Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5′-pyrophosphohydrolase (RppH) and bis(5′-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.It has been more than 50 years since the discovery of dinucleoside polyphosphates (Np nNs) exist as Escherichia coli RNA caps which can be cleaved by 5′-pyrophosphohydrolase (RppH) and bis(5′-nucleosyl)-tetraphosphatase (ApaH).Nicotinamide adenine dinucleotide and coenzyme A serve as a 5′-cap of prokaryotic RNA. Here the authors report that methylated and non-methylated dinucleoside polyphosphates (Np NpnNs are ubiquitous molecules15 present in both prokaryotic and eukaryotic organisms.Their intracellular concentrations can increase from the μM to the mM range under stress conditions16, which is why they are often called alarmones. Nevertheless, the molecular targets of the alarm signaled by NpnNs have not been identified yet. The main source of the NpnNs is the back reaction of an aminoacyl adenylate with an acceptor nucleotide catalyzed by various aminoacyl-tRNA synthetases17. But there is evidence that e.g., the ubiquitinylation process can lead to Ap4A or Ap3A (ref. 18). Even though they have a similar chemical structure to known RNA caps, such as NAD (refs. 20) and the 7-methylguanylate cap21, they were never detected as a part of RNA.The role and chemical structure of the 5′-end of prokaryotic RNA is still unclear. The discovery of nicotinamide adenine dinucleotide (NAD)nNs can be accepted by two types of RNA polymerases  as non-canonical initiating nucleotides (NCINs) in in vitro transcription. To prove the existence of NpnN-RNA caps in vivo, we develop an liquid chromatography–mass spectrometry (LC–MS) technique for the detection of NpnNs in isolated and digested RNA. We detect six previously unknown NpnN-caps  in fractions of short RNA (sRNA) from Escherichia coli harvested in exponential phase and additional three NpnN-caps  in late stationary phase. Some of the detected NpnN-caps are mono- or dimethylated. We identify two enzymes, RppH from the NudiX family and bis(5′-nucleosyl)-tetraphosphatase (ApaH), as decapping enzymes that can cleave NpnN-RNA. While the methylations of NpnN-caps protect the RNA from decapping by RppH, ApaH cleaves even the methylated forms of NpnN-caps. We propose that bacteria use methylated caps as protection against RNA degradation under starvation conditions.In this work, we show that NpnNs , we performed in vitro transcriptions in the presence of different NpnNs  and E. coli RNAP (multi-subunit enzyme). T7 RNAP was selected as a first tool to explore the capability of NpnNs to be substrates of RNAPs as it was previously shown to be able to use comparably sized non-canonical initiating substrates23. Consistently, based on three-dimensional structures, the nucleotide-binding pockets for initiation phase of T7 (ref. 24) and E. coli22 RNAP are spacious enough to accommodate such substrates.To investigate whether NpnNs Fig.  can servTM 5′-phosphate-dependent exonuclease  prepared for a translation inhibition study25.The in vitro transcription produced a mixture of capped and uncapped RNA  ATP and GTP concentrations  to NpnN was 1, we observed between 27% (for Ap6A) and 46% (for Ap4G) of capped products. The NAD-capped RNA was produced in a comparable amount to the majority of NpnNs. The similar behavior of NpnNs to NAD supports the theory that these molecules might be present as 5′-RNA caps in cells.To identify the best substrate for T7 RNAP under in vitro conditions, we varied the concentrations of the Npons Fig. . The amoE. coli RNAP that is known to accept NAD as an NCIN both in vitro and in vivo27  for E. coli RNAP. In both cases, we observed a higher production of NpnN-RNA compared to NAD-RNA .Subsequently, we tested the RNA Fig. . NeverthnNs exist as 5′-RNA caps in vivo in E. coli. We established an LC–MS method for their detection in RNA. As the intracellular concentration of NpnNs is known to grow under stress conditions28, we collected cells from exponential  and late stationary (STA) phases of growth. We focused on sRNA where the NAD-cap2 and the CoA-cap3 have also been detected. The purified sRNA was washed to remove all non-covalently interacting molecules and digested by Nuclease P1 into the form of nucleotides , Ap3G ([M−H]− at m/z 771.071), and Ap5A , dimethyl-Gp4G ([M−2 H]2− at m/z 447.017), and methyl-Ap5G , methyl-Ap5A ([M−2 H]2− at m/z 463.992), and dimethyl-Ap5G  and 2′-O (Gm) similarly to eukaryotic m7Gp3Nm RNA caps. We used custom-synthesized m7Gp4Gm, m1Gp4Gm, and m2Gp4Gm. In parallel, we synthesized all possible mono-methylated Ap3A: m6Ap3A, m1Ap3A, and Amp3A  helped us to identify m6Ap3A as another cap in E. coli RNA , the concentration of m7Gp4Gm was significantly higher (1200 fmol per µg of sRNA) and comparable to NAD (1900 fmol per µg of sRNA).We compared the amounts of mEXP Fig. . In geneSTA Fig. . While tnN-capped RNAs are produced in E. coli, degradation mechanisms of the capped RNA in E. coli must also exist. ApnA have been reported to be in vitro substrates for the E. coli NudiX enzyme NudH (RppH (refs. 29)) and ApaH (ref. 30). RppH is an E. coli decapping enzyme of 5′-ppp RNA31 and 5′-diphosphate RNA8. To assess whether ApnA-capped RNA can be an RppH or ApaH substrate in vivo, we prepared NpnN- and NAD-capped RNA by in vitro transcription, and tested the products as substrates for both enzymes. First, we added RppH or ApaH, to cleave the 5′-capped RNA  are poor substrates for the T7 RNAP  on the mixture of isolated sRNA with a spiked model Gp1 h Fig. .nN-caps to RppH cleavage, we performed molecular dynamics (MD) simulations of the interaction between RppH and two models of NpnN-capped RNA (Gp4G-G and m7Gp4Gm-G). In the MD simulations, we observed that the interactions with the arginines R28 and R86 were lost when the methyl groups were present  hampers the cleavage of mRNA by NudiX decapping enzyme Dcp2 (ref. 33). Our proposed mechanism correlates with this finding and suggests that the strategy, through which cells protect their RNA against decapping by methylation, is general and may also be found in higher organisms. Concurrently, a work reporting the existence of Ap4N-RNA caps in E. coli was published34. However, we did not detect any of the caps reported therein by our LC–MS technique, as the Ap4N-RNA caps were detected under different stress conditions using a different detection technique.In summary, we identified Npess Fig. . In exponNs can be explained via their RNA capping potential. Moreover, the 5′-terminal NpnNs may interact with a wide range of cellular partners and influence, e.g., cellular response to starvation. In the near future, besides searching for the methyltransferases responsible for the methylation of the NpnN-caps, the key challenge lies in the development of specific techniques to identify the NpnN-capped RNA.In conclusion, it is intriguing to consider the possibility that many functions of NpAll chemicals were either purchased from Merck or Jena Biosciences and used without further purification. Oligonucleotides were purchased from Generi Biotech. The list of sequences is present in Supplementary Table Denaturating polyacrylamide gels (PAGE) were visualized by a Typhoon FLA 9500 imaging system.23 in a 25 μL mixture containing: 80 ng/μL of template DNA (35 A or 35 G), 1 mM UTP, 1 mM CTP, 0.8 mM GTP, or 0.8 mM ATP, respectively, and 0.2 μL α 32P GTP or α 32P ATP (activity: 9.25 MBq in 25 μL), respectively, 1.6 mM (0.2–1.6 mM for incorporation efficiency experiments) NpnNs, 5% dimethyl sulfoxide (DMSO), 0.12% triton X-100, 12 mM dithiothreitol (DTT), 4.8 mM MgCl2 and 1× reaction buffer for T7 RNAP and 62.5 units of T7 RNAP . The mixture was incubated for 2 h at 37 °C. The samples (3 μL) were mixed with 3 μL of 2× RNA loading dye (NEB) and analyzed by 12% PAGE .In vitro transcription was performed using a standard protocol2, 0.5 mM CaCl2, pH 7.6 at 25 °C, supplied with the enzyme), and 4 units of DNAse I (NEB) were incubated at 37 °C for 60 min. The enzyme was heat deactivated at 75 °C for 10 min followed by immediate cooling on ice. All samples were purified on RNA mini Quick Spin Columns (Merck) for further use.The DNA template was digested by DNAse I to obtain pure RNA. A total of 25 μL of the transcription mixture, 3 μL of 10× reaction buffer for DNAse I  were treated with 20 units of 5′-polyphosphatase (Epicenter) in the solution of 1× buffer in a total volume of 20 μL for 1 h at 37 °C. For the negative control, the enzyme was replaced by the same amount of water. The samples (3 μL) were mixed with 3 μL of 2× RNA loading dye and analyzed by 12% PAGE .Because of the incompatibility of the buffers, all samples were purified on RNA mini Quick Spin Columns before the reaction. The RNA (500 ng) was treated with 1 unit of Terminator™ 5′-phosphate-dependent exonuclease (Epicenter) in the solution of 1× reaction buffer A and the mixture was incubated at 30 °C for 1 h.rrnB P1 or 10 ng/μL of the amplicon FV21 possessing rnaI promoter, 8 μL of 5× buffer, 90 mM NaCl, 0.2 mM ATP, 0.8 mM NpnNs, and 1 unit of E. coli RNAP holoenzyme (NEB). This mixture was preincubated for 10 min at 37 °C. The transcription was started by addition of an initiation mixture containing 0.2 mM CTP and UTP, 0.15 mM GTP and 0.5 μL α 32P GTP (activity: 9.25 MBq in 25 μL) and incubated at 37 °C for 1 h.In vitro transcription was performed in a volume of 40 μL. At first, the mixture contained 10 ng/μL of template plasmid 458 with promoter 35. The sum of areas under the peaks corresponding to the 5′-ppp RNA (Arp) and 5′-capped RNA (Arcap) were integrated. The percentage of 5′-capped RNA species were calculated according to: (Arcap/ Arp + Arcap) × 100.The experiments were performed in triplicates and the amount of capped RNA was calculated from PAGE analysis using the software ImageJE. coli ApaH (EG10048) was prepared by PCR  using primers #3071 and #3072. Subsequently, the gene was inserted into expression vector pET22b (Novagen) via unique NdeI and XhoI restriction sites. The additional six histidine residues at the C-terminus served as the His-tag facilitating the protein purification process, and the two residues (amino acids L and E) preceding the His-tag, were inserted because of the restriction enzyme (XhoI) used for cloning. The resulting construct (LK2505) was verified by sequencing. The verified plasmid was transformed into expression strain E. coli DE3 resulting in strain LK2508.The gene for 600 reached 0.6 and the culture was allowed to grow subsequently for 3.5 h at 25 °C. ApaH was purified similarly to RNAP36. Briefly, 1 L grown culture was harvested by centrifugation  and cells were washed with sonication buffer . The cells were then resuspended in 25 mL sonication buffer, sonicated 12 × 10′′  on ice with 1 min intervals between sonications, and subsequently centrifuged . The supernatant containing ApaH was mixed with 1 mL Ni-NTA agarose  and incubated for 2 h on ice (constant mixing). The mixture was then applied on a Poly-Prep chromatography column, washed with 30 mL sonication buffer and then with 30 mL sonication buffer containing 30 mM imidazole. ApaH was eluted with sonication buffer containing 400 mM imidazole (5 × 0.5 mL) and fractions were analyzed by sodium dodecyl sulfate–PAGE. Purified ApaH was dialyzed into storage buffer  and stored at −20 °CThe expression strain LK2508 was grown in rich lysogeny broth (LB) medium containing ampicillin (100 μg/mL) at 37 °C. Expression of ApaH was induced by IPTG (0.8 mM) when OD600 reached 0.5 and the culture was allowed to grow subsequently for 3 h at 25 °C. The pellet was sonicated 12 × 10′′  in Tris buffer (50 mM Tris-HCl (pH 8), 1 M NaCl, 5 mM MgSO4, 5 mM 2-ME, 5% glycerol, and 5 mM imidazole). The supernatant was mixed with Ni-NTA agarose and washed with 30 mL Tris buffer and 30 mL Tris buffer with 30 mM imidazole. NudC was eluted with Tris buffer with 400 mM imidazole. Purified NudC was dialyzed into storage buffer (50 mM Tris-HCl (pH 8), 0.5 M NaCl, 50 mM KCl, 1 mM MgCl2, 5 mM DTT, and 50% (v/v) glycerol) and stored at −80 °C.The expression strain 1827 was grown in rich LB medium containing kanamycin (50 μg/mL) at 37 °C. Expression of NudC was induced by IPTG (0.8 mM) when OD2, 10 mM DTT, pH 7.9 at 25 °C, supplied with the enzyme) and 650 nM RppH (NEB). For ApaH or NudC cleavage: 2 μL of 10× buffer NudC  and 650 nM ApaH or 6.5 μM NudC. RNA was replaced by water for the negative control. The mixtures were incubated at 37 °C for 1 h and purified on RNA mini Quick Spin Columns. A total of 3 μL of the purified samples were mixed with 3 μL of 2× RNA loading dye for analysis and 6 μL were used for the Terminator treatment.To test the cleavage of the 5′-caps, the RNA samples were divided into two parts. The positive control contained ~700 ng of the RNA , for RppH cleavage: 2 μL of 10× buffer 2  after in vitro transcription with α E. coli strain DH5α containing pUC18 was used for studying RNA modification in two different growth conditions: exponential phase and stationary phase. The growths were performed in LB medium (Merck) in the presence of 0.1 mg/mL of Ampicillin . Parallel cultures of E. coli were inoculated in Erlenmeyer flasks from cultures grown on LB Agar. Cultures (1 L) were grown at 37 °C until an optical density at 600 nm (OD600) of 0.3 was reached . Cultures (1 L) were harvested after 2 h they reached an OD600 of 1.6 . All cells were harvested by centrifugation at 5000 × g for 10 min. The pellets were washed once with PBS and stored at −80 °C.The E. coli strains DH5α containing pUC18 were grown in M9 minimal medium containing 1× M9 salts , 2 mM MgSO4, 0.1 mM CaCl2, and 0.4% glucose). The growth was performed in the presence of 0.1 mg/mL of Amp. Parallel cultures (1 L each) containing 14NH4Cl or 15NH4Cl (as sole source of nitrogen) were inoculated in Erlenmeyer flasks from cultures grown overnight in M9 medium (50 mL). All cells were harvested by centrifugation at 5000 × g for 10 min after 48 h of growth (OD = 0.5), washed once with PBS and stored at −80 °C.E. coli was isolated using the RNAzol protocol (Merck). The pellets  were suspended in 4 mL lysozyme solution  each and incubated for 1 h at 7 °C. RNAzol (10 mL) was added to the lysate to isolate the RNA. After shaking vigorously for 15 s, the mixture was incubated for 15 min on ice and centrifuged at 6000 × g for 45 min. In this step, the DNA, proteins, and most polysaccharides formed a semisolid pellet at the bottom of the tube. The RNA, which remained in the supernatant, was mixed with 0.4 volumes of 75% ethanol (v/v). After storing on ice for 10 min, the sample was centrifuged at 6000 × g for 16 min for pelleting the long RNA. The supernatant containing sRNA was mixed with 0.8 volumes of isopropanol. After storing on ice for 30 min, the sample was centrifuged at 6000 × g for 40 min for pelleting the sRNA. The pellets containing the long and sRNA were washed twice with 75% ethanol (v/v) and 70% isopropanol (v/v), respectively. Both pellets were dissolved in small amounts of water and stored at −80 °C. To remove possible contaminants, all sRNA samples were submitted to size-exclusion chromatography for five times using an Amicon Ultra 3 KDa cut-off (Merck). All experiments were performed in at least triplicates.sRNA from 4G-RNA or Ap5G-RNA and divided into two halves. The first part was diluted to a final volume of 300 μL with 30 μL of 10 × buffer 2 (for RppH) or with 10 × buffer NudC (for ApaH) and RppH (NEB), or ApaH were added to reach the final concentration of 5 μ. The second half was used as a negative control and also diluted to a final volume of 300 μL using buffer without the addition of RppH. The mixtures were incubated at 37 °C for 1 h, and further used for LC–MS analysis.sRNA (1 mg) from stationary phase was spiked with 10 μg of model GpA total amount of 1 mg of sRNA was divided into two aliquots; one aliquot of 0.5 mg was digested by 10 U of Nuclease P1 (Merck) in 50 mM ammonium acetate buffer (pH 4.5) at 37 °C for 1 h. The second aliquot of 0.5 mg was incubated without enzyme and used as negative control. The digest was purified over Amicon-Millipore filters 10 kDa (Merck). The flow through was dried up on a Speedvac system and dissolved in 10 μL of a mixture of acetonitrile (10%) and ammonium acetate . The final pH was adjusted to 10 using a solution of NaOH.In vitro transcription was performed in the 50 μL mixture containing 120 ng/μL of template DNA (2ntG), 2.0 mM GTP, 5% DMSO, 0.12% triton X-100, 12 mM DTT, 4.8 mM magnesium chloride, and 1 × reaction buffer for T7 RNAP and 125 units of T7 RNAP. The mixture was incubated for 2 h at 37 °C. The mixture was filtered on an Amicon Ultra 0.5 Centrifugal Filter 3 kDa (Merck), which was then washed with water (2 × 150 μL). The combined flow through was evaporated on a Speedvac system.N,N-dimethylacetamide (20 mL). The reaction mixture was stirred overnight at room temperature (RT). Celite (200 mg) was added and the resulting suspension was stirred at RT for 30 min. Afterward, the suspension was filtered and the solids were washed with dry acetone (100 mL). Product precipitated at 5 °C. After filtration and washing with cold acetone, the product was isolated as an off-white powder in 81% yield . 1H nuclear magnetic resonance (NMR)  δ 8.56 , 8.55 , 6.15 , 4.81 , 4.46 , 4.28 , 4.02 − 3.80  ppm. 13C NMR  δ 151.73, 148.49, 147.56, 143.87, 120.05, 89.21, 86.15, 74.83, 70.80, 61.75, 38.51 ppm. MS (ESI+) m/z (%): 282 (100), 304 . High resolution mass spectrometry (HRMS) (ESI) m/z: [(M)+] (C11H16 O4N5) calc.: 282.11940, found: 282.11968.Iodomethane  was added dropwise to a suspension of adenosine  in N1-methyladenosine  was suspended in an aqueous solution of NaOH  and heated at 110 °C for 75 min. Then, the solution was neutralized by addition of 10% water solution of p-toluenesulfonic acid. Then, water was removed under reduced pressure . Methanol was added to the solid residue and resulting suspension was heated at 80 °C for 5 min. Methanol was removed under reduced pressure. Finally, the solid residue was suspended in EtOAc and the resulting suspension was refluxed at 80 °C overnight. After filtration, EtOAc from the liquors was removed on rotary evaporator. Solids were dried under vacuum to get the N6-methyladenosine in 50% yield . 1H NMR  δ 8.24 , 8.19 , 6.03 , 4.78 – 4.77 , 4.46 − 4.37 , 4.29 , 3.97 − 3.80 , and 3.07  ppm. 13C NMR  δ 154.72, 152.16, 151.50, 139.76, 125.86, 88.27, 85.77, 73.69, 70.62, 61.50, 29.76 ppm. MS (ESI+) m/z (%): 282 (100), 304 . HRMS (ESI) m/z: [(M)+] (C11H16 O4N5) calc.: 282.11968, found: 282.11938.3 (2.0 eq.) was added dropwise. Reaction mixture was stirred at 0 °C until complete consumption of the starting material according high-performance liquid chromatography (HPLC). Then, water was added and the resulting solution was neutralized with NaOH 6 M and HCl 1 M. The respective methyladenosine monophosphates were isolated after preparative HPLC . Then, water from each fraction was removed under reduced pressure. Final compounds were obtained after lyophilisation.The respective methyladenosine derivative (1 eq.) was suspended in trimethyl phosphate . Resulting suspension was cooled at 0 °C and POClN1-methyladenosine 5′-monophosphate (yield 63%). 1H NMR  δ 8.63 , 8.52 , 6.17 , 4.77 , 4.51 , 4.39 , 4.22 − 4.06 , 3.93  ppm. 13C NMR  δ 151.03, 147.95, 147.16, 142.82, 118.98, 88.01, 84.52, 74.76, 70.44, 64.35, 37.76, 8.28 ppm. MS (ESI+) m/z (%): 362 (100), 384 . HRMS (ESI) m/z: [(M)+] (C11H17 O7N5P) calc.: 362.08601, found: 362.08607.N6-methyladenosine 5′-monophosphate (yield 31%)39. 1H NMR  δ 8.25 , 7.99 , 5.94 , 4.60 , 4.38 , 4.27 , 4.03 , 2.94 − 2.85  ppm. 13C NMR  δ 154.30, 152.04, 150.72, 139.16, 118.50, 87.00, 84.05, 83.96, 70.46, 64.48, 29.66 ppm. MS (ESI−) m/z (%): 360 (100), 382 . HRMS (ESI) m/z: [(M)−] (C11H15 O7N5P) calc.: 360.07146, found: 360.07108.O-methyladenosine 5′-monophosphate (yield 47%)40. 1H NMR  δ 8.39 , 8.11 , 6.07 , 4.56 , 4.37 , 4.28 , 4.03 , 3.36  ppm. 13C NMR  δ 155.09, 152.22, 148.87, 140.07, 118.51, 85.47, 84.72, 83.26, 69.13, 64.46, 58.22 ppm. MS (ESI−) m/z (%): 360 (100). HRMS (ESI) m/z: [(M)−] (C11H15 O7N5P) calc.: 360.07146, found: 360.07077.2′-+) m/z (%): 500 , 522 , 544 . HRMS (ESI) m/z: [M+Na+] (C13H17O9N7NaP2) calc.: 500.04552, found: 500.04558.Adenosine 5′-diphosphate , imidazole , and 2,2′-dithiopyridine  were suspended in dimethylformamide . Then, trimethylamine  and triphenylphosphine  were added. Resulting suspension was stirred at RT for 24 h. The resulting clear solution was poured into a flask containing anhydrous sodium perchlorate (1 eq.) dissolved in dry and cold acetone (8 mL per 1.0 mL of DMF). After cooling at 4 °C for 30 min, liquids were separated from solids by centrifugation. Resulting solid pellet was washed four times with dry and cold acetone, and was centrifuged each time. Finally, solids were washed with diethyl ether and were centrifuged. Product was isolated as an off-white solid in 45% yield . Solids were dried overnight under vacuum and used in the next step without further purification. MS (ESI2 (3 eq.) were suspended in dry DMF. Resulting suspension was stirred at RT for 6 h. The reaction was stopped by addition of H2O. Products were isolated after preparative HPLC . Co-distillations with water followed by several freeze-drying from water gave off-white solid products.Respective methyladenosine 5′-monophosphate (1.2 eq.), adenosine 5′-diphosphoimidazolide (1.0 eq.), and dry MgCl1H NMR  δ 8.50 , 8.45 , 8.31 , 8.15 , 6.08 , 6.03 , 4.74 , 4.70 , 4.53 , 4.39 − 4.21 , 3.86  ppm. 13C NMR  δ 157.91, 155.55, 153.17, 151.45, 150.71, 149.42, 145.38, 142.31, 121.06, 120.73, 90.68, 89.61, 86.79 − 86.72 , 86.42 − 86.34 , 77.83, 77.54, 72.94, 72.91, 67.85 − 67.81 , 67.69 − 67.65 , 40.54 ppm. 31P NMR  δ −10.41 , −21.89  ppm. MS (ESI+) m/z (%): 771 (100). HRMS (ESI) m/z: [M+] (C21H30 O16N10P3) calc.: 771.10486, found: 771.10593.1H NMR  δ 8.26 , 8.21 , 8.09 , 6.01 , 5.99 , 4.63 , 4.59 , 4.49 , 4.37 − 4.30 , 4.27 , 2.97  ppm. 13C NMR  δ 157.66, 157.06, 155.39, 155.07, 150.90, 141.74, 141.21, 120.63, 90.13, 90.02, 85.91 − 85.83 , 85.79 − 85.71 , 77.85, 77.81, 72.50, 72.36, 67.49 − 67.44 , 67.39 − 67.35 , 25.86 ppm. 31P NMR  δ −10.42 , −21.89  ppm. MS (ESI−) m/z (%): 769 (100), 791 , 813 . HRMS (ESI) m/z: [M−] (C21H28 O16N10P3) calc.: 769.09031, found: 769.08953.1H NMR  δ 8.30 , 8.28 , 8.09 , 8.09 , 6.03 , 5.97 , 4.59 , 4.57 , 4.48 , 4.35 − 4.24 , 4.21 , 3.50  ppm. 13C NMR  δ 157.89, 157.86, 155.40, 155.34, 151.24, 150.98, 142.18, 142.10, 120.88, 89.85, 88.27, 86.44, 86.26 − 86.21 , 86.20 − 86.16 , 77.60, 72.80, 71.08, 67.77 − 67.73 , 67.39 − 67.36 , 61.13, 32.43 ppm. 31P NMR  δ −10.38 , −21.93  ppm. MS (ESI−) m/z (%): 769 (100), 791 , 813 . HRMS (ESI) m/z: [M−] (C21H28 O16N10P3) calc.: 769.09031, found: 769.08968.LC–MS was performed using a Waters Acquity UPLC SYNAPT G2 instrument with an Acquity UPLC BEH Amide column . The mobile phase A consisted of 10 mM ammonium acetate, pH 9, and the mobile phase B of 100% acetonitrile. The flow rate was kept at 0.25 mL/min and the mobile phase composition gradient was as follows: 80% B for 2 min; linear decrease to 68.7% B over 13 min; linear decrease to 5% B over 3 min; maintaining 5% B for 2 min; returning linearly to 80% B over 2 min. For the analysis, electrospray ionization (ESI) was used with a capillary voltage of 1.80 kV, a sampling cone voltage of 20.0 V, and an extraction cone voltage of 4.0 V. The source temperature was 120 °C and the desolvation temperature 550 °C, the cone gas flow rate was 50 L/h and the desolvation gas flow rate 250 L/h. The detector was operated in negative ion mode. For each sample, 8 μL of the dissolved material was injected.nNs. Ions with <50 counts were not considered for further analysis. MassLynx software was used for data analysis and quantification of the relative abundance of dimethyl-Gp4G.Triplicates of Nuclease P1-digested RNA samples were used to identify NpThe fragmentations studies were performed using SCIEX QTRAP 6500+ instrument with an Acquity UPLC BEH Amide column . Mobile phase A was 10 mM ammonium acetate pH 9, and mobile phase B was 100 % acetonitrile.Ap3A: For the determination of Ap3A structure the flow rate was a constant 0.25 mL/min and the mobile phase composition was as follows: 80% B for 2 min; linear decrease over 3 min to 50% B; and maintain at 50% B for 1 min before returning linearly to 80% B over 2 min. ESI was used with curtain gas of 35 (arbitrary units), Ionspray voltage of 4.5 kV. The ion source gas was 50 (arbitrary units), and the drying gas temperature was 400 °C. The declustering potential was −200 V, the entrance potential −10 V, collision energy −46 V, excitation energy 0.1 V. The detector was operated in negative ion mode. For the confirmation of Ap3A structure, the first precursor ion was [M − H]− at m/z 754.96 and as second ion the ATP fragment was selected [M − H]− at m/z 487.8. For each sample, 8 μL of the dissolved material was injected.mAp3A: For the determination of mAp3A structure the flow rate was a constant 0.20 mL/min and the mobile phase composition was as follows: 80% B for 2 min; linear decrease over 12 min to 50% B; and maintain at 50% B for 1 min before returning linearly to 80% B over 2 min. ESI was used with curtain gas of 20 (arbitrary units), ionspray voltage of 4.5 kV. The ion source gas was 50 (arbitrary units), and the drying gas temperature was 400 °C. The declustering potential was −300 V, the entrance potential −10 V, collision energy −54 V, and excitation energy 0.1 V. The detector was operated in negative ion mode. For the identification of mAp3A structure, the first precursor ion was [M − H]− at m/z 768.908 and as second ion the dehydrated mAMP fragment was selected [M − H]− at m/z 341.8. For each sample, 8 μL of the dissolved material was injected.nNs , NAD, and CoA. After LC–MS analysis of all the aliquots the area under the peak was calculated and plotted against the concentration. The experimental points were fitted with a liner regression and the intercept with X-axis represent the concentration of the cap in the sample. The quantification was repeated three times and the final value was obtained from the average of the three measurement with relative SD.A total of 2 mg of sRNA from STA was divided in four aliquots. After digestion with NuP1, each aliquot was spiked with increasing concentration of NpE. coli RppH in complex with modified ppcpAG 5′-capped dinucleotide (PDB ID 4S2Y). We modified the adenine to guanine and extended the 5′-nucleotide triphosphate into Gp4G or the methylated m7Gp4Gm. Hydrogens were added so that the amino acids were present in the standard protonation state at pH 7. Three catalytic magnesium ions were kept in place. The complex was solvated in an 83  × 68 × 71 Å box of water molecules. Some water molecules were replaced by sodium and chlorine ions to neutralize the complex charge and mimic the cellular ionic strength (0.15 M). The MD simulations were performed using the NAMD software41 and AMBER force field with ff14SB (ref. 42) parameters for the protein, OL3 (ref. 43) parameters for RNA, the SPC/E model of water44, and corresponding parameters for monovalent ions45 and magnesium46. Parameters for non-canonical RNA caps were constructed using existing RNA parameters and available parameters for polyphosphates47. Partial atomic charges were fitted based on quantum-chemical HF/6-31 G* calculations using Gaussian 09 (ref. 48) using the RESP procedure AmberTools49. We used periodic boundary conditions in the MD simulations to emulate a bulk solvent. An isobaric-isothermal ensemble (NPT) scheme with Langevain temperature and pressure control was applied. The following equilibration protocol was used: (1) 1000 steps of conjugate gradient minimization with restraints on heavy atoms of the protein and RNA, (2) heating from 0 to 310 K followed by 10 ps of MD with above restraints, (3) 1000 steps of minimization without restraints, and (4) heating from 0 to 310 K followed by 100 ps of unrestrained equilibration. We used 1 fs MD integration steps for the equilibration phase and 2 fs for the 200 ns production phase. The final snapshots were visualized using PyMol50.All models were based on a crystal structure of Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


In this manuscript, we report the the crystal structure of diphenylbis(mesitylimidazole)borane trifluoromethanesulfonate. Weak interactions, such as π–π stacking are present in the structure. H-imidazole-κN3)diphenylboron trifluoromethanesulfonate, C36H38BN4+·CF3SO3− or (2B(MesIm)2OTfPh), is reported. Bis(1-mesityl-1H-imidazole-κN3)diphenylboron (2B(MesIm)2+Ph) is a bulky ligand that crystallizes in the orthorhombic space group Pbcn. The asymmetric unit contains one 2B(MesIm)2+Ph cationic ligand and one trifluoromethanesulfonate anion that balances the positive charge of the ligand. The tetrahedral geometry around the boron center is distorted as a result of the steric bulk of the phenyl groups. Weak interactions, such as π–π stacking are present in the crystal structure.The solid-state structure of bis(1-mesityl-1 The synthesis of 2B(MesIm)2OTfPh started from the reaction of 1 eq. of Ph2BCl with 2 eq. of 1-mesityl-1H-imidazole. The product was further reacted with 1 eq. of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to yield the title compound.In this paper, we discuss the synthesis and crystal structure of Pbcn. The asymmetric unit consists of one 2B(MesIm)2+Ph ligand and one triflate anion that balances the total positive charge of 2B(MesIm)2+Ph  is 116.7 (3)° and larger than the typical tetrahedral angle (109°), whereas the angle between the imidazole moieties and the boron center (N1—B1–N3) is smaller at 105.8 (3)°. The remaining two angles are 107.4 (3) and 109.3 (3)°. The bulky mesityl groups point away from each other, creating a pocket in which the triflate molecule is located Fig. 4. The dihAlthough no classical hydrogen bonds were found in the structure, weak intermolecular interactions between the triflate anion and the protons on the imidazole groups are present Table 1. The triet al., 20162B(MesIm)2OTfPh. One example is the structure of (3-butylimidazole)triphenylboron  ] 2OTfPh is shown in Fig. 62BCl , 1-mesityl-1H-imidazole  and toluene (10 mL). The mixture was stirred at room temperature for 2 h. During the course of the reaction, a white precipitate formed. Then TMS OTf  was added as a brown liquid. The mixture was further stirred at 383 K overnight. The toluene was evaporated under vacuum, affording a white residue that was washed with Et2O (3 × 10 mL) to obtain 2B(MesIm)2OTfPh as a white powder . Single crystals suitable for X-ray diffraction were grown by vapor diffusion using diethyl ether and DCM. 1H NMR : δ (ppm) 7.81 , 7.47 , 7.35 , 7.24–7.27 , 7.10 , 6.93 , 2.25 , 1.99 . 13C NMR : δ (ppm) 104.92 (s), 137.99 (s), 134.31 (s), 133.06 (s), 131.05 (s), 129.66 (s), 128.99 (s), 128.28 (s), 127.85 (s), 126.45 (s), 124.23 (s), 21.03 (s), 17.38 (s).The synthesis of Uiso(H) = 1.2 or 1.5 × Ueq(parent atom)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020005058/jj2222sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020005058/jj2222Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020005058/jj2222Isup4.cdxSupporting information file. DOI: 1983246CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Butyrophilin and butyrophilin-like proteins select γδ T cells and direct the migration of γδ T cell subsets to distinct anatomical sites. γδ T cells expressing Vδ2 paired with Vγ9 (Vγ9Vδ2 T cells) are the predominant γδ T cell type in human peripheral blood. Vγ9Vδ2 T cells, which cannot be studied easily in vivo because they do not exist in rodents, are often referred to as innate-like T cells. The genetically recombined γδ T cell receptor (TCR) that responds to isoprenoid-derived pyrophosphates (phosphoantigens) produced by infected and malignant cells in a butyrophilin-dependent manner qualifies them as therapeutically relevant components of the adaptive immune system. On the other hand, cell-surface proteins such as the C-type lectin CD161 mark a functional phenotype of Vγ9Vδ2 T cells that mediates TCR-independent innate-like responses. Moreover, CD56  and the G protein-coupled receptor GPR56 define Vγ9Vδ2 T cells with increased cytolytic potential and, like CD161, may also be expressed by dendritic cells, principally facilitating the generation of an innate-like immunological synapse. In this review, we summarise current knowledge of Vγ9Vδ2 T cell functional phenotypes that are critical to lymphoid stress surveillance. B cells, αβ and γδ T cells represent the three cell lineages that use unique heterodimeric antigen-binding surface receptors through the recombination of V(D)J genes . AlthougDeveloping independently of MHC  in the tThe examination of γδ T cell biology including tissue-specific localisation through distinct γδ TCRs has revealed fundamental contributions of these cells to tissue homeostasis. Many forms of microbial as well as nonmicrobial stress upregulate surface expression of ligands for γδ TCRs or for NKG2D, which are therefore collectively referred to as stress antigens . By respγδ T cells also sustain tissue integrity by supporting repair processes. For instance, when skin is injured, DETC use their Vγ5 TCR to specifically respond to damaged keratinocytes and contribute to wound healing and re-epithelialisation ,18. ThisWhilst Vγ5 DETC are absent in humans, Vγ9Vδ2 TCR-expressing γδ T cells are unique to primates and not found in rodents . ExpressVγ9Vδ2 T cells predominantly respond to a phosphorylated metabolite of the non-mevalonate methylerythritol phosphate (MEP) pathway, named hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) . This alVγ9Vδ2 T cells display considerable functional plasticity, combining adaptive and innate features including high cytotoxic potential normally associated with NK cells. The invariant Vγ9Vδ2 TCR effectively recognises various bacteria and parasites, whilst with lower affinity also binds mevalonate-derived metabolites accumulating in infected or hypermetabolic, transformed cells. In this sense, the adaptive Vγ9Vδ2 TCR, although indirectly through BTN3 molecules, serves a pattern-recognising purpose allowing for immediate, MHC-independent responses. Supplementary to their strong cytolytic potential, Vγ9Vδ2 T cells secrete various cytokines ,28. MoreAlthough Vγ9Vδ2 T cells differ from Vδ1 T cells in terms of antigen recognition and tissue residence , includiIntracellular sensing of danger-associated pAgs through BTN3A1 allows for the recognition of metabolically hyperactive cells by Vγ9Vδ2 T cells as in the case of viral infection  or maligConsistent with their MHC class II-independent activation, Vγ9Vδ2 T cells lack CD4 expression but may express CD8 although the functional purpose of CD8 on Vδ2 T cells remains unclear. In accordance with their residence in peripheral blood, Vγ9Vδ2 T cells also lack C-C chemokine receptor type 2 (CCR2), which enables other leukocytes to extravasate from blood vessels. + conventional αβ T cells (Th2 cells), Vγ9Vδ2 T cells also display some cytokine plasticity. Vγ9Vδ2 T cells have for instance been shown to produce the Th2 cytokine IL-4 [Mycobacterium tuberculosis antigens [When activated through their TCR, Vγ9Vδ2 T cells show cytotoxic responses through the release of granzymes, perforin and IFN-γ. Although less versatile than helper subsets of CD4ine IL-4 , prefereantigens . Furtherantigens . Interesantigens , IL-17 pantigens .EMs, CD45RA−CD27−) predominate [EM and with terminal differentiation towards effector/memory T cells re-expressing CD45RA  [Although Vγ9Vδ2 T cells perform innate-like responses, they may also generate long-lived memory populations  and therdominate . MoreoveA+CD27−) . In addi+ and CD8+ αβ T cell subsets as well as subpopulations of NK cells. Mucosal-associated invariant T (MAIT) cells are also characterised by high expression of CD161 [+ innate-like T lymphocytes as well as NK cells is the ability to respond to the interleukin (IL) combination IL-12 plus IL-18 in the absence of αβ or γδ TCR engagement [high population of both, αβ and γδ T cells [+ and CD161– T cells, including γδ T cells, revealed a conserved transcriptional signature consistent with the functional phenotype. The genes encoding the subunits of the IL-12 and the IL-18 receptor were conserved across CD161+ T cells of all lineages, constituting the ability of these cells to respond to IL-18 plus IL-12 by producing IFN-γ in a TCR-independent manner.While Vγ9Vδ2 T cells express genetically recombined T cell receptors (TCRs), the hallmark of adaptive immunity, they can also respond in an unconventional, TCR-independent way, i.e., innate-like manner and participate in lymphoid stress surveillance for example through NKG2D . Additioof CD161 . MAIT ceof CD161 . MAIT ceof CD161 . Howevergagement ,55. IFN- T cells . Gene exIL-15 has been suggested to function as a danger signal during tissue stress surveillance . In thisCD56, also known as a neural cell adhesion molecule (N-CAM or NCAM1), belongs to the immunoglobulin (Ig) superfamily of cell adhesion molecules, which are large cell-surface glycoproteins containing Ig-like and fibronectin type III-homologous (FnIII) domains . Three m+ leukocytes, a role in the cytolytic interaction between CD56+ NK cells and CD56/NCAM-expressing tumour cell targets has previously been excluded [In hematopoietic tissues, CD56 is expressed on several lymphoid cell types, including NK cells as well as αβ and γδ T cells. In addition, CD56 has been detected on primary and leukaemic myeloid cells. Immunoprecipitation from NK cells using the anti-Leu-19 monoclonal antibody followed by enzymatic deglycosylation has been shown to result in a 137-kDa protein . Moreoveexcluded .+ effector Vγ9Vδ2 T cells upregulated surface expression of lysosomal-associated membrane protein 1  and exhibited strong cytotoxicity against MOLT-4 tumour cells in vitro. When Vγ9Vδ2 T cells were expanded from peripheral blood mononuclear cells of healthy donors by stimulating the cells with isopentenyl pyrophosphate (IPP) and IL-2, 30–70% of expanded Vγ9Vδ2 T cells expressed CD56 on their surface [+ Vγ9Vδ2 T cells were capable of killing squamous cell carcinoma and other solid tumour cell lines. This effect was suggested to be mediated by the enhanced release of cytolytic granules, because CD56+ γδ T cells expressed higher levels of CD107a compared with their CD56– counterparts following exposure to tumour cell lines. The lytic activity of CD56+ Vγ9Vδ2 T cells appeared to involve the perforin-granzyme pathway and was mainly Vγ9Vδ2 TCR/NKG2D dependent. Importantly, CD56-expressing Vγ9Vδ2 T cells were also found to be resistant to Fas ligand and chemically induced apoptosis [Effector Vγ9Vδ2 T cells differentiated with IL-15 express CD56 . In resp surface . Interespoptosis .+ αβ T cells has been demonstrated to closely correlate with CD56 surface expression [+CD8+ αβ T cells were shown to contain high amounts of perforin and granzyme B and to exhibit strong anti-CD3 mAb redirected cytotoxicity against Fcγ receptor-bearing P815 target cells. These findings, which indicate that CD56 marks γδ T cells and CD8+ αβ T cells with increased cytolytic potential, have been corroborated using a reverse approach. To distinguish CD56+ T cells from CD56– T cells, Chan et al. performed an immunophenotyping array using 41 NK cell and T cell biomarkers related to activation, co-stimulation, inhibition, cytotoxicity, adhesion, and development [– T cells, CD56+ T cells expressed higher levels of γδ TCR and CD8. Additional markers of cytotoxic cells such as CD16, NKG2A, NKG2D, CD122, DNAM-1, and granzyme B were also expressed at higher levels in CD56+ T cells. Collectively, these observations suggest that CD56 expression defines a functional phenotype that marks effector cells equipped with increased cytotoxicity and effector cytokine-producing capacity.Likewise, the cytolytic effector function of human circulating CD8pression . CD56+CDelopment . Compare+ [+ and CD56– Th9-type Vγ9Vδ2 with regard to cytolytic potential and IL-9 production has not been performed so far but would certainly be of interest.The differentiation of Vγ9Vδ2 into cytotoxic effector cells is promoted strongly by IL-15 and correlates with upregulated expression of CD56 . Interes+ . A compa+ DC-like subset in human blood that expresses HLA-DR and CD56 [+ monocytes in response to GM-CSF and type 1 IFN [CD56 can also be expressed by myeloid cells. We and others have previously detected a CD14and CD56 ,66. Evidpe 1 IFN . In addipe 1 IFN .+ subset of peripheral blood mononuclear cells (PBMCs) containing both, DC-like cells and abundant γδ T cells, with the potent N-BP zoledronate and IL-2 elicited rapid expansion of CD56+ Vγ9Vδ2 T cells as well as the production of γδ T cell effector cytokines  and the monokine IL-1β [+ Vγ9Vδ2 T cells, which strictly depended on CD56+ DC-like cells, since depletion of CD14+ cells prior to stimulation with zoledronate/IL-2 almost completely abolished cytokine production and dramatically impaired expansion of Vγ9Vδ2 T cells. This is particularly noteworthy as CD56 can facilitate homophilic interactions in cis and trans. Along the same line, CD56brightCD11c+ cells have been shown to play a key role in the IL-18–mediated proliferation of Vγ9Vδ2 T cells [Stimulation of the CD56ne IL-1β . The abs T cells .+ DC-like cells could also activate NK cells, when statins  were used instead of zoledronate [+ PBMCs. As a consequence, zoledronate not only activated CD56+ Vγ9Vδ2 T cells but also NK cells [Interestingly, in the presence of IL-2 co-stimulation, CD56edronate . Treatmeedronate . Of noteNK cells , a previ+CD14+DC-like cells from CD56–CD14+DC-like cells, we previously performed a microarray analysis using blood samples from four different donors . The expression of NCAM1 (CD56), KLRB1 (CD161), IL2RB (CD122) and GPR56 was significantly enriched in CD56+ DC-like cells from all four donors. While the emergence of NCAM1/CD56 served as a useful internal control, the association of CD161 with CD56 was well in accordance with our finding that IL-15 induces the expression of both, CD161 and CD56 [+ DC subset. Altogether, the co-expression of CD161 and CD56 not only on Vγ9Vδ2 T cells but also on DC-like antigen-presenting cells suggested that CD161 and CD56 mark a functional phenotype of immune cells involved in innate-like responses characterised by cytotoxicity and Th1-type cytokine production, most likely facilitated through CD56+ cell-to-cell interactions and a positive feedback loop mediated by IL-15.To distinguish CD56and CD56 . Interesand CD56 , and DCsand CD56 ,73. Bothand CD56 . Expressand CD56 , which mIntriguingly, GPR56 also emerged from our gene expression analysis as a cell-surface receptor that is co-expressed with CD56. GPR56 belongs to the adhesion class of G protein-coupled receptors (GPCRs), which comprises 33 members in humans with a broad cellular distribution in the developing embryo, the reproductive tracts, and the nervous as well as the immune system . Adhesios, αi, αq und α12/13), Gβ, and Gγ subunits to promote numerous cellular responses [q) or adenylyl cyclase . Canonical downstream signalling includes cyclic adenosine monophosphate (cAMP) formation by AC resulting in the activation of protein kinase A (PKA) as well as PLCβ-catalysed production of second messengers, which mobilise calcium  and activate protein kinase C . In addition to these signalling events, Gαq-coupled receptors also stimulate the small GTPase Rho, which has a key role in cell migration through the stimulation of ROCK, and the expression of growth-promoting genes through the stimulation of mitogen-activated protein kinases (MAPKs) including ERK [GPCRs can be activated by ligands or are transactivated by receptor tyrosine kinases . In addiesponses . GPCRs cding ERK .12/13 and induce cell migration in a Rho-dependent manner [q/11 and Gβ subunits [q/11 association [GPR56 has for instance been shown to couple to Gαt manner . In addisubunits . In thisociation . Tetraspociation ,81.Among lymphocytes, expression of GPR56 has previously been shown to be restricted to cytotoxic cell populations such as NK cells and T cells including γδ T cells . Of noteEMRA) after antigenic stimulation and expands during infection with cytomegalovirus and dengue virus [EMRA has been shown to express GPR56, and GPR56+ TEMRA cells displayed a transcriptional and proteomic program with cytotoxic features that are distinct from effector/memory T cells. Moreover, GPR56+ TEMRA cells displayed higher levels of clonal expansion and contained the majority of virus-specific TEMRA cells.Although CD45RA expression is generally associated with naive T cells, a subset of effector/memory T cells re-expresses CD45RA (termed Tue virus . A subpoq/11 association [The tetraspanin CD81, which promotes/stabilises the GPR56-Gαociation , was alsociation . CD81 isCD161, CD56 and GPR56 define a functional phenotype of Vγ9Vδ2 T cells and can also be expressed by myeloid DCs ,82. CD56Vγ9Vδ2 T cells contribute to lymphoid stress surveillance by recognising cells that accumulate either self-pAgs as a consequence of metabolic dysregulation or microbial pAgs during infection. Even in the absence of pAgs, Vγ9Vδ2 T cells with a distinct functional phenotype defined by CD161 expression can respond to DC-derived IL-12 and IL-18 by producing large amounts of IFN-γ. Interactions between Vγ9Vδ2 T cells and DCs are driven by IL-15, a stress-related cytokine that serves as an alarmin and upregulates CD161 and CD56 on either cell type. IL-15 also stimulates its own production and may, therefore, be central in the formation of a potential innate-like immunological synapse that may especially promote lymphoid stress surveillance. Intriguingly, the adhesion class GPCR, GPR56, is associated with CD56 and CD161 expression on both, Vγ9Vδ2 T cells and DCs. Although little is known about GPR56, it appears to be associated with cytotoxic potential. Moreover, GPR56 is expressed by terminally differentiated effector αβ T cells that may be critical for the control of viral disease . Since VAs innate-like T cells that combine adaptive properties of conventional αβ T cells and innate features of NK cells, γδ T cells are highly attractive candidates for cell-based immunotherapy of infectious and malignant disease . Recentl


Their structures were determined on the basis of extensive spectroscopic data analysis and ECD calculations. Among them, the compound 7 (6-hydroxy-3-methoxyviridicatin) was first reported as a natural product. Inhibition on LPS-induced NO production in RAW 264.7 murine macrophages found that compounds 5, 7 and 8 showed significant inhibitory effects on NO production, with IC50 values of 49.85, 22.14 and 46.02 μM respectively.Two new quinazoline derivatives versicomides E (The online version of this article (10.1007/s13659-020-00287-5) contains supplementary material, which is available to authorized users. It is also the first report of the presence of compound 7 (6-hydroxy-3-methoxyviridicatin) as a natural product. Furthermore, anti-inflammatory assay with lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophages found that compounds 5, 7 and 8 showed strong inhibitory effects on NO production with IC50 values of 49.85, 22.14 and 46.02 μM respectively.Endophytic fungi are defined as organisms that live in healthy internal tissues of plants without causing any immediate, obvious and negative effect , 2, and 1) was isolated as faint yellow powder with the molecular formula C19H25N3O4 by means of HRESIMS . The 1D NMR and HSQC spectra of compound 1 indicated that there was a methoxy  and a 1,3,4,5-tetrasubstituted benzene . Its 13C NMR and HMBC spectra confirmed the existence of a Val moiety and a similar Ile moiety, which was also validated by COSY. Then, compared with signals of quinazoline derivatives D20 =  − 88.5 ; UV (MeOH) λmax (log ε) 209 (2.66), 242 (3.11) nm; IR (KBr) νmax 1662, 1629, 1590, 1467, 1383 cm−1; ECD (MeOH): 200 (Δε + 3.49), 229 (Δε − 5.13) nm; 1H and 13C NMR data: Table m/z 360.1919 for [M + H]+ .Faint yellow powder; D20 =  − 32.4 ; UV (MeOH) λmax (log ε) 226 (2.62), 279 (0.95) nm; IR (KBr) νmax 1677, 1617, 1597, 1492, 1361 cm−1; ECD (MeOH): 219 (Δε + 2.59), 238 (Δε − 4.83) nm; 1H NMR and 13C NMR data: Table m/z 360.1919 for [M + H]+ .Faint yellow jelly; [− 32.4 c .0, MeOH;50 values.The inhibition of NO production assay was performed according to the reported procedures . DiDOX w(DOCX 6850 kb)


Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation , selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis  In the, SFTPB; . No sing, SFTPB;  or R124H, SFTPB;  is delet, SFTPB; , CLDN10 , SFTPB; , FGF10, , SFTPB; , NGLY1 -ω-hydroxy fatty acid (OAHFA) class of amphiphilic lipids appears to be downregulated in dry eye  similar to the N- and C-termini of processed lipid stabilizing pulmonary surfactant protein B  reservoir of PBS (40 ml) with surface tear film that is compressed or expanded by symmetric movement of opposing barriers. As area changes, surface pressure is monitored by a Wilhelmy wire probe with sensitivity exceeding 0.01 mN/m. Surface pressure represents the surface tension of the buffer minus the surface tension of the film. We floated onto PBS normal human tears (3 μl) pooled from over 50 different individuals, or the same tear pools passed over immobilized anti-C-term lacritin antibodies , or over a preimmune Ig column . Each was subjected to compression and expansion isocycling  that mimics blinking. Isocycling is the synchronized repeated inward or outward movement of barriers at constant speed. Isocycling was performed at 35 °C, the surface temperature of the human eye (D–E) were more stable as per a higher “lift off” area , D inset, right) and maximum surface pressure , D inset, left) achieved with compression. “Lift off” is when surface pressure first reaches 1 mN/m as compression progressively reduces film area , D inset, right and left) and expansion . Film stability is considered in terms of molecular packing density at the interface , the laFig. 1A; , 26, 29.uman eye . Normal ilm area —an indicnterface .Figure 1B: “lacrt tear proteoforms”). Lacritin dimers, trimers, and larger polymers are attributable to cross-linking by tear tissue transglutaminase, a calcium-dependent glutamine γ-glutamyltransferase that is active in normal tears . Film stability under compression was unaffected per “lift off” area , D inset, right) and maximum surface pressure , D inset, left) suggesting that C-terminal proteoforms maybe contributory. To test this possibility, we synthesized C-terminal synthetic peptides “N-94,” “N-94/C-6,” and “N-64/C-31” that together span all detected C-terminal proteoforms in tears  and therefore serve as surrogates. N-terminal 24 amino acid peptide “C-95” lacking 95 C-terminal amino acids  was included as a negative control. “N-94” represents lacritin's C-terminal 25 amino acids with two amphipathic α-helices and a six amino acid C-terminal random coil domain, whereas “N-94/C-6” lacks the latter. “N-64/C-31” is a more internal amphipathic α-helix . In total, 6 μM of each was added to anti-C-term lacritin-depleted tears . In total, 6 μM is the suspected concentration of C-terminal lacritin proteoforms in human basal tears, versus ∼18 to 27 μM for all anti-Pep Lac N-Term detectable lacritin  largely restored film stability under compression (G–H) and expansion (B). Not beneficial were N-64/C-31 and C-95 .Normal basal tears contain different forms of lacritin as monomer, polymer, and proteoforms  - of whiFig. 1al tears  and whosal tears . Which fFig. 1A; ). We subFig. 1C; A. Film sα-helix ) and tears collected without anesthesia from 21 Secondary Sjögren's syndrome dry eye patients ). C-terminal proteoforms and monomer were deficient or absent in most non-Sjögren's and Sjögren's syndrome dry eye tears . Sjögren's syndrome samples were kindly provided by the Sjögren's International Collaborative Alliance for which volume was limiting. Non-Sjögren's dry eye tears  were sufficient for compression and expansion isocycling. Compression values  were essentially identical to anti-C-term lacritin-depleted normal tears, as was the benefit of supplementation with 6 μM N-94 or N-94/C-6 . Similar benefit was apparent in expansion profiles (B). Thus, lacritin C-terminal proteoforms, but not apparently lacritin monomeric or polymeric forms, lower tear surface tension and when selectively absent or deficient in dry eye tears contribute to decreased film stability upon compression that can be largely rescued with N-94 or N-94/C-6.Lacritin monomer is selectively deficient in tears of almost all forms of dry eye . Are C-et al. . We testprofiles B. Thus, A–B, inset). The initial maximal surface pressure as the instantaneous stress response differed substantially between normal (∼1.7 mN/m) and both anti-C-term lacritin-depleted normal (∼0.6 mN/m) and dry eye tears . As per compression/expansion isocycling, spiking N-94 or N-94/C-6 into anti-C-term lacritin-depleted normal tears was fully corrective (A), but only partially so in dry eye tears in which the initial reading was elevated (respectively ∼1.2 and 1.6 mN/m) but then fell off precipitously with relaxation (B). Viscoelasticity was assessed by Fourier transform of the relaxation data ('ER') and loss modulus ('EIM') (C–E)—the latter a measure of viscous behavior. N-94 and N-94/C-6 elevated the stored elastic modulus and diminished the loss modulus of both anti-C-term lacritin-depleted normal tears and dry eye tears (D–E). The “dilatational elastic modulus” (“E∗”) is the sum of ER and EIM, and the quotient of EIM/ER, (“tan ϕ”) is the “loss factor” such that values less than or greater than 1 are respectively elastic or viscous. At all frequencies, normal tears  as well as N-94 or N-94/C-6 supplemented anti-C-term lacritin-depleted normal  and supplemented dry eye tears  are elastic (F–G). This contrasts with anti-C-term lacritin-depleted normal and dry eye  tears that are respectively viscous between 10−3.5 and 10−2.2 Hz, and 10−3.7 and 10−2.3 Hz (F–G). Curious about the role of N-94/C-6 amino acids with nonpolar side chains, we synthesized “N-94/C-6-ser” in which seven of eight  were replaced with serine. Serine is identical to alanine, but with polar –OH group. Also, serine is uncharged and neither hydrophobic nor hydrophilic. Lacritin-depleted tears supplemented with N-94/C-6-ser  were surprisingly elastic . Arrective A, but onlaxation B. Viscoeion data ,44 there ('EIM') , C–E—theye tears , D–E. Th elastic , F–G. Th0−2.3 Hz , F–G. Cu elastic , suggest elastic  such thaA) from four normal individuals for adsorption , relaxation , compression (D–E)/expansion isocycling, Brewster microscopy  studies—again all at 35 °C. We also performed Raman microscopy (I–K) using in part meibum collected from 27 other normal individuals. N-94 and N-94/C-6 introduced into the PBS subphase rapidly penetrated into the overlying meibum film with kinetics  in keeping with a two-step reaction model:If N-94 and N-94/C-6 are capable of acting as a tear lipid surfactant, one likely destination is the aqueous/lipid interface thereby accommodating respective hydrophilic and hydrophobic faces of their two amphipathic α-helices—as per pulmonary surfactant protein B , 33, 34,Fig. 3Fig. 3F; , and visA to B could be due to docking and B to C to incorporation. The equation describing such a mechanism —in keeping with N-94/C-6's superior performance in tear viscoelasticity studies (D–E). Yet both peptides are film stabilizing, as per elevated compression values (D–E), superior film thickness . To confirm this interaction, we flowed 250 μM N-94/C-6 in PBS onto C18-functionalized fused-silica coverslips dosed with sufficient meibum to form a 10 to 20 μm film for analysis by Raman microscopy at 35 °C. Higher concentration N-94C-6 was necessary for detection. Raman microscopy monitors inelastic light scattering at frequencies less than or greater than incident light with the difference from incident known as a Raman shift . A Raman shift not prominent in meibum but characteristic of aromatic groups contributed by N-94/C-6's three phenylalanines provided evidence for incorporation  that in turn transformed meibum into a continuous, thicker film —the latter in agreement with an increase in the meibum CH2-twisting mode at 1300 cm−1 . Thus, N-94 and N-94C-6, as surrogates for natural C-terminal lacritin proteoforms in normal tears but substantially lacking in dry eye tears, rapidly interact with and stabilize the tear lipid layer.in which echanism  derives han N-94 B—in keep studies , D–E. Yen values , D–E, suFig. 3F; , and a sA). We then reconstituted peptides to 6 μM in PBS and overlaid each with meibum or (O-acyl)-ω-hydroxy fatty acid (OAHFA) film. OAHFAs are thought to reside at the meibum/aqueous interface , in keeping with their amphiphilic (both lipophilic and hydrophilic) nature. Here they are presumed to contribute to an elastic monolayer  as per Balas et al. (13 16-(O-oleoyloxy)hexadecanoic acid molecules should be required to cover a PBS subphase with a surface area of ∼9.5 × 1013 nm2 assuming ∼1.5 molecules per nm2—as determined for dipalmitoylphosphatidylcholine or phosphatidylcholine . Topping this with OAHFA or meibum under gentle rotation for 30 min at 35 °C tested the affinity of peptides for each film, as inversely reflected by the level of Cy3 fluorescence in the PBS subphase (“T1”) according the equation below. Ka is the association constant:Surfactant protein B is thought to superficially associate with underlying anionic phospholipids of pulmonary surfactant , whereasFig. 4onolayer  essentiaonolayer , and traonolayer . We synts et al.  and estilcholine . Prior ta's of Cy3-N-94/C-6 (respectively 0.35 ± 0.03 and 0.21 ± 0.04 for OAHFA and meibum) exceeded those of N-94/C-6-Cy3 (0.14 ± 0.03 and 0.03 ± 0.03) by 2.5- to 7-fold (D–E), implying preferential association of the C-terminal half. This is in keeping its C-terminal net positive charge  and superior hydrophobicity (five versus three amino acids with nonpolar side chains). In contrast, C-95 Ka's were at background levels similar to Cy3 alone (D).Ko 7-fold , D–E, imy3 alone D.C). Our purpose was to ask whether peptide dissociation from OAHFA or meibum was detectable although not apparent by Raman microscopy (J) nor by Langmuir surface balance (not shown). After 30 min of gentle rotation at 35 °C, Cy3 fluorescence of the PBS subphase was assessed (“T2”). The dissociation constant (Kd) of N-94/C-6 was estimated as:While not  disturbing the OAHFA or meibum film, we next replaced 2/3 of the subphase with fresh PBS C. Our pucroscopy J nor by d of 1.1 from meibum and OAHFA (D–E).This suggested a Knd OAHFA , D–E.A) that is accordingly absent from N-94/C-6. In PBS, N-94 displays a fluorescence optimum of 361 nm after excitation at 280 nm implying exposure (A) and a thin film formed at the interface suggesting affinity but inability to partition. Studies were accordingly performed with OAHFA as described above in which 52% of N-94 and only 15% of equimolar “Ctrl pep” were quenched (F). This corresponded to respective (T0 − T1)/T1 Ka's of 1.21 ± 0.45 and 0.2 ± 0.09 and an N-94 1/(T1 − T2)/T2 Kd of 1.20 ± 0.27 (G). “Ctrl pep” was syndecan 1 “Pep30-50” peptide (A) but with PSIPRED-predicted random coil structure and fewer amino acids with nonpolar (30 versus 44%) or basic (5 versus 25%) side chains. The fluorescent contribution of 1/(T1 − T2)/T2 disassociated N-94 was calculated via the equation below that takes into consideration peptide in residual PBS. Dissociated N-94 was intact by matrix-associated laser desorption/ionization (MALDI) mass spectrometry (B) and restored homeostasis to interferon-γ and TNF-stressed human corneal epithelial cell cultures (H), as performed after calculating the quantity released per the equation:For validation, advantage was taken of N-94’s penultimate C-terminal tryptophan A that isexposure  appropriexposure . Chlorofexposure A and a tquenched F. This c0 ± 0.27 G. “Ctrl  peptide , also wi peptide A but wittrometry B and rescultures H, as perC). Thus N-94 and N-94/C-6, as surrogate C-terminal lacritin proteoforms, appear to preferentially associate with meibum and OAHFA of the tear lipid layer through their C-termini and (at least in nonstructured films) are subject to release at a level not detected by Raman microscopy (J) nor by Langmuir surface balance (not shown) but sufficient to restore epithelial homeostasis.Molar amounts were then obtained by coupling this value to the extinction coefficient, as confirmed by immunodot blot analysis C. Thus Ncroscopy J nor by in vivo. 4 μM 125I-N-94 or 44 μM 125I-N-94/C-6 were applied twice daily to rabbit eyes for 3 and 4 days, respectively (A–C), or respectively as three 1.3 μM doses over 10 min or as a single 44 μM dose (D). Concentrations chosen were in support of a phase 2 human trial. Turnover was exponential with data best fitting the equation:0 = baseline radioactivity, A is a coefficient, t = time and τ = release (turnover) time. Accordingly, N-94 and N-94/C-6 release times were respectively 400 ± 95 and 2000 ± 500 min for multiday dosing, and 13 ± 2 and 22 ± 4 min following 10 min of dosing. The same was true for the meibomian gland although at lower levels that were not statistically different from baseline (B–C). Radioactivity in other tissues and fluids remained near or at baseline (B–C). Thus, tear release (presumably from lipid) of 125I-N-94 and 25I-N-94/C-6 is respectively 4- to 20-fold slower than 5-Dodecanoylaminofluorescein, although only after repeated application. Repeated application achieved respective 125I-N-94 and 25I-N-94/C-6 totals of 1.1 or 14 nmol (seven or nine 35 μl doses of 4 or 44 μM). This contrasts with a single 94 nM dose of 5-dodecanoylaminofluorscein (one 1 μl dose of 94 mM (F)—perhaps aided by their helical structure that makes backbone amide bonds less accessible to proteases and/or by relevant inhibitors resident in tears. Thus N-94 and N-94C-6 are relatively protease-resistant, a property possibly enhanced by their association with the tear lipid layer.Topical application of 5-Dodecanoylaminofluorescein (94 mM) and sodium fluorescein (0.13 mM) onto human eyes suggests respective turnover rates of 0.93 ± 0.36% and 10.3 ± 3.7% per minute, respectively  over a tectively , A–C, or μM dose D. Concenbaseline , B–C. Rabaseline , B–C. Thof 94 mM ). If N-9of 94 mM ; predictof 94 mM F—perhapsA and 6A; amino acids 65–88 of lacritin's 119 amino acids). In silico analysis by PROSPER . To assess which ones may contribute to processing, we first optimized tear volume such that all N-64/C-31 (50 μM) was fully hydrolyzed by 48 h at 35 °C and neutral pH (B). The assay was then repeated in the absence or presence of eight different proteolytic inhibitors at standard (1×) or fivefold higher concentrations (except EDTA at 0.5 and 1x) for endpoint analysis by semiquantitative MALDI mass spectrometry in which 4 μM N-94/C-6 was spiked into the digest immediately before mixing with MALDI matrix sinapinic acid  to be sutral pH B. The asnic acid . Process−3.7 and 10−2.3 Hz as a measure of viscosity by restoring elasticity to dry eye tears that otherwise are subject to premature collapse. We further report that C-terminal lacritin proteoforms are selectively deficient in dry eye. Interestingly, as proxy proteoforms gradually cycle off OAHFA in nonpressured films, bioactivity sufficient to restore homeostasis of corneal epithelial cells is retained—a slow release role for extracellular lipid films never previously appreciated.How and which proteins help prevent premature collapse of the complex lipid film at the air/liquid interface of the eye necessary for vision? Using proxy synthetic peptides N-94 and N-94/C-6, we report that proteoforms from the C-terminus of the tear glycoprotein lacritin are essential through their rapid and stable insertion into tear lipids, including C16:1 OAHFA presumed to reside at the lipid/aqueous interface. This minimizes the loss factor between 10et al. (B) paralleled a smaller library of lacritin synthetic peptides and recombinant fragments previously generated to dissect lacritin's ocular mitogenic  were manufactured by PolyPeptide Group  with amino terminal acetylation and carboxy terminal amidation  or only amino terminal acetylation (N-94), both under GMP conditions, and with trifluoracetic acid removed in place of acetate. Purity was respectively 98.9 and 97.2%. Numbering of synthetic peptides (A) and recombinant proteins excludes the signal peptide. N-94/C-6-Y and N-94-Y with added C-terminal tyrosine, control lacritin peptides N-64/C-31 (LKSIVEKSILLTEQALAKAGKGMH) with amino terminal acetylation and carboxy terminal amidation and C-95 (EDASSDSTGADPAQEAGTSKPNEE) with carboxy terminal amidation, as well as additional N-94 and N-94/C-6—both with terminal modifications as noted above—were synthesized by Genscript  and completed as acetate salts with respective purity of 95.9, 95.4, 98.1, 97.9, 95.7, and 96.4%. Also, Genscript synthesized Cy3-labeled N-94/C-6  and “N-94/C-6” (KQFIENGSEFAQKLLKKFS), respectively representing the C-terminal active 25 or 19 amino acids of human lacritin A were mapeptides A and recKDTQLLT; ) and lamKDTQLLT; ) - all wg for 30 min. A pool of 50 collected tears was subdivided into four pools. One was passed over a preimmune rabbit Ig column (“mock depletion”). Others were subjected to lacritin immunodepletion with rabbit polyclonal antibody “ab C-term” with specificity for lacritin's C-terminal 54 amino acids  with validation by ab C-term western blotting using LI-COR. No column leaching of antibody was detected by secondary alone western blotting. Dry eye, normal, or normal depleted tears were lyophilized for shipment on dry ice (−79 °C) for Langmuir surface balance experiments. Also shipped in this manner were lyophilized N-94, N-94/C-6, N-64/C-31, and C-95. Quantitation of lacritin monomer and C-terminal proteoforms was done by densitometery on tear Western blots immunostained with “anti-C-term” lacritin antibodies. All data were normalized to the maximum monomer per blot.Collection of all human samples was approved by Institutional Review Boards as specified below and abides by the Declaration of Helsinki ethical principles. Eighty-five basal tear samples from over 50 individuals  were collected after 0.5% proparacaine anesthesia from both eyes by wicking onto a paper “Schirmer” strip that had been carefully inserted under the center lower lid of the eye for 5 min. Wicking of >15 mm or ≤6 mm of tears was respectively considered normal or evidence of dry eye. Tears on strips were immediately stored at −80 °C. Approval was from the Walter Reed National Military Medical Center Institutional Review Board with informed consent. Tears collected on Schirmer strips without anesthesia from 21 patients with Secondary Sjögren's Syndrome were kindly provided by the Sjögren's International Collaborative Clinical Alliance  with Institutional Review Board approval at each of the Alliance collection sites. Only samples that had wicked ≤6 mm were tested. Tears were eluted from Schirmer strips immediately prior to experiments, by soaking in 25 μl of ice-cold PBS with protease inhibitors  for 30 min on ice, and subsequent centrifugation at 20,000no acids , as prevno acids . AnotherMeibum was collected from four normal individuals under the auspices of the Kyoto Prefectural University of Medicine Institutional Review Board (per a collaboration with GAG) and 27 other normals per the University of Virginia Insitutional Review Board, both with informed consent. For this purpose, the lower lid was squeezed using opposing cotton applicators or meibomian gland expressor forceps. Collection was into glass vials with 500 μl chloroform  for flash freezing on dry ice and storage at −80 °C.et al. (1H NMR spectra were recorded on a Varian Inova 600 (600 MHz) spectrometer in CDCl3 with chemical shifts referenced to internal standards (CDCl3: 7.26 ppm 1H).16-(O-oleoyloxy)hexadecanoic acid palmitic acid) was synthesized as described by Balas et al.  using a 2 per second over ten consecutive cycles, surface pressures were compared during compression and expansion isocycling. In other experiments, time-dependent relaxation of surface tension was monitored after pre-equilibrated films at a surface pressure of ∼15 mN/m were subjected to a sudden step compression of less than 5% of the prior surface areas. Fourier transformation made possible comparative appreciation of dilatational elastic moduli as per Loglio et al. . After removal of 500 μl of the PBS subphase for determination  of the T1 fluorescence, fresh PBS was gently injected into the subphase for an additional 30 min rotation at 35 °C. A 100-μl aliquot of 500 μl of the subphase provided the T2 fluorescence value. Fluorescence of 100 μl of PBS was subtracted as background signal. T0, T1, and T2 values facilitated calculation of the association (Ka) and dissociation (Kd) constants of Cy3 or Cy3-labeled peptides into and from the OAHFA or meibum layer, as described in the Cy3 fluorescence signals  of 100 μl of Cy3 or Cy3-labeled N-94/C-6  or Cy3-labeled C-95  in PBS were collected in a black 96-well plate at 35 °C in a SpectraMax M3 microplate reader. Low photomultiplier tube power and 1 flash per read (reading from bottom) were applied to minimize bleaching. Wavelength cutoff was 570 nm. After a T0 fluorescence value of 6 μM peptide in PBS was obtained, 6 μM peptide in PBS was added to a final volume of 750 μl in a glass tube. The fluid surface area was 95 mmSimilarly, tryptophan fluorescence signals  of N-94 or SDC1 “Pep30-50” peptide (“Ctrl pep”) in PBS were collected in a quartz cuvette (282 QS 1.000) at 35 °C in the cuvette chamber of the same SpectraMax M3 microplate reader at low photomultiplier tube power, 1 flash per read, and wavelength cutoff of 325 nm. Area under the emission spectra ≥325 nm was used as signals. T0, T1, and T2 fluorescence values were collected as described above for Cy3-labeled peptides but with the whole 500 μl sample. Fluorescence of 500 μl of PBS was subtracted as background signal. Only OAHFA films were used.4 cells/ml and then treated with interferon-γ  and tumor necrosis factor  with or without equal molar amounts of T0 or T2-released N-94, or with C-95, every 2 days for 7 days. Viability was assessed by the alamarBlue assay . Quantity of T2 released N-94 was quantitated from fluorescence values fitted into the equation noted in the Results section with mass values obtained via the extinction coefficient. This value was confirmed by immunodot blot analysis versus an N-94 standard curve (C).For cell culture studies, T0 and T2 subphase samples containing N-94 were filter-sterilized for inclusion in human corneal epithelial (HCE-T) cell viability experiments and MALDI mass spectrometry. HCE-T cells were validated by short-tandem repeat profiling. Cells were seeded overnight in 96-well plates at a density of 1.5 x 10rd curve C.125I-N-94 in PBS repeated three times over 10 min or a single 35 μl (10 μCi) dose of 44 μM 125I-N-94/C-6 in 10 mM sodium citrate, 137 mM sodium chloride (pH 6.5) were topically added to each eye of respectively 12 and 14 female pigmented New Zealand White/New Zealand Red F1 cross rabbits . 0.25, 0.5, 1, 3, 6, or 12 h after the first dose, tears were collected (30 s from inferior cul de sac) using Tear Flo Test strips  without prior anesthesia. Blood was also collected  via an auricular artery canula into tubes containing K2EDTA and centrifuged to obtain plasma. Later in the day, 125I-N-94/C-6 treated eyes were further treated with 35 μl (10 μCi) of 44 μM of 125I-N-94/C-6 twice daily for three (125I-N-94/C-6) additional days. The final treatment was on the morning of the fifth day after which tears were collected at 0.5, 1, 3, 6, 12, and 24 h postdose from two animals per time point who were then euthanized for collection of blood and ocular tissues. Eyes of a second group of 14 rabbits were treated twice daily with 35 μl of 4 μM 125I-N-94 (8 μCi) for 3 days with the final treatment on the morning of the fourth day after which tears were collected at 0.5, 1, 3, 6, 12, and 24 h postdose from two animals per time point who were then euthanized for collection of blood and ocular tissues. Radiopurity determined upon dosing by size-exclusion chromatography was 67.2% on Day 1 and 45.2% on Day 4 for 125I-N-94 and 98.8% on Day 1 and 97.9% on Day 5 for 125I-N-94/C-6. TCA precipitable radioactivity of formulated 125I-N-94 and 125I-N-94/C-6 respectively averaged 78% and 94% over the 4 and 5 days of dosing. TCA precipitable radioactivity of samples was assessed by scintillation counting and expressed per tissue wet weight.Rabbit pharmacokinetic studies were performed by Covance Laboratories Inc using Covance standard operating procedures in accordance with the Wisconsin Department of Health Services, Radiation Protection Section, as licensed to Covance, and in compliance with Animal Welfare Act Regulations (9 CFT 3). 44 μM stocks of N-94-Y and N-94/C-6-Y were radioiodinated by Perkin-Elmer  to a final specific activity of respectively 24.2 and 2.72 μCi/μg with initial radiopurity respectively 69.62 and 97.65%. At Covance Laboratories Inc , 35 μl (3.3 μCi) of 1.3 μM 2 (pH 7.4), or instead with 5% DMSO in the same buffer as vehicle control. Samples were then processed as above after spiking in fresh 4 μM N-94/C-6 during the 0.1% TFA dilution for combination with sinapinic acid matrix.Stability of 50 μM N-94 and N-94/C-6 in 10 μl of human basal tears for 4 h at 37 °C was assessed by MALDI TOF mass spectrometry (Bruker MicroFlex) by diluting the digest 1:20 with 0.1% trifluoracetic acid for 1:1 application in a sinapinic acid matrix in 50% acetonitrile and 0.1% trifluoracetic acid for ionization. Mass measurements were taken in linear, positive mode. To appreciate how C-terminal proteoforms may be processed, 50 μM N-64/C-31 was incubated at 35 °C for 48 h with 38 μl of basal normal tears without or with individual inhibitors  diluted from stock as AEBSF (0.2 or 1 mM), antipain (74 or 370 μM), bestatin (130 or 650 μM), chymostatin (100 or 500 M), EDTA (2.5 or 5 mM), leupeptin (10 or 50 μM), and pepstatin (1 or 5 μM) in a reaction volume of 50 μl buffered by 20 mM HEPES, 150 mM NaCl, 10 mM CaCl125I-N-94 and -N-94/C-6 pharmacokinetic studies. Data are reported as the mean ± SD with statistical approaches detailed in figure legends, as performed in Prism 8.4.3.All experiments were performed at least three times, with the exception of some Brewster angle microscopy and Source data for G. W. L. is cofounder and CSO, and MGO is CMO of TearSolutions, Inc. G. A. G., M. S., J. R., K. D. T., C. S., D. S. R., R. K. S., J. P. K., J. M. H., K. L. H., A. L., T. S., R. L. M. declare no conflict of interest.