plasmids, ranging from 31 to 50 kb, were classified as IncX1-type plasmids. The rest of the plasmids ranged from 12 to 294 kb and were classified as IncQ1 type, IncA/C2 type, IncFIB type, IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid type, IncHI1B(R27)/IncFIA(HI1)/IncHI1A/IncX1 hybrid type, and IncFIB(K)/IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid type  (see tet(X4)-harboring plasmids were detected in the same slaughterhouse. These types of plasmids were more abundant than the existing 15 tet(X4)-bearing plasmids of different replicons in the NCBI database (see tet(X4). In addition, most of these tet(X4)-harboring plasmids were conjugative, showing that the plasmid-mediated trait greatly enhanced transmissibility of the tigecycline resistance gene tet(X4).According to S1-PFGE, a total of 27 ngth see . The majrge) see . In totabase see , which i10.1128/mSystems.00134-20.6TABLE S3tet(X4)-bearing plasmids in this study. Download Table S3, DOCX file, 0.02 MB.Basic information on 27 Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00134-20.7TABLE S4tet(X4)-bearing plasmids in the NCBI nr database and this study. Download Table S4, DOCX file, 0.03 MB.Basic information on 42 Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the tet(X4)-bearing plasmids, 15 tet(X4)-bearing plasmids from the NCBI nonredundant (nr) database were retrieved as of 1 December 2019 (see tet(X4)-bearing plasmids sequenced in this study to determine the distribution of resistance genes, replicons, and insertion sequences among the 42 plasmids (CR2 was associated with tet(X4) in all plasmids, no matter what the plasmid types were. This observation was consistent with the idea that ISCR2 could mediate the generation of the circular form, facilitating transmission of tet(X4) between different genetic contexts , being absent from only four plasmids, i.e., three IncQ plasmids and one IncA/C2 plasmid (tet(X4)-bearing plasmid. It is 87,445 bp in size and carries genes related to plasmid replicon, maintenance, conjugative elements, and resistance, including erm(42) and tet(X4) flanked by ISCR2 and IS26. Online BLASTn analysis showed that it was most similar to the online IncA/C plasmids found in Salmonella spp. and other Enterobacteriaceae at 88% coverage with more than 99% identity -bearing IncA/C2 plasmid may be mediated by ISCR2 and the circular intermediate ISCR2-tet(X4)-abh detected previously (tet(X4) in this classical MDR plasmid would draw wide attention, and surveillance on tet(X4)-bearing IncA/C2 plasmids in environmental microbiotas and pathogens should be performed.To investigate the characterization of all available 2019 see . These wplasmids . It was contexts , 43. ISCand floR . The res plasmid . To the identity . IncA/C volution , and theeviously . The occtet(X4)-bearing plasmid identified in this study was pRF25-1_12k_tetX_flye, belonging to IncQ1, which was most similar to pLHM10-1-p6, with the genetic structure ISCR2-catD-tet(X4)-△ISCR2 (see mph(A), aac(3)-VIa, aadA1, tet(M), qnrS1, blaTEM-1A, and floR. IncQ plasmids were found distributed in environmental microbiomes such as wastewater (tet(X4)-bearing IncQ plasmids may exist in other environmental microbiomes.The smallest SCR2 see  31). In. Intet-bearing plasmids with similar online plasmids. a, b, c, d, e, and f represent different tet(X4)-bearing plasmids with similar backbones. Download FIG S1, JPG file, 0.8 MB.Circular comparison of different Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mcr-harboring plasmids and tet(X4)-harboring plasmids belonging to the IncX1 type and sharing similar backbones were found (tet(X4)-harboring IncX1 plasmids ranged from 31 to 59 kb and were characterized by two variable regions, including a multidrug resistance region and a type IV secretion system (T4SS) gene cluster containing different virB genes (tet(X4)-harboring and mcr-1-harboring IncX1 plasmids, the co-occurrence of tet(X4) and mcr-1 in the same conjugative IncX1 plasmid should be put under close surveillance for risk assessment.Three IncX1 plasmids were also found among these 27 plasmids. In the NCBI database, some re found , 50 -bearing plasmid type distribution, IncFIB(K)/IncFIA(HI1)/IncX1 hybrid plasmids were the most widespread in the sequenced plasmids. This type of plasmid was also found in plasmids deposited in the online database (see tet(X4)-bearing plasmids, pYPE12-101k-tetX4 (CP041443.1) and pG3X16-2-3 (CP038140.1). Among those plasmids, pRF10-1_119k_tetX, pRW8-1_122k_tetX, pRW8-2_117k_tetX_flye, and pRF155_129k_tetX_flye showed a backbone similar to that of pG3X16-2-3 , and dfrA5 , which was the first identified tet(X4)-bearing plasmid in E. coli  were identified in these 27 plasmids see . BLASTn  E. coli  -bearing plasmids belonged to different incompatibility types (tet(X4)-bearing plasmids, a tet(X6)-bearing integrative and conjugative element (ICE) was found in RF14-2, which is discussed below.The rest of ty types . Apart ftet(X)-positive strains were detected in the same sample for seven samples (see tet(X4)-positive isolates suggested that tet(X4) had spread in the same microbiota. We also found that the tet(X4)-harboring plasmids carried by these strains were diverse (see tet(X4) gene were found between tet(X4)-harboring plasmids in strains RF45-1 and RF45-2 (see tet(X4) were also found in pRF108-1_107k_tetX_flye, but only one tet(X4) copy was found in pRF108-2_97k_tetX, indicating that polymorphism of tandem repeats happened during bacterial division of the same clone. The resistome in a single sample was investigated previously (tet(X4)-bearing plasmidome was not studied. Our initial attempt to recover different tet(X4)-bearing plasmids in a single microbiota was successful and proved the complex transmission routes of tet(X4) among environmental and fecal microbiota. This method is limited because of the number (only two in this study) of tet(X4)-bearing strains isolated from one sample; more colonies harboring tet(X4) should be recovered and analyzed phenotypically and genomically, and a long-read metagenomics method could be performed to analyze the polymorphism of tet(X4)-bearing structures in single-microbiota samples.Interestingly, two different ples see . These serse see . Meanwhierse see . Copy nu45-2 see . A helpe45-2 see . Meanwhieviously , 28, but10.1128/mSystems.00134-20.8TABLE S5tet(X)-positive strains each. Download Table S5, DOCX file, 0.02 MB.Basic information on the seven samples detected with two Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00134-20.9TABLE S6Table S6, DOCX file, 0.02 MB.Plasmids participate in reorganization in the process of conjugation. Download Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the tet(X)-bearing isolates, strains RF14-1 and RF14-2 belonged to E. coli and Providencia rettgeri, respectively (see tet(X4) variant carried by an IncX1 plasmid, with a single nucleotide mutation compared with the tet(X4) gene (tet(X4) gene, which was confirmed by measuring the MICs of E. coli harboring the cloning vector via the TA cloning method. Also, the IncX1-type plasmid carrying the tet(X4)-like gene could be successfully transferred into E. coli C600. The other two IncX1 plasmids carrying tet(X4) isolated in this study have no transferability  variant, designated tet(X6), was detected in an SXT/R391 integrative and conjugative element (ICE). The function of tet(X6), i.e., the ability to confer tigecycline resistance, was confirmed by the TA cloning method. It was homologous to tet(X4) (87.3%) and other tet(X) variants -containing region was integrated into variable region III of the ICE (PmiFra1 in Proteus mirabilis in the NCBI database (tet(X6)-bearing structure was in the variable region III of the ICE, sharing a similar structure with other ICEs with different accessory regions characterized by ISCR2 (tet(X6)-bearing ICE was low compared with the high incidence of tet(X4)-bearing plasmids among tet(X)-bearing strains in this study, the existence of a tet(X6)-bearing ICE located on the chromosome implied that tet(X) transmission occurred via multiple routes. The co-occurrence of different tet(X) variants in different genetic backgrounds of diverse bacteria highlighted the complex tet(X) evolution in microbiotas.Within the seven samples positive for two vely see . A tet(XX4) gene , was foulity see . Furtherlity see , which slity see . In Provvariants . Coincid isolate  and was  isolate –55. The 1 family . This noe system . Structu the ICE . In addidatabase . The tetby ISCR2 . Althougtet(X4)-harboring genetic contexts from tet(X4)-bearing plasmids in the online database and in this study were analyzed and categorized into four major groups (CR2-tet(X4)-abh was the prominent structure observed in tet(X4)-bearing plasmids (see CR2 (abh-tet(X4)-ISCR2-virD2-floR, was categorized into three subtypes with different genes—IS26, △ISCR2, and ISCR2—in the upstream region. The last genetic structure type (G4) had the longest genetic region, abh-tet(X4)-ISCR2-yheS-cat-zitR-ISCR2-virD2-floR. The first structure type was mainly distributed in small plasmids, including IncQ and ColE2-like plasmids (see tet(X4)-bearing structures existed in plasmids of different replicons and chromosomes (tet(X4)-bearing structures.All the r groups . ISCR2-tmids see . The sec, 57tet-bearing plasmids, it was still impossible to obtain complete plasmid sequences for some tet(X4)-bearing plasmids, even with different assembly strategies (see tet(X4). For tet(X4)-bearing plasmids with low copy numbers of tet(X4), Nanopore long reads could cover the repeat region, and complete plasmid sequences could be finished, such as those for pRF108-1_107k_tetX_flye and pRW8-2_117k_tetX_flye. However, for tet(X4)-bearing plasmids with tet(X4) tandem repeat regions larger than long reads, or with heterogenous numbers of tet(X4) tandem repeats, accurate copy numbers were difficult to obtain, resulting in failure of plasmid assembly. This demonstrated that the repeat regions containing tet(X4) were diverse and in a polymorphic state. To summarize, four kinds of repeat regions were detected, which ranged from 4.6 to 20 kb in length (abh-tet(X4)-ISCR2 in 4,606 bp, which was the reported tet(X4)-bearing circular intermediate  (tet(X4) tandem repeat structures. An IncQ tet(X4)-bearing plasmid, pRB3-2_un_11k_tetX_flye in RB3-2, similar to pLHM10-1-p6 (tet(X4)-bearing ColE2-like plasmid p16EC-9K, which was also observed in a polymorphic state of tandem plasmid repeats (tet(X4) (26-aadA2-lnu(F)-IS26-abh-tet(X4)-ISCR2-floR-tet(A)-△IS26 in 14 kb and △ISCR2-erm(42)-ISCR2-abh-tet(X4)-ISCR2-floR-IS26 in 20 kb, were found in RF45-2 and RF15-1, respectively. Certain long reads harboring tandem repeats of tet(X4) were illustrated to infer the complex structures (see tet(X4)-bearing tandem repeat, implying the complex structures of tet(X4) among plasmids. Although the multiple repeats of tet(X4) were common in the samples, the MICs of tetracycline, including tigecycline, were not affected significantly. The reason for the frequent occurrence of tet(X4) repeats in natural isolates, compared with the low prevalence of duplications of other resistance genes, warrants further investigations.Although Nanopore long-read data were generated to perform gies see . After sgies see , all then length . They we,608 bp) , 43. ThiM10-1-p6 , was fouM10-1-p6 . This ph repeats , and thi(tet(X4) . Two addures see . The 20-10.1128/mSystems.00134-20.2FIG S2tet(X4)-bearing tandem repeat structures resolved by Nanopore long reads. The three parts represent the three types of tet(X4)-bearing repeats observed in two Nanopore long reads for each sample. Download FIG S2, JPG file, 0.3 MB.Three major Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the tet(X4), especially the tandem repeats of the ISCR2-tet(X4) structure, probably generated by rolling-circle transposition, in E. coli was reported  based on WGS data were analyzed, and the results showed that ISCR2 copy numbers were equal to or higher than that of tet(X4)  transmission. The observation of multiple copy numbers of tet(X4) in this study suggests that ISCR elements may play important roles in the enrichment of resistance genes in microbiota.Recently, the polymorphism of reported . Similaranalysis . Further tet(X4) , enhancitet(X4)-bearing plasmids of six transconjugants showed different plasmid sizes compared with that of their parental strains after conjugation (tet(X4)-bearing strains, plasmid DNA was extracted from the six transconjugants, and long-read sequencing was performed. All the plasmids were finished in complete and circular forms except RF15-1 and TRF15-1 (see 26 (26-ΔTnAs1 (26-mph(A)-orf-orf-ΔIS6100 -bearing plasmid reorganization by replicative transposition. For the plasmid reorganization in TRF15-1, the plasmids were not successfully assembled even with the Nanopore long-read data, which implies that possible underlying complex structures exist, and this warrants further investigation.Among the conjugative plasmids, the jugation . From thjugation , 60, 61,15-1 see . Linear  (see 26 . In TRF16-ΔTnAs1 . The pla-ΔIS6100 . SimilarA1-aadA2 . Distinc-1_389kb . The rolbacteria . This watet(X4)-bearing plasmid reorganization would incorporate more resistance genes and replicon genes in the novel large MDR hybrid or cointegrate plasmids, which expanded the host range, causing a severe public health concern. Although the reorganizations were observed during conjugation under laboratory conditions, the prevalence of such MDR hybrid or cointegrate plasmids in the natural environment, with a focus on the MDR plasmidome among different environments, should be investigated.The tet(X4)-bearing plasmids, tet(X4)-bearing genetic contexts, and complex tet(X4)-bearing plasmid reorganization and highlight the wide distribution of various tet(X4)-bearing structures in different E. coli clones. Identification of tet(X6)-bearing ICEs in the same tet(X4)-positive microbiota spotlighted the existence of coevolution of multiple tet(X) variants. Nanopore long reads significantly enhanced the characterization of polymorphism of tet(X4)-bearing plasmids from the perspective of single-plasmid-molecule analysis. The importance of plasmids and ISCR2 in facilitating the transmission of tet(X) was confirmed. In summary, this work demonstrates the significant role of the tet(X)-bearing plasmidome in the swine slaughterhouse for tet(X) transmission along the pork production chain, and stringent surveillance of tet(X)-bearing microbiotas of animals, humans, and the environment should be conducted to evaluate the risk posed by the emerging plasmid-mediated tigecycline-resistant tet(X) variants.The data presented in this study expand the understanding of diversity of tet(X) was checked using PCR with the primers tet(X)-F (5′-TGA ACC TGG TAA GAA GAA GTG-3′) and tet(X)-R (5′-CAG ACA ATA TCA AAG CAT CCA-3′) (581 bp). The PCR amplicons were subsequently sequenced by Sanger sequencing. 16S rRNA gene sequencing was performed to confirm species identification of the tet(X)-positive isolates using universal primers .In May 2019, 240 samples consisting of 182 swine fecal samples, 22 swine carcass samples, 11 ground blood samples, 10 wastewater samples, and 15 soil samples were collected from a slaughterhouse in Jiangsu Province, China. The samples were stored at low temperature during rapid transfer to our lab for further processing. Solid and liquid samples (1 g each) or cotton swabs (surface samples) were incubated in 5 ml LB broth supplemented with tigecycline (2 mg/liter) for 6 h to enrich the tigecycline-resistant microbiota. The enriched cultures were streaked onto MacConkey agar plates containing tigecycline (2 mg/liter) to screen single colonies, and colonies with different morphology characteristics were further purified and stored in LB broth with 15% glycerol at −80°C. Genomic DNA of samples was prepared by the boiling method. The presence of http://www.eucast.org/clinical_breakpoints/) (tet(X)-positive isolates were measured by the agar microdilution method and interpreted in accordance with the CLSI standard  guidelines  and werepoints/) . The MICstandard . E. colitet(X)-carrying genetic elements, a conjugation assay with a filter mating method (tet(X)-positive strains as the donor strains and rifampin-resistant E. coli C600 as the recipient (1:4). Transconjugants were selected on LB agar plates supplemented with rifampin (300 mg/liter) and tigecycline (2 mg/liter). The transconjugants harboring tet(X4) were confirmed by PCR and antimicrobial susceptibility testing as described above. To characterize the tet(X)-bearing plasmid profiles, tet(X)-positive strains and their transconjugants were digested with S1 nuclease  followed by pulsed-field gel electrophoresis (PFGE) with the CHEF Mapper XA system . The Salmonella Braenderup H9812 standard strain restricted with XbaI was used as the molecular marker.To investigate the transferability of g method  was carrGenomic DNA of the tigecycline-resistant strains was extracted using the TIANamp bacterial DNA kit , following the manufacturer’s instruction. The plasmids of strains were extracted using the Qiagen plasmid midi-kit  after overnight culture in 100 ml LB broth. The genomic DNA of selected strains with different resistance phenotypes was subjected to short-read sequencing (2 × 150 bp) with the Illumina HiSeq 2500 platform. Subsequently, genomic DNA of certain strains and plasmid DNA were sequenced with the Oxford Nanopore Technologies MinION long-read platform with the RBK004 barcoding library preparation kit and MinION R9.4.1 flow cells to obtain the complete sequences, as described previously , 67.https://github.com/tseemann/mlst) based on assembled contigs. The draft genomes were annotated using the software Prokka (E. coli strains was constructed using Roary and FastTree based on SNPs of core genomes (de novo assembly of Nanopore long-read MinION sequences of plasmids DNA and genomic DNA (tet(X4)-bearing structures were analyzed using the BLASTn program against the nr database. Genomic DNA with short-read Illumina and long-read Nanopore data were used to perform de novo assembly with the hybrid strategy as described previously (http://rast.nmpdr.org/) automatically and modified manually. Plasmid replicons, insertion sequences, and antimicrobial resistance determinants were determined using online tools (https://cge.cbs.dtu.dk/services/). BRIG and Easyﬁg were used to generate the genetic comparison ﬁgures (tet(X4)-bearing plasmids available in the NCBI nr database were downloaded for further analysis. The diversity and heterogeneous status of the tet(X4)-bearing plasmidome were investigated based on the Nanopore single-molecule analysis method -bearing plasmids generated in this study were deposited in the National Center for Biotechnology Information database (see https://figshare.com/s/6077f70a0ec952ee2796) for reference. Other data that support the findings of this research are available upon request.The base see . The ass10.1128/mSystems.00134-20.3FIG S3tet(X4)-bearing plasmids in transconjugants and their parental plasmids in donor strains to visualize molecular mechanisms of plasmid reorganizations among the five tet(X4)-bearing strains. The gray regions denote the homologous regions between the plasmids. The red arrow signifies the tet(X4) gene, and blue indicates gene clusters involved in plasmid reorganization. Download FIG S3, JPG file, 2.6 MB.Linear comparisons between Copyright © 2020 Li et al.2020Li et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Increased intratumoral mast cells foster immune suppression and gastric cancer progression through TNF-α-PD-L1 pathway. J ImmunoTher Cancer 2019;7:54. doi: 10.1186/s40425-019-0530-3Lv Y, Zhao Y, Wang X, Since the online publication of this article, the authors noticed that the results of tumor-infiltrating mast cells in inhibiting IFN-γ production was mistakenly duplicated in figure 5. The correct is figure is below:


In vitro studies from 3 established Doc resistant CRPC (DocRPC) cell lines are consistent with the clinical findings showing DocRPC patients had decreased Enz-sensitivity as well as accelerated development of Enz-resistance via enhanced androgen receptor (AR) splicing variant 7 (ARv7) expression. Mechanism dissection found that Doc treatment might increase the generation of ARv7 via altering the MALAT1-SF2 RNA splicing complex. Preclinical studies using in vivo mouse models and in vitro cell lines proved that targeting the MALAT1/SF2/ARv7 axis with small molecules, including siMALAT1, shSF2, and shARv7 or ARv7 degradation enhancers: Cisplatin or ASC-J9®, can restore/increase the Enz sensitivity to further suppress DocRPC cell growth. Therefore, combined therapy of Doc-chemotherapy with anti-ARv7 therapy, including Cisplatin or ASC-J9®, may be developed to increase the efficacy of Enz to further suppress DocRPC in patients.Anti-androgen therapy with Enzalutamide (Enz) has been used as a therapy for castration resistant prostate cancer (CRPC) patients after development of resistance to chemotherapy with Docetaxel (Doc). The potential impacts of Doc-chemotherapy on the subsequent Enz treatment, however, remain unclear. Here we found the overall survival rate of patients that received Enz was significantly less in patients that received prior Doc-chemotherapy than those who had not.  Chemotherapy with Docetaxel (Doc) has been established as the standard-of-care to treat patients with castration resistant prostate cancer (CRPC) at the metastatic stage , 2. MechThe average period of Doc-chemotherapy efficacy is 18 months before development of Doc-chemotherapy resistance (Doc-resistance). The androgen-deprivation-therapy (ADT) with Enzalutamide (Enz) may extend survival of CRPC patients by several more months , 5 beforvia increasing the ARv7 expression, which may then accelerate the development of Enz-resistance, thus reducing the efficacy of subsequent Enz treatment. Therefore, the potential combination of Doc-chemotherapy plus anti-ARv7 therapy may help increase the efficacy of subsequent Enz treatment to suppress Enz-resistant (EnzR) cell growth.We found that Doc-chemotherapy may induce adverse effects After PCa patients developed CRPC, the current standard therapy involves either Doc-chemotherapy or ADT with either Enz or Abiraterone . The potvs Doc-pretreated patients. PSA response was defined as 50% PSA decline from baseline during Enz treatment, PSA-PFS as the length of time from Enz initiation until time of 25% increase from baseline/nadir value, PFS as the length of time from Enz initiation until clinical or radiographic disease progression, and overall survival (OS) as the length of time from Enz initiation until death from any cause.We examined whether Doc-chemotherapy reduces the efficacy of subsequent Enz treatment, and retrospectively examined clinical outcomes in 114 consenting patients with metastatic CRPC that received Enz at Johns Hopkins Hospital between March 2012 and August 2014. Of those, 52 men received Enz prior to Doc-chemotherapy (Doc-naïve) and 62 men received Enz following Doc-chemotherapy (Doc-pretreated). We investigated prostate specific antigen (PSA) response rates, PSA progression-free survival (PSA-PFS), progression-free survival (PFS), and overall survival (OS); comparing outcomes in Doc-naïve patients As shown in the in vitro CRPC cells, we first established DocR CRPC (DocRPC) cell lines from CWR22Rv1 (named DocR1_CWR22Rv1) and C4-2 cells (named DocR3_C4-2), . We obtained another DocR_CWR22Rv1 cell line (named as DocR2-CWR22Rv1) from Dr. Carlos Cordon-Cardo -2-hydroxy-3-phenylpropanoate}) was purchased from LC Laboratories .ASC-J9PLKO.1-puro-shARv7 and PLKO.1-puro-shSF2 were constructed with target sequence 5′-GCGAGTAACAAGGGCATGGAA-3′ and 5′-CTTCAAGGTTTCGAGAGTTAA-3′, respectively, according to Addgene's pLKO.1 protocol. Lentiviral particles were generated by calcium phosphate transfection of lentivirus expressing plasmids, packaging plasmid psPAX2, and envelope plasmid pMD2.G into HEK 293T cells and lentiviral particles were collected to infect target cells according to Add gene's pLKO.1 protocol. The siMALAT1 was purchased from IDT. The transfection procedure was according to the instructions.GAPDH.  and 2 μgs of total RNA was subjected to reverse transcription using Superscript III transcriptase . Quantitative real-time PCR (qRT-PCR) was conducted using a Bio-Rad CFX96 system with SYBR green to determine mRNA expression levels of genes of interest. Expression levels were normalized to expression of Cells were lysed in lysis buffer and proteins (50 μg) were separated on 10% SDS/PAGE gel and then transferred onto PVDF membranes . After blocking membranes with 5% non-fat milk, they were incubated with appropriate dilutions of specific primary antibodies anti-AR , anti-ARv7 , anti-SF2 , anti-GAPDH , or anti-α-tubulin . The blots were then incubated with HRP-conjugated secondary antibodies  and visualized using the ECL system (Thermo Fisher Scientific).For EnzR clones, the DocR1_CWR22Rv1, DocR2_CWR22Rv1 and DocR3_C4-2 cells were selected by culturing cells with 20 μM Enz for 1-2 months and then prepared for MTT assays to analyze cells proliferation rates to check for resistance development.6 luciferase labeled PCa  cells were orthotopically injected into both anterior prostates. After 2 weeks of implantation, each set of mice were randomly assigned into experimental groups and treated with DMSO, 35 mg/kg Enz, 75 mg/kg ASC-J9®, or the combination of Enz+ASC-J9®, by i.p. injection 3x/wk/2wks. Tumors were monitored weekly by In Vivo Imaging System (IVIS).Male 6-8-week-old nude mice were purchased from NCI . The Matrigel mixture with 1-2x10t test or ANOVA. p ≤ 0.05 was considered statistically significant.Experiments were performed at least 3 times with data points in triplicate. Statistical analyses were carried out with Prism 6. The data values were presented as mean ± SD. Differences in mean values between two groups were analyzed by two-tailed Student’s Supplementary Figures


The aim of this study was to investigate the function of miR-3150b-3p in malignant behaviors of colorectal cancer (CRC). The tumor-inhibitive effect of miR-3150b-3p was determined by cell viability, invasion, and migration assays. The influence of miR-3150b-3p on the expression of Golgi phosphoprotein 3 (GOLPH3) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway was evaluated by luciferase reporter, qRT-PCR and western blot analysis. MiR-3150b-3p was markedly decreased in CRC cell lines compared with colonic mucosal epithelial cell line (FHC). Furthermore, miR-3150b-3p inhibited malignant biological behaviors by targeting GOLPH3, an oncogene in CRC. Additionally, we suggested that miR-3150b-3p ameliorated CRC tumorigenesis in vitro through GOLPH3-mediated JAK2/STAT3 pathway. MiR-3150b-3p might function as a promising tumor suppressor in CRC. MiR-3150b-3p expression was significantly decreased in colorectal cancer (CRC) tissues.The activation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) was implicated in CRC progression.Golgi phosphoprotein 3 (GOLPH3) is a novel oncogene in CRC.MiR-3150b-3p was lowly expressed in CRC cell lines.MiR-3150b-3p inhibited CRC cell growth via decreasing GOLPH3 expression.MiR-3150b-3p ameliorated CRC tumorigenesis via GOLPH3-mediated JAK2/STAT3 pathway.MiR-3150b-3p might be a valuable target for developing therapeutic strategy against CRC.Colorectal cancer (CRC) remains one of the most common malignancies worldwide.et al have observed higher levels of methylated miR-3150b in non-small-cell lung cancer tissues.MicroRNAs (miRNA) play crucial roles in a variety of biological processes,2 at 37°C.The human fetal colonic mucosa cell line (FHC) and CRC cell lines   were cultured in RPMI-1640 medium with 5% COHCT116 and SW480 cells in the logarithmic growth phase were seeded in 6-well plates. When these cells reached 30%–50% confluence, they were transfected with miR-3150b-3p mimic/inhibitor or their negative controls using Lipofectamine 2000 .Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay  as previously described.Briefly, transfected cells were wounded using a sterile micropipette tip, incubated in serum-free RPMI-1640 medium, and photographed under a microscope  at 0 and 48 hours after wounding.5 cells per well) were added to the upper chamber, while serum-supplemented culture medium was added to the lower chamber. Following 48 hours of incubation, the number of stained cells was calculated under a microscope.Cell invasion ability was assessed using transwell chambers coated with 40 µL Matrigel as previously described.The indicated luciferase plasmids  along with mimic NC or miR-3150b-3p mimic were co-transfected into HEK293T cells. Luciferase activities were analyzed 24 hours after transfection.Following standard quantitative PCR procedure, quantitative PCR was carried out for detecting miR-3150b-3p and GOLPH3 mRNA expression levels using U6 and β-actin as the internal controls.Protein concentrations were determined using a BCA assay kit . The rabbit antihuman antibodies against GOLPH3 , p-JAK2 (No 4406), JAK2 (No 3230), p-STAT3 (No 9145), STAT3 (No 12640), survivin (No 2808), c-myc (No 5605), matrix metalloproteinase (MMP)-2 (No 40994), MMP-9 (No 13667) and GAPDH (No 5174)  and secondary antibodies were used in this study. The expression of proteins was determined using the enhanced chemiluminescence reagent .All data were analyzed by one-way analysis of variance. Significant differences were indicated as p<0.05 or p<0.01.As shown in Then, miR-3150b-3p was overexpressed in HCT116 cells following transfection with miR-3150b-3p mimic and was knocked down in SW480 cells following transfection with miR-3150b-3p inhibitor. Transfection efficiency was confirmed as shown in We then performed the luciferase assay. The results revealed that miR-3150b-3p mimic could significantly decrease the luciferase activity of wild-type GOLPH3 3′-UTR vector in HEK293T cells . Figure The results indicated that ectopic expression of GOLPH3  could pa10.1136/jim-2019-001124.supp1Supplementary dataAs above-mentioned, miRNA-3150b-3p might inhibit the malignant phenotypes of CRC cells by targeting GOLPH3. However, whether miR-3150b-3p exerted its anticarcinogenic function via the JAK2/STAT3 signaling pathway remains unclear. As demonstrated by 10.1136/jim-2019-001124.supp2Supplementary dataIn the present study, we first found that miR-3150b-3p was frequently downregulated in human CRC cells. The overexpression of miR-3150b-3p inactivates the JAK2-STAT3 axis by downregulating the target gene GOLPH3, thereby inhibiting CRC tumorigenesis.et al showed that miR-4319 overexpression suppressed CRC carcinogenesis by regulating cell cycle distribution.et al reported that overexpression of let-7e significantly delayed cell proliferation, migration, epithelial-mesenchymal transition process and stemness, and promoted cell apoptosis in CRC cells.In recent years, abundant studies provide strong evidence that miRNAs act as tumor suppressor genes in CRC. For example, Huang et al found that miR-134 might directly target GOLPH3, thereby inhibiting cell proliferation in gastric cancer.GOLPH3 is a well-known oncogene in several solid tumors, such as hepatocellular carcinoma,Several lines of evidence suggest that abnormal activation of the JAK2/STAT3 signaling pathway is critical for the development and progression of various cancers, including CRC.In conclusion, miR-3150b-3p might be the potential target for treatment of CRC. Several limitations were included in our study. First, the in vivo experiments were excluded. Second, the other molecular mechanisms may be involved need to further investigation.


The vaccine efficacy of standard-dose seasonal inactivated influenza vaccines (S-IIV) can be improved by the use of vaccines with higher antigen content or adjuvants. We conducted a randomized controlled trial in older adults to compare cellular and antibody responses of S-IIV versus enhanced vaccines (eIIV): MF59-adjuvanted (A-eIIV), high-dose (H-eIIV), and recombinant-hemagglutinin (HA) (R-eIIV). All vaccines induced comparable H3-HA-specific IgG and elevated antibody-dependent cellular cytotoxicity (ADCC) activity at day 30 post vaccination. H3-HA-specific ADCC responses were greatest following H-eIIV. Only A-eIIV increased H3-HA-IgG avidity, HA-stalk IgG and ADCC activity. eIIVs also increased polyfunctional CD4+ and CD8+ T cell responses, while cellular immune responses were skewed toward single-cytokine-producing T cells among S-IIV subjects. Our study provides further immunological evidence for the preferential use of eIIVs in older adults as each vaccine platform had an advantage over the standard-dose vaccine in terms of NK cell activation, HA-stalk antibodies, and T cell responses. Immunization with the standard-dose seasonal inactivated influenza vaccine (S-IIV) remains the most effective public health intervention against infection by seasonal influenza A and B viruses for older adults. Yet, vaccine effectiveness can be lower in older adults compared to younger age groups2. Enhanced inactivated influenza vaccines (eIIV) that induce greater hemagglutinin inhibition (HAI) titers to the immunodominant surface HA glycoprotein and confer superior immunogenicity and/or vaccine efficacy compared to S-IIV in preventing influenza-related medical complications have recently become available5. These eIIVs, include Fluad , fluzone-high-dose , and Flublok .Older adults (≥65 years) account for the majority of influenza-related morbidity and mortality each year+ T cells6 and CD4+ T cell helper activity7. MF59 is also a potent inducer of germinal center (GC) reactions and increases the magnitude, diversity, affinity8, and persistence of influenza virus-specific antibodies. Fluzone-high-dose contains four times the amount of HA protein than standard-dose S-IIV. The benefit of high-dose over S-IIV varies depending on seasonal strain dominance and appears strongest during A(H3N2) dominant seasons9. In older adults, this vaccine stimulates greater T follicular helper (TFH) cell activation and plasmablast recruitment10, while IFN-γ+ T cell responses are boosted but comparable to S-IIV11. Flublok contains three times the amount of HA as S-IIV. Although not originally designed solely for older populations, Flublok provided improved protection against laboratory-confirmed infection in older adults during a A(H3N2) dominant season despite an antigenic mismatch between the vaccine and circulating A(H3N2) strain4. As a recombinant protein vaccine produced via insect cell culture-based baculovirus expression systems, Flublok also has an advantage in that it can be made without egg-based adaptations.Fluad contains the MF59 adjuvant, which is an oil-in-water emulsion of squalene. MF59-adjuvanted influenza vaccines have been shown to boost IFN-γ12 despite representing only a fraction of the total immune response to vaccination. Instead, protection likely requires a multi-pronged immune response involving a range of humoral and cellular mechanisms that cannot be assessed alone by traditional HAI assays. Furthermore, influenza infection may occur in individuals despite high HAI responses following vaccination or infection13, or in some cases, individuals may not become seropositive (HAI titer ≥ 1:40)14. Universal influenza vaccine development is currently a global priority to improve protection against diverse influenza virus strains and across all age groups. This will first require the identification of additional CoPs with known clinical efficacy. Hence, a deeper understanding of how currently available vaccines stimulate multiple arms of the immune system is necessary.Antibodies measured by the HAI assay have traditionally been used as the gold standard of vaccine-induced correlate of protection (CoP)15 and age-related impairment in the recruitment and helper capacity of TFH cells have been associated with suboptimal antibody responses in older adults16. Antibody-dependent cellular cytotoxicity (ADCC) and memory T cell responses have been associated with protection against symptomatic infection19 and may represent additional correlates of protection in older adults. Meanwhile, there remains unresolved concerns for immune interference by prior or repeat vaccination and infection on current season vaccine effectiveness based on observational studies20, which is most relevant to older adults who may typically receive multiple consecutive vaccinations.Recently, advances have been made in identifying additional CoPs. TFH cells have been shown to play a crucial role in the generation of antibody responses following S-IIV vaccination21. We previously reported that all three vaccines induced greater day 30 HAI titers and mean fold rise of HAI antibodies against A(H3N2) vaccine-matched, egg-derived virus compared to S-IIV21. R-eIIV was superior when neutralizing antibody titers were assessed using cell-derived A(H3N2) virus, followed by H-eIIV and A-eIIV, and all eIIVs stimulated some boosting of T cell responses to multiple influenza virus strains. In this current study, we further analyzed a subset of participants to determine whether improved immunogenicity following enhanced vaccination may be attributable to other humoral and cellular parameters besides HAI. Since the A(H3N2) virus undergoes antigenic drift more rapidly than A(H1N1) virus subtypes, it poses a more significant hurdle for vaccine-mediated protection22 and we, therefore, focused our study on assessing responses against A(H3N2).We conducted a randomized controlled trial to compare the immunogenicity of three vaccines, Fluad, fluzone-high-dose, and Flublok versus standard Fluquadri (S-IIV)+ TFH cells and multiple-cytokine-secreting CD4+ and CD8+ T cells to study the interplay between humoral and cellular responses. These immune parameters were selected due to their contribution in mediating the immune response against the influenza virus as well as their relevance for universal vaccine development. Finally, we explored whether enhanced vaccination has the potential to revive immune responses among individuals with extensive prior vaccination history.For a more comprehensive insight into humoral immune responses, we measured the magnitude of antibody responses directed against HA protein as well as the quality of response by assessing polyclonal antibody avidity, stalk-binding activity, and IgG subclass distribution related to Fc receptor-mediated (FcR) effector functions. We also measured the recruitment of CD4n = 37), A-eIIV (n = 34), H-eIIV (n = 30), and R-eIIV (n = 23)  and cellular assays (n = 13–24 per group) were performed in parallel for most participants, where matched specimens and complete time-points were available (Table We examined antibody and cellular responses following S-IIV 23) Fig.  vaccinat23) Fig.  randomly3 Fig.  v21 and determined using vaccine-matched, egg-derived A(H3N2) A/Hong Kong/4801/2014 (H3-2014) virus. Subsequent humoral assays were performed using A(H3N2) A/Switzerland/9715293/2013 (H3-2013) HA0 recombinant protein, as only the HA1 domain of the vaccine A/Hong Kong/4801/2014 was commercially available at the time of study onset. H3-2013 was last used during the 2015/2016 vaccination season. Therefore, measurement of H3-2013-specific responses resulting from the 2017/2018 vaccination may also represent back-boosting of 2015/2016 vaccine responses for H3-2013-specific B cells. Nevertheless, the IgG response against both HA proteins was significantly correlated , with IgG1 and IgG3 being the predominant mediators of anti-viral effector functionsnes Fig. . Whereasnes Fig. . plays a crucial role in helping B cell plasmablasts (CD27hiCD38hiCD19+)   Fig.  and a pe6−) Fig.  at day 7+PD1+ TFH1 cells (p < 0.05)  HAI responders (≥4-fold rise seroconversion) had significantly higher recruitment of ICOS05) Fig.  comparedy 7 Fig. , and ICOIgG Fig.  titers a26 and cytotoxic CD8+ T cells correlate with protection from influenza17. We, therefore, analyzed the frequencies of single-positive , double-positive , and triple-positive (IFN-γ+TNF-α+IL-2+) cytokine-secreting CD4+ and CD8+ T cells following vaccination  CD8+ T cells in A-eIIV (p = 0.03) and R-eIIV (p = 0.0006), a subset of T cells previously described for its importance in reducing disease severity during pandemic H1N1 infection17. Moreover, the emergence of double (IFN-γ+TNF-α+IL-2−) and single (IFN-γ+TNF-α−IL-2−) CD8+ T cells in the A-eIIV group also positively correlated with HAI titers  or “not regularly immunized” (≤2 vaccinations) in the past 5 years       Fig.  and sign04) Fig. , high-av02) Fig.  and ADCC03) Fig. . In cont03) Fig.  and ADCCp = 0.002)  Fig. .28. We found that enhanced vaccines consistently outperformed standard-dose vaccination across a range of immune parameters. A lack of confirmed CoPs for influenza virus infection beyond HAI and microneutralization (MN) means that despite our systematic dissection of immunogenicity, we are unable to ascertain a single enhanced vaccine for preferential use in older adults.Multiple enhanced seasonal influenza vaccines are now available for older adults, yet a consensus on the prioritization of a preferred vaccine has not been determined due to a lack of consistent data from different studies29. A previous study also observed that an early peak in IgG avidity coincided with the recruitment of activated ICOS+PD-1+ TFH1 cells leading to rapid recruitment of memory B cells (MBC) within 7 days post vaccination, possibly in extrafollicular sites30. In addition, previous studies have shown that TFH1 cells help MBCs but not naïve B cells differentiate into antibody-secreting plasmablasts and plasma cells in vitro15 and that plasmablasts induced by influenza vaccination are often derived from MBCs31. It is possible that the advantage we observed with the MF59 adjuvant is due to an early TFH1 rise which leads to more rapid and efficient MBC selection, clonal expansion, and superior antibody quality. Similarly, a MF59-adjuvanted monovalent pandemic H1N1 vaccine also increased the avidity of HA1-specific antibodies when assessed by surface plasmon resonance and ELISA8. In our study we observed an increase in anti-H3-HA IgG antibody avidity for A-eIIV recipients, which may be driven by pre-existing HA head-specific MBCs, as there was no increase in the avidity of stalk-specific antibodies.We found that A-eIIV stimulated an early (day 7) increase in the proportion of high-avidity antibodies compared to S-IIV but it is not maintained long term, which has also been observed by others+ TFH cells are essential for the selection, expansion, and differentiation of memory MBCs into antibody-secreting plasmablasts and plasma cells. Prior studies have detected transient HA-specific circulating TFHs following influenza vaccination32 that are clonally related to GC-derived TFH cells33. Therefore, we quantified total circulating TFHs as a surrogate for influenza virus-specific TFH response occurring in the lymphoid compartment. We found a positive correlation between the recruitment of TFH1 cells and the increase of day 30 anti-HA antibodies following vaccination with A-eIIV and H-eIIV  CD8+ T cells were readily boosted by A-eIIV and R-eIIV vaccines concurrently with polyfunctional cytokine responses . Increased antigen dosage may factor into more efficient cross-presentation, although the intrinsic ability for cross-priming of exogenous antigens is reported to be generally low49. It may be possible that improved IgG responses lead to greater antibody-mediated opsonization of viral proteins or immune complex formation, which in turn promote antigen uptake and cross-presentation by APCs leading to improved CD8+ T cell responses50.We also observed an increase in multiple-cytokine-secreting CD851. Furthermore, approximately 60% of the serum antibody repertoire following vaccination consists of pre-existing clonotypes and not de novo responses toward the vaccine strain52. Therefore, our use of the A/Switzerland-lineage H3-2013 HA protein and virus remains relevant. Secondly, we were limited by the number of participants that were able to provide additional blood for PBMC isolation resulting in a limited amount of matched serological and cellular data. This also restricted our ability to stratify data and we have presented pooled datasets when studying vaccination history. Almost two-thirds of our selected participants were previously vaccinated in the prior season and nearly three-quarters had at least one vaccination in the last 5 years. Thus, assessing the full impact of vaccination history and vaccine responsiveness will require further longitudinal analysis in subsequent seasons of our trial. Finally, vaccine immunogenicity is not equivalent to protective efficacy and larger studies are needed to determine immune-mediated protection. As there is yet to be defined, quantitative thresholds for CoPs beyond HAI and MN, we cannot ascertain whether responses we have identified reach protective levels.Our study had a number of limitations. Firstly, our experiments used recombinant A(H3N2) A/Switzerland/9715293/2013 HA protein and were mismatched for the vaccine strain A(H3N2) A/Hong Kong/4801/2014, although responses had substantial cross-reactivity in vitro , and all of the patients provided informed consent prior to inclusion in the study. The study protocol was approved by the Institutional Review Board of the University of Hong Kong (UW:16-2014).Following randomization, a total of 800 donors (200 per group) received either Fluquadri , Fluad , Fluzone-High-Dose  or Flublok . All vaccines contained antigens from A/Michigan/45/2015 (H1N1), A/Hong Kong/4801/2014 (H3N2), and B/Brisbane/60/2008 (Victoria lineage); quadrivalent vaccines contained additional antigens from B/Phuket/3073/2013 (Yamagata lineage). Five years of prior vaccination history for each subject was based on clinical records or self-reporting (Table 21. Heparinised blood was collected from a subset of donors immediately before and days 7 and 30 post vaccination (see Table 5 live cells during FACS (n = 23–24 per vaccine group). TFH and plasmablast staining was performed on fresh blood at the time of collection. However, there were technical issues with immunostaining optimization and complete data across all three time-points was only available for a subset of subjects (n = 13–24 per vaccine group). HAI experiments were performed for all study participants who donated additional blood samples for plasma and PBMC isolation (n = 23–37 per vaccine group), as previously described21.Clotted blood was collected immediately before and 30 days post vaccination. Sera was isolated for HAI assays, as previously described21. Briefly, RDE-treated and HI sera were serially titrated in phosphate-buffered saline (PBS) and incubated with four hemagglutinating units of egg-propagated A/Hong Kong/4801/2014 (H3N2) antigen for 1 h at room temperature and then with 0.5% turkey red blood cells for 30 min at room temperature. HAI titers was recorded as the reciprocal of the highest sera dilution that inhibited hemagglutination.The HAI assay was carried out as described previouslyEscherichia coli expressed HA-stalk proteins from A/Hong Kong/1/1968  or A/Puerto Rico/8/1934 53 . Plates were rinsed, blocked with 1% fetal bovine serum (FBS) in PBS, incubated with 1:100 HI plasma diluted in 0.05% Tween-20/0.1% FBS in PBS for 2 h then rinsed again, and incubated with detection antibodies for 2 h using IgG-HRP , IgG1-HRP , IgG2-HRP  IgG3-HRP  or IgG4-HRP (1:4000 HP6025) (IgG1/2/3/4 detection antibodies from SouthernBiotech). For detection of high-avidity antibodies, an additional 8 M urea wash step (3× 5 min) was performed on a separate plate before the addition of anti-IgG-HRP detection antibody54. The color was developed with equal amounts of stabilized hydrogen peroxide and tetramethylbenzidine (R&D systems) for 20 min, stopped with 2N sulfuric acid and absorbance values were recorded at 450 nm on a spectrophotometer (Tecan Life Sciences). The avidity index was calculated as a percentage of the optical density with urea-treatment versus without treatment. A 4-parameter logistic regression (4PL) method was used to interpolate absorbance values from a standard curve generated from purified human IgG .Plates  were coated with one representative influenza virus protein at a time. Plates were coated with either 1 μg/ml of purified baculovirus-expressed HA protein from A/Switzerland/9715293/2013 (H3-2013), nucleoprotein (NP) from A/Switzerland/9715293/2013, HEK293-expressed HA protein from A/California/07/09 (H1-2009), HEK293-expressed neuraminidase (NA) protein from A/Hong Kong/4801/2014 (N2-2014), A/Michigan/45/2015 (N1-2015) , Plates were coated with 4μg/ml of H3-2013 or G2-stalk protein, rinsed and blocked, incubated with 1:400 HI plasma diluted in PBS for 2 h, then incubated with NK-92-FcRγIIIA-bearing cells (Fox Chase Institute for Cancer Research) at 37 °C for 5 h. Cells were then stained using anti-human CD56-PE (5.1H11) and CD107a-APC (H4A3) (Biolegend), fixed with 4% paraformaldehyde (PFA) and acquired by flow cytometry (AttuneNxT). Purified anti-human LEAF CD16 antibody (3G8) and irrelevant HIV-1 gp120 protein were used as positive and negative controls, respectively. Responses were subtracted for background and normalized to the percentage of maximum positive controls.Whole blood samples were stained with indicated antibodies . The TFH panel contained: anti-human CD3-PE/Dazzle 594 (UCHT1), CD4-AlexaFluor700 (SK3), CXCR5-PerCPCy5.5 (J252D4), CD45RA-FITC (HI100), CCR6-BV605 (G034E3), CXCR3-APC (G025H7), PD-1-BV711 (EH12.2H7), and ICOS-PE (C398.4A). A separate plasmablast panel contained: CD19-BV510 (HIB19), CD27-FITC (M-T271), and CD38-BV421(HIT2). Cells were acquired by flow cytometry (BD LSRFortessa).Cryopreserved PBMCs were thawed and restimulated with a multiplicity of infection (MOI) 4 of UV-irradiated A/HK/4851970/2014 virus (H3N2 Switzerland lineage), PMA/ionomycin or RPMI alone for 6 h at 37 °C. Brefeldin A and monensin were then overnight. Cells were stained with Zombie-NIR (Biolegend) followed by anti-human CD3-PE/Dazzle 594 (UCHT1), CD4-BV605 (OKT4), CD8-AlexaFluor700 (SK1), CD107a-PacificBlue (H4A3), CCR5-PE (J418F1), CCR7-PerCP/Cy5.5 (G043H7), and CD45RA-APC (HI100) and a dump channel containing CD19-BV510 (HIB19), CD56-BV510 (HCD56), and CD14-BV510 (M5E2). Cells were then permeabilized and fixed (BD Cytofix/cytoperm) and further stained for anti-human IFN-γ-FITC (4S.B3), IL-2-PECy7 (MQ1-17H12), and TNFα-BV711 (MAb11). Stained cells were acquired via flow cytometry (AttuneNxT). Background no virus responses were subtracted from each sample.t test (unpaired) was used. Correlations were performed using Spearman’s test. Differences in baseline characteristics were detected with the chi-square test. Adjusted p values < 0.05 were considered statistically significant.Statistical analysis was performed on Prism 7 (Graphpad). For multiple-group comparisons, a Friedman (paired) or Kruskal–Wallis (unpaired) test, followed by the Dunn-Bonferroni post-hoc test was used. For two-ground comparison, the Wilcoxon signed-rank test (paired) or Mann–Whitney Further information on research design is available in the Supplementary figuresReporting Summary


C. elegans . Consistent with the role of a terminal selector, unc-42(e419) loss-of-function mutants were shown to lack the expression of the putative chemoreceptor genes sra-6 and srb-6 specifically in the ASHs, while, for example, srb-6 expression in the ADL and ADF sensory neurons was unaffected . unc-42(e419) mutants also lack ASH expression of several other terminal identity markers: three Gα encoding genes , the neuropeptide encoded by flp-21, and the eat-4 vesicular glutamate transporter . Furthermore, loss of UNC-42 function disrupted behavioral responses to high osmolarity and nose touch , which are both detected primarily by the ASHs . However, the severity of the nose touch defect was likely due to developmental defects in both the ASH sensory neurons as well as some downstream interneurons, where altered glutamate receptor expression accompanies loss of UNC-42 .The genes encoding transcription factors that initiate the terminal differentiation programs within individual neurons have been termed “terminal selectors” (Hobert 2008). They are considered master regulators of neuronal identity because they integrate upstream lineage inputs to ultimately drive (directly and/or indirectly) the expression of all terminal differentiation features of a neuron . The paired-like homeodomain transcription factor UNC-42 has been proposed to function as the terminal selector in the developmental specification of the ASH polymodal nociceptors in Table 1: Summary of the effect of unc-42(gk598) on ASH terminal marker gene expression*sra-6 expression in ASH was previously reported to be affected by unc-42(e419) .unc-42(gk598) allele, we confirmed the role of UNC-42 in regulating ASH terminal markers, and identified seven additional genes whose ASH expression strongly depends upon UNC-42  mutants, with only ~20% of animals losing expression in one ASH (image not shown). In addition, ASH expression of some genes  was found to be unaffected in unc-42(gk598) animals (Table 1). We note that several of these are either expressed broadly throughout the nervous system, or function in several sensory neurons. As such, they may not necessarily be constituents of the terminally differentiated gene battery of the ASHs. Combined, UNC-42 broadly regulates the expression of ASH identity markers, although additional transcription factors are likely to also contribute.Using the DiD was purchased from Molecular Probes (Invitrogen).unc-42(gk598) strain was generated by the C. elegans Reverse Genetics Core Facility at the University of British Columbia, which is part of the International C. elegans Gene Knockout Consortium. The gk598 allele contains a 1430 basepair deletion . VC1444 was outcrossed 6x to N2 to generate FG498 for use in this study. Some of the strains used in this study were obtained from the Caenorhabditis Genetics Center, which is funded in part by the National Institutes of Health – National Center for Research Resources.The VC1444 unc-42(gk598), CX3465 kyIs39 [sra-6::gfp + lin-15(+)], HA1695 rtIs27 [osm-10p::gfp], PY5163  line 1, NK885 unc-119(ed4);qyIs174 [hlh-2p::gfp::hlh-2 + unc-119(+)], CX2565 kyIs4 lin-15A(n765) [ceh-23p::unc-76::gfp + lin-15(+)], RW10748 unc-119(ed3); zuIs178; stIs10024; stIs10549 [stIs10549 = nhr-79::H1-wCherry + unc-119(+)], HA357 rtEx251 [nlp-15p::gfp + lin-15(+)]; lin-15B lin-15A(n765), AL132 icIs132 [unc-40::gfp], HA341 lin-15; rtEx235 [nlp-3::gfp], LX990 lin-15B lin-15A(n765); vsEx494 [ocr-2p::gfp::ocr-2 3’utr + lin-15(+)], CX3716lin-15B lin-15A(n765) kyIs141 [osm-9::gfp5 + lin-15(+)], IC692 quEx162 [sax-3p::gfp + (pRF4) rol-6]. Transgenes were examined in the wild-type andunc-42(gk598) background. For the generation of extrachromosomal arrays, germline transformations were performed as previously described . Plasmids injected for analysis include: sri-14p::venus, pFG258 srd-33p::gfp and pFG233 srd-10p::gfp. Transgenic lines were examined in the N2 wild-type background, and at least one array was chosen to be crossed into the unc-42(gk598) background for comparison. Strains generated in our lab for this study have not been sent to the CGC.Strains used in this study include: N2 Bristol wild-type, FG498