Cmax increased in an approximately dose-proportional manner in the dose range of 25–800 mg. T1/2 was within 4.80–6.95 h. In MAD, the accumulation ratio for Cmax and AUC indicated a slight accumulation upon repeated dosing of VV116. In FE, the standard meal had no effect on Cmax and AUC of VV116. No serious adverse event occurred in the studies, and no subject withdrew from the studies due to adverse events. Thus, VV116 exhibited satisfactory safety and tolerability in healthy subjects, which supports the continued investigation of VV116 in patients with COVID-19.VV116 (JT001) is an oral drug candidate of nucleoside analog against SARS-CoV-2. The purpose of the three phase I studies was to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116. Three studies were launched sequentially: Study 1 , Study 2 , and Study 3 . A total of 86 healthy subjects were enrolled in the studies. VV116 tablets or placebo were administered per protocol requirements. Blood samples were collected at the scheduled time points for pharmacokinetic analysis. 116-N1, the metabolite of VV116, was detected in plasma and calculated for the PK parameters. In SAD, AUC and  The global pandemic of COVID-19 caused by the SARS-CoV-2 has been lasting for over 2 years and caused a significant impact on human life and human society. As the pandemic continues, how to return life to normal comes to be the top priority for the whole world. In the past year, remdesivir (RDV) , and COVNucleoside analogs are one of the most important classes of antivirals that target the highly conserved active site of viral polymerase and function by being incorporated into new viral genomes to cause defective genomes , 11. TheVV116 has been approved for the treatment of COVID-19 in Uzbekistan based on the positive clinical trial results. The phase I clinical trial of VV116 in China has been completed and advanced to global phase II/III clinical trials . Herein, we report the results of three phase I studies to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116.Three studies  were launched sequentially  and Good Clinical Practice (GCP). The study protocol and informed consent forms were approved by the Ethics Committee of the Shanghai Xuhui Central Hospital. All of the subjects provided written informed consent before any study-related procedure was performed. The studies were registered on ClinicalTrials.gov .Subjects who met the following inclusion criteria, while did not meet the exclusion criteria, were eligible to participate in the study.aged 18–45 years, regardless of sex;2 (inclusive);body weight≥50 kg for males, ≥45 kg for females, body mass index (BMI) of 19–26 kg/mnormal vital signs, physical examination, 12-lead ECG, laboratory tests, ophthalmological examination and B-mode ultrasound examination, or abnormality with no clinical significance;willing to use effective contraceptive measures during the study and within 3 months after the last dose of investigational products.Inclusion criteria:allergy history;history of any systemic disorders or diseases requiring medical intervention;blood donation or blood loss≥400 mL within 3 months, or history of blood product use;participation in any clinical trial within 3 months;any concomitant medication within 2 weeks prior to screening;drug or alcohol addicts, or heavy smokers within 1 year;positive test for hepatitis B, hepatitis C, HIV, or syphilis;pregnant or lactating female, or partners of male subjects who have a birth-giving plan within 3 months.Exclusion criteria:Based on the nonclinical toxicology data of VV116, the estimated maximum recommended starting dose was selected at 25 mg, which was equivalent to 1/100 and 1/48 of no observed adverse effect level (NOAEL) in the 14-day studies in rats and dogs, respectively. In the mean time, the estimated maximum dose was selected at 1200 mg. Initially, eight ascending-dose groups  were designed in the protocol. According to the safety results of the 25 mg dose group, as well as the ongoing clinical trial in Uzbekistan, adjustment was made for the dose group settings to 25, 200, 400, 800, and 1200 mg under the communication with Center for Drug Evaluation (CDE), NMPA, which was also approved by the Ethics Committee.Eligible subjects were admitted to the Phase I Clinical Research Center 1 day before drug administration. On the morning of day 1, after overnight fasting for ≥10 h, subjects were administered VV116 tablet or placebo at the respective dose level with 240 mL water under fasting condition. All the subjects stayed at the Phase I Clinical Research Center until 48 h after dosing with close medical monitoring, except for subjects in 400-mg dose group, who stayed until 72 h after dosing for exploratory mass balance evaluation. A follow-up visit by telephone was performed by investigators on day 7 (±1 day).Three ascending-dose groups  were designed in the protocol. Twelve subjects were enrolled in each dose group at a 3:1 ratio to receive the study drug or the placebo. Eligible subjects were admitted to the Phase I Clinical Research Center one day before drug administration. VV116 tablets or placebo were administered twice a day (12 h apart), continuous administration was given for 5.5 days (days 1–6). All the subjects stayed at the phase I unit until 48 h after the last dose. A follow-up visit by telephone was performed by investigators on day 12.In this part, 12 eligible subjects were randomized and equally assigned into three groups . Each group experienced three treatment periods  with a 3-day washout period in between. For the fasting period, a single oral dose of 400 mg VV116 tablet was administered after an overnight fasting period (≥10 h). For the two fed periods, a single oral dose of 400 mg VV116 tablet was administered within 30 min after the consumption of a standard meal , or a high-fat meal . All the subjects stayed at Phase I Clinical Research Center until 48 h after the last dose. A follow-up visit by telephone was performed by investigators on day 13.N-acetyl-beta-D-glucosaminidase (NAG), coagulation function, thyroid function, and hormone test of reproductive system), thyroid B ultrasound, ophthalmology examination, and monitoring for adverse events (AEs) throughout the study. AEs were evaluated according to National Cancer Institute Common Terminology Criteria for Adverse Events . All AEs were coded using Medical Dictionary for Regulatory Activities .Safety was assessed by vital signs, physical examinations, 12-lead ECG, clinical laboratory tests  no need to continue the trial based on the obtained trial data.The dose-escalation termination criteria were determined by AEs. Dose escalation should be terminated when any of the following criteria is met: (1) AEs related to study drug of CTCAE Grade 2 and above occur in ≥1/2 subjects of one dose group; (2) AEs related to study drug of CTCAE Grade 3 and above occur in ≥1/3 subjects of one dose group; (3) SAE related to study drug occurs in at least one subject; (4) ALT or AST > 3×ULN and total bilirubin (TBiL) >2×ULN, non-biliary elevation  occurs in a dose group, and could not be explained by other diseases; (5) urine crystallization is observed in at least two subjects in a certain dose group, with glomerular filtration rate <60 mL/min/1.73 mApproximately 3 mL blood samples were collected at the scheduled timepoint for analysis. In 25 mg group of single ascending-dose study, blood samples were collected at the following 15 time points: 0 (pre-dose), 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, 36 and 48 h post dose. Afterward, the time points were slightly adjusted depending on the pharmacokinetic profile of the 25-mg group. In other dose groups of single ascending-dose study, as well as food-effect study, blood samples were collected at the following time points: 0 (pre-dose), 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h post dose.In multiple ascending-dose study, blood samples were collected at the following time points: 0 (pre-dose), 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h post first dose at day 1; each pre-dose at day 5; 0 (pre-dose), 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h and 48 h post dose at day 6.g for 10 min, then were divided into two centrifuge tubes (at least 0.6 mL plasma each), frozen, and stored under −80 °C until analysis.The collected blood samples were separated by centrifuging at 4 °C, 1500 × Exploratory mass balance evaluation was conducted in 400 mg dose group of single ascending-dose study. Random urine sample within 24 h to 0 h prior to administration on day 1, and total excreted urine volumes in the time intervals of 0–6, 6–12, 12–24, 24–48, and 48–72 h after administration were collected respectively. Random fecal samples within 24 h to 0 h prior to the administration on day 1, and within 0–72 h after administration were collected, respectively.A simple, precise and accurate LC-MS/MS method was developed and validated to determine the concentration of VV116 and its metabolite 116-N1 in human plasma.For VV116, VV116-D4 was chosen as the internal standard. Under the calibration range (2–2000 ng/mL), the regression coefficient was 0.9998. The intra-run precision ranged from 1.1% to 6.6%, and the inter-run precision from 4.6% to 9.6%. The accuracies were 85.6%–102.3%.For the metabolite 116-N1, 116-N1-D4 was chosen as the internal standard. The calibration curves were linear over 10–10,000 ng/mL, of which the regression coefficient was 0.9998. The intra-run precision ranged from 2.2% to 12.4%, and the inter-run precision from 2.5% to 8.2%. The accuracies were 90.5%–100.1%.Cmax), area under the plasma concentration–time curve (AUC), AUC from time zero (pre-dose) to the time of the last measurable concentration (AUC0-t), AUC from time zero (pre-dose) extrapolated to infinity (AUC0-∞), area under the concentration–time curve during a dosing interval (AUC_TAU), time of maximum plasma concentration (Tmax), terminal plasma elimination half-life (t1/2), apparent volume of distribution (Vd/F), apparent clearance (CL/F), terminal phase elimination rate constant (Ke), mean residence time (MRT), cumulative excretion (Ae), and fractional excretion (Fe). AUC0-t and AUC0-∞ were calculated using a Linear Up Log Down rule method. Tmax and Cmax were based on the actual measured values. In multiple ascending-dose study, accumulation ratio after repeated dosing (Rac) was also analyzed.PK parameters were calculated using a non-compartmental (NCA) model by WinNonlin Software version 8.3.1 . Main PK parameters included maximum observed plasma concentration . Descriptive statistics were expressed as arithmetic mean, standard deviation, coefficient of variation, median, maximum, minimum, and geometric mean of each dose group. Frequency and percentage were calculated to summarize categorical variables.In the single ascending-dose study, the dose-exposure relationship was assessed using confidence interval criteria. The linear regression was carried out. The regression equation was expressed as ln(PK) = α + β×ln(Dose) where logarithmic transformation was used for PK parameters and doses. The 90% confidence interval of the estimated slope (β) was calculated. PK parameters were considered to be linearly associated to dose, in the condition that the 90% confidence interval of β was within the judgment interval.0-t, AUC0-∞, and Cmax under different dietary conditions were analyzed. The mean, mean difference and 90% confidence interval of logarithm transformed PK parameter were estimated using generalized linear mixed model, where the dietary condition, sequence, and period were included as a fixed effect, while the subject was included as a random effect. The pairwise comparisons  were carried out for each PK parameter. Food intake was considered to have no effect on the bioavailability of VV116, in the condition that the 90% confidence interval of the geometric mean ratio of AUC0-t, AUC0-∞, and Cmax under fed condition was within 80%–125% of that under fasting condition.In the food-effect study, PK parameters including AUCA total of 86 eligible subjects  were enrolled and completed the study. No subject was discontinued from the study. Subject distribution is displayed in Fig. VV116 was hydrolyzed rapidly to its metabolite 116-N1 after oral administration. The prototype drug was not detected (lower limit of quantitation was 2 ng/mL) in plasma, while its metabolite 116-N1 was detected, and calculated for the PK parameters in the three studies.The main PK parameters of 116-N1 in each dose group after a single dose of VV116 are summarized in Table Cmax and AUC were dose-dependent. The mean ± SD of Cmax was 165 ± 74.0, 1096 ± 412, 1898 ± 701, 2796 ± 225 and 3086 ± 778 ng/mL for 25 mg, 200 mg, 400 mg, 800 mg and 1200 mg dose groups, respectively. AUC0-t was 744 ± 244, 6631 ± 1603, 12759 ± 2747, 25886 ± 5904 and 28057 ± 5145 h·ng/mL, while AUC0-∞ was 904 ± 301, 6986 ± 1683, 13064 ± 2727, 26233 ± 5897 and 28325 ± 5272 h·ng/mL for the above five dose groups. Confidence interval (CI) criteria were used to assess the dose linear relationship. Table Cmax, AUC0-t, and AUC0-∞ was 0.85 (0.73–0.97), 1.03 (0.95–1.11), and 0.98 (0.90–1.06) for 25–800 mg dose interval, which was not completely within the judgment interval (0.94–1.06). Even though, we could draw a conclusion that AUC parameters and Cmax increased in an approximately dose-proportional manner in the dose range of 25–800 mg. However, Cmax, AUC0-t, and AUC0-∞ did not show significant change with dose escalation from 800 to 1200 mg. The median of Tmax was within 1.00–2.50 h, and the mean t1/2 was within 4.80–6.95 h . As for drug exposure, Cmax, AUC0-t, and AUC0-∞ increased after repeated dosing. The accumulation ratios (Rac) of Cmax were 1.34, 1.18, and 1.24, and Rac of AUC were 1.53, 1.41, and 1.42 for 200, 400, and 600 mg dose groups, respectively, which showed a slight accumulation upon repeated dosing of VV116.The mean The key PK parameters of 116-N1 after a single oral dose of 400 mg VV116 under different diet conditions are listed in Table Cmax, AUC0-t, and AUC0-∞ between standard meal and fasting were 106.60% (93.40%–121.67%), 119.52% (114.50%–124.76%), and 118.21% (113.53%–123.08%), respectively, which was within the equivalent range 80%–125%. The GMR (90% CIs) of Cmax, AUC0-t, and AUC0-∞ between high-fat meal and fasting were 107.92% (94.56%–123.18%), 126.32% (121.01%–131.85%), and 124.67% (119.74%–129.81%), respectively. AUC0-t and AUC0-∞ increased by 26.32% and 24.67% with a high-fat meal compared to the fasting condition.The geometric mean ratios (GMR) and their 90% CIs of Tmax of 116-N1 under fasting condition, fed condition with a standard meal, fed condition with a high-fat meal was 1.5, 3.0, and 2.5 h, respectively. We figured out that VV116 administration under fed condition could prolong the time to the peak as compared with the fasting condition, but had little effect on the system exposure of the study drug.The median No deaths, serious adverse event (SAE), AEs of Grade 3 or above, or AEs leading to drug discontinuation/interruption were reported throughout the three studies. All AEs were recovered without any treatment or intervention.The number (incidence) of subjects experiencing AEs for 25, 200, 400, 800, and 1200 mg dose group and placebo group was 2 (50%), 3 (50%), 3 (50%), 3 (50%), 0 (0), and 5 (50%), respectively Table . No relaThe number (incidence) of subjects experiencing AEs for 200 mg, 400 mg, 600 mg dose group and placebo group was 3 (33.3%), 5 (55.6%), 6 (66.7%), and 5 (55.6%), respectively Table . The incThe number (incidence) of subjects experiencing AEs under fasting condition, fed condition with a standard meal, fed condition with a high-fat meal was 0 (0), 2 (16.7%), and 4 (33.3%). Two subjects under fed condition with a standard meal were observed atrioventricular block with first degree, while four subjects under the fed condition with a high-fat meal experienced positive results in urine bacterial test, presence of crystal urine, increase in blood pressure, and atrioventricular block with first degree. All AEs were CTCAE Grade 1 in severity.Only one subject in 400 mg dose group of Study 3 experienced mild increase in transient blood thyroid-stimulating hormone, which recovered spontaneously without any treatment. No clinically significant abnormality was discovered in sex hormone test, ophthalmological examination, and thyroid B ultrasound test.VV116 is a prodrug of nucleoside analog, intended for the treatment of COVID-19. RDV is the first FDA-approved drug for the treatment of COVID-19, which is also a nucleoside analog. Compared with RDV, VV116 exhibits better in vitro antiviral activity and selectivity . In addiTmax 1.00–2.50 h). In the single ascending-dose study, AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25–800 mg. However, the parameters did not show significant change with dose escalation from 800 to 1200 mg , indicating the probability of drug absorption saturation. Drug solubility is an important factor affecting the drug absorption and maximum drug absorption occurs when the drug has maximum concentration (saturation solubility) at the site of absorption. It was suspected that limited solubility of VV116 might be the reason for drug absorption saturation. The fractional excretion of 116-N1 in urine was 53.6% in 0–72 h after administration, while that of 116-N1 and VV116 in feces was 5.25%, which indicated that VV116 was principally excreted through kidney in the form of metabolite 116-N1.VV116 was hydrolyzed rapidly to its metabolite 116-N1 after oral administration. 116-N1, instead of the prototype drug VV116, was detected in plasma, and calculated for the PK parameters. Peak plasma 116-N1 concentration reached quickly after oral administration  for 5.5 days (days 1–6) was adopted in the multiple ascending-dose study. The accumulation ratio of AUC parameters and Cmax indicated a slight accumulation of VV116 after continuous dosing. The trough concentrations of 116-N1 following multiple administration of 200 mg at day 5 and day 6 were within 242–345 ng/mL (Table 90 (186.5 ng/mL) of 116-N1 against the omicron variant in a preclinical anti-SARS-CoV-2 assay. Therefore, the dosage regimen of 200 mg BID and above can continuously maintain the effective antiviral concentration, and is recommended for subsequent clinical studies in patients with COVID-19.The mean Tmax under fasting, standard meal and high-fat meal condition was 1.50, 3.00, and 2.50 h, respectively, indicating that fed condition could prolong the time to the peak. Compared with fasting condition, the GMR (90% CIs) of Cmax under fed condition with both standard meal and high-fat meal was within the equivalent range 80%–125%; the GMR (90% CIs) of AUC for standard meal was also within the range 80%–125%, however for high-fat meal, AUC0-t and AUC0-∞ slightly increased by 26.32% and 24.67%, respectively. Since food intake has no effect on Cmax of VV116, while high-fat meal slightly increases AUC, it is recommended that VV116 could be taken under fasting condition or fed condition with regular meal in the treatment of COVID-19.The median In the single ascending-dose study, there was no apparent dose-related trend, with a greater proportion of subjects reporting AEs following administration of placebo (50.0%) than following administration of VV116 (39.3%). The severity of AEs was CTCAE Grade 1 with the exception for one case of Grade 2 neutropenia. In the multiple ascending-dose study, the incidence of AEs in the VV116 group was comparable with that in the placebo group (51.9% vs. 55.6%). AE occurrence was slightly dose-related. Only 1 subject in 400 mg dose group reported one case of increased alanine aminotransferase and increased aspartate aminotransferase, respectively. All AEs in subjects administered VV116 were Grade 1 in severity, and were recovered without any treatment. No serious adverse event occurred throughout the study, and no subject withdrew from the study due to AE. In the preclinical animal toxicology study, it was discovered that VV116 might have toxicity on eyes, thyroid, and gonads. In our studies, ophthalmology examination, thyroid function, thyroid B ultrasound, and sex hormone tests were performed on healthy subjects before and after VV116 administration. No obvious toxicity was observed in the above organs. Overall, VV116 demonstrated satisfactory safety profiles in healthy subjects throughout the three studies.14C-GS-5734) after a single intravenous administration of 10 mg/kg [14C]RDV at 4 h was 57.8 [Hepatotoxicity is the primary adverse drug reaction (ADR) of RDV, manifested as transaminase elevation. In phase I clinical study (Study GS-US-399-5505), subjects received one loading dose of 200 mg RDV followed by 100 mg for up to 9 days, transient ALT elevation of Grade 1 or 2 was observed in 9 of 20 subjects (45%) . Transamwas 57.8 , while tTmax 1.00–2.50 h). AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25–800 mg, while drug absorption saturation was probably achieved at the dose of 800 mg. Standard meal had no effect on Cmax and AUC of VV116. Effective antiviral concentration was achieved at dose levels between 200 and 600 mg BID following multiple administration.VV116 exhibited satisfactory safety and tolerability in healthy subjects. Peak plasma drug concentration of 116-N1 reached quickly after oral administration of VV116 (median In conclusion, the safety data and PK profile from these studies support the continued investigation of VV116 in patients with COVID-19.


Lys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC–nevirapine, and RTIC–efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA–tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNA Lys3, which forms a complex with the 5’ end of the HIV-1 viral RNA and reverse transcriptase (RT). Here, the authors present the 2.8 Å cryo-EM structure of a minimal HIV-1 RT–vRNA–tRNALys3 initiation complex (miniRTIC), and miniRTIC structures with the bound non-nucleoside reverse transcriptase inhibitors nevirapine and efavirenz at 3.1 and 2.9 Å resolution, respectively.Initiation of HIV-1 reverse transcription occurs at the host tRNA A packaged viral enzyme, reverse transcriptase (RT), initiates DNA synthesis at the 3′-end of a host tRNALys3 that is part of a binary complex preassembled with the 5′-end of the HIV-1 viral RNA, called the primer-binding site (PBS). Kinetic investigations have shown that reverse transcription initiation on this vRNA–tRNALys3 template–primer complex is slow (~500-fold slower than elongation), nonprocessive, and displays a distinct pausing pattern along with the viral genomic RNA (vRNA) template6. By contrast, the elongation phase of reverse transcription, in which RT associates with DNA–DNA and DNA–RNA substrates, occurs with rapid and processive polymerase kinetics and has been well-characterized by a wide range of structural, biophysical, and biochemical approaches. RT in complex with nucleic acid substrates (DNA–DNA and DNA–RNA duplexes) has been captured in multiple structural states representative of the RT elongation polymerase catalytic cycle and RNase H engagement7.Reverse transcription of the HIV-1 single-stranded (ss) RNA genome into double-stranded (ds) DNA is an essential early step in viral replication and a major target for current antiretroviral therapies10. By encompassing the essential conserved features within the tRNALys3 primer and 101 nucleotides within the vRNA template that modulate RT initiation in vitro and in vivo, cryo-EM and X-ray crystallographic investigations have provided the first structural glimpses of how RT recognizes vRNA–tRNALys3 template–primer complexes14. A cryo-EM reconstruction revealed the overall architecture of the complex12. The 18 base-pair PBS helix sits in the RT cleft, and is extended by an additional four base pairs between vRNA and tRNA. The tRNA 5′-end refolds to form a coaxially stacked, long helical structure protruding from the RNase H domain of RT. The vRNA forms two helical regions H1 and H2 above the RT polymerase-active site, and a single-stranded connecting loop that bridges from RNA located in the RNase H domain to that near the RT polymerase domain. The structures of the core region of the RTIC revealed features that could explain the slow nature of initiation: RT adopts an open conformation with a hyperextended thumb, and the tRNA primer terminus is deviated away from the active site relative to structures representative of elongation14. However, the resolution of these structures, limited to 4.1 Å 13 and 3.95 Å 14, has prevented accurate mapping of protein–RNA contacts, and modeling of ions and small-molecule ligands, which is required to understand drug binding.In contrast, RT initiation has been challenging to investigate using structural methods7. Two of the most well-characterized NNRTIs include nevirapine (NVP), a first-generation inhibitor, and efavirenz (EFZ), a more efficacious and higher-affinity compound. Structures of RT in complex with elongation substrates and NNRTIs have uncovered the mechanism by which these drugs inhibit the chemistry step of polymerization, by allosteric conformational changes that destabilize the RT–nucleic acid complex15. NNRTI binding distorts the active site, repositions the primer grip, displaces the 3′-primer terminus, and loosens the thumb and finger clamp18. NNRTIs can also increase RNase H activity when RT is bound to an RNA–DNA elongation intermediate7. NNRTIs substantially reduce the buildup of early reverse transcription products, implying that NNRTIs may also impede initiation19. Notably, the recently determined low-resolution structures of the RTIC all exhibit a partially open RT-NNRTI-binding pocket, a feature previously absent in all RT structures lacking a bound NNRTI14. In addition, the complex studied by cryo-EM was also shown to be inhibited by NVP in biochemical assays12. However, whether or how NNRTIs perturb the conformation of an RTIC remains unknown. The slow nature of initiation suggests an inherent vulnerability of the RTIC to disruption, making it a particularly appealing target for the discovery of antivirals.Reverse transcription is a key target for antivirals, including nucleoside analogs that act as chain terminators and non-nucleoside inhibitors (NNRTIs) that allosterically disrupt enzyme functionLys3 initiation complex (miniRTIC) to 2.8-Å resolution, allowing for de novo modeling and definition of RT–RNA contacts. Using a similar approach, we also determined structures of the miniRTIC–NNRTI complexes with NVP and EFZ . The structural and biochemical data confirm the potent inhibitory activity of these drugs against initiation. Our results provide a high-resolution view of the RTIC and suggest how drugs might selectively target initiation.Here, we apply a combined biochemical and structural approach to delineate the protein–RNA contacts within the RTIC and determine the origins of NNRTI action on initiation. Using a minimal, active RNA initiation complex that spans the portions of vRNA and tRNA that reside within the RT cleft, while eliminating dynamic peripheral RNA elements that do not interact with the protein Fig. , we solv14. The resulting minimal vRNA–tRNA template–primer complex contained a 26-nucleotide fragment of the vRNA genome and a 39-nucleotide truncated tRNALys3. This bimolecular complex encompasses 4 nt of single-stranded template vRNA, the 22-bp extended PBS helix, and 6 bp of the tRNA primer helix capped by a GNRA loop  ~threefold faster than an equivalent un-cross-linked complex 12. The addition of NNRTIs substantially reduced the rate of nucleotide incorporation by the miniRTIC: EFZ slowed incorporation by ~20-fold, whereas NVP slowed incorporation by ~16-fold  system that maintains the protein–dsRNA core while eliminating regions of segmental flexibility, which included peripheral elements of the vRNA  and tRNAoop Fig. . To circlex Fig. , and simold Fig. . Togethe13. The reconstruction of the ~138-kDa miniRTIC was resolved at an overall resolution of 2.8 Å, an ~1.3-Å improvement in the resolution from our previous +1 RTIC core structure13 . Globally, the RT protein backbone is nearly identical to that of previous RTIC structures (p66 backbone r.m.s.d. = 1.2 Å (6WAZ [10.2210/pdb6waz/pdb]) and 0.8 Å (6HAK [10.2210/pdb6hak/pdb]), p51 backbone r.m.s.d. = 0.96 Å (6WAZ [10.2210/pdb6waz/pdb]) and 0.93 Å (6HAK [10.2210/pdb6hak/pdb])). Density consistent with the size of an octyl-β-glucoside molecule (β-OG), a cryo-EM freezing additive, was observed adjacent to the NNRTI-binding pocket , away from the catalytic triad , and resides in the P′-site. This displacement is accompanied by an ~3-Å shift of the primer grip toward the thumb and away from the polymerase-active-site residues. Collectively, these movements shift the primer terminus away from the palm subdomain, disrupting protein–nucleic acid contacts responsible for coordinating the polymerization reaction. In addition, the p66 thumb opens by ~6 Å and adopts a hyperextended conformation reminiscent of RT–DNA–NNRTI structures as reported in past analyses of lower-resolution RTIC structures5 Å Fig.  to facil5 Å Fig.  in a man14. In addition, the wider dsRNA PBS helix makes additional contacts with the connection domain of the p51 subunit  compared to RT bound to a dsDNA substrate  reside along the minor groove of the PBS helix between tRNA187 Fig. . The tRN187 Fig. , while t187 Fig. . Notably187 Fig. .2+ ion bound by D443, D549, and the phosphate of vRNA nt 200 .A second set of contacts between RT and the PBS helix arise approximately one RNA helical turn away toward the RNase H domain Fig. . In a siWe next sought to investigate the mechanism of NNRTI action on reverse transcription initiation. Cross-linked, minimal RTICs were purified and imaged by cryo-EM using similar methods described above; however, prior to freezing complexes, we incubated the miniRTIC with either 1 mM NVP or 200 µM EFZ for an hour at room temperature to ensure drug saturation. The structures of the miniRTIC–NVP and miniRTIC–EFZ complexes were resolved at overall resolutions of 3.1 and 2.9 Å, respectively  and a 101-nucleotide vRNA template construct. This construct, previously employed during low-resolution cryo-EM studies, includes all peripheral RNA elements required for efficient initiation13. RT extension reactions were initiated in the presence or absence of 3 µM NVP or 3 µM EFZ and quenched at time points of 5, 10, and 30 min. Extended tRNALys3 products were resolved at the single-nucleotide resolution on a denaturing Urea-PAGE sequencing gel 13. The shortened tRNA helix of the miniRTIC, which coaxially stacks with the extended PBS helix in the context of a larger RTIC, exhibits poor density, reflecting disorder at the periphery of the complex  the primer grip must return to its canonical conformation and the tRNA 3′ primer terminus must translate into the polymerase-active site, and (ii) the RTIC must reside in this conformation long enough to bind an incoming dNTP and perform the critical chemistry step of polymerization. Critically, this second step must occur prior to RT dissociation or shifting of the tRNA primer terminus back into the P′ site. Alternatively, the distributive nature of initiation suggests that this second step could also be achieved by the dissociation and subsequent rebinding of RT in a polymerase competent conformation.Lys3 template–primer complex. Hyperextension of the thumb and inherent rigidity of the A-form PBS helix causes the displacement of the tRNA 3′ primer terminus away from the palm domain and the catalytic triad. Compared to elongation complexes, this conformation, which is unique to the RTIC, features fewer contacts between the PBS helix and the thumb and palm subdomains. The dearth of stabilizing interactions between the RNA and the RT polymerase domain helps to explain the slow kinetics of polymerization and lack of active-state RTIC structures. The RNase H domain, despite featuring a more substantial contact landscape during initiation, is slightly deviated away from the PBS vRNA template strand compared to its position in RT–dsDNA and RT–DNA–RNA complexes, likely reducing the strength of electrostatic and van der Waals interactions in this region. Yet additional contacts within the RNase H domain may stabilize the extended PBS helix and, in turn, the coaxially stacked tRNA within the full RTIC. Recent work on the +3 extended RTIC suggested that RNase H contacts can cause the RTIC to adopt off-pathway conformations, further highlighting their importance during initiation13. While the miniRTIC features primarily van der Waals and electrostatic RT–dsRNA contacts 12. Two expression vectors, one containing p66 (ampicillin resistance) and the other containing p51 (kanamycin resistance) were constructed. The C-terminus of p66 contains an unstructured linker and a cleavable six-histidine tag. A cysteine mutation for cross-linking was introduced into alpha-helix H of p66 (Q258C). The protein used in this study also had the C280S mutation, introduced in prior structural work, and the E478Q mutation, introduced to eliminate RNase H activity as RT has been shown to cleave dsRNA when stalled for long periods31. Cells were grown in LB medium at 37 °C until an optical density at 600 nm reached a value of 0.6. Cells were induced with the addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside and grown overnight at 19 °C. Cell pellets were lysed through sonication , 5 mM β-ME, 5% glycerol, and 1 mM imidazole), and the enzyme was purified by gravity Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography , 5 mM β-ME, 5% glycerol, and 10 mM imidazole. Elution buffer: 300 mM NaCl, 50 mM Tris-HCl (pH 8.0), 20 mM β-ME, 5% glycerol, and 100 mM imidazole) followed by an initial size-exclusion chromatography (SEC) step using Superdex 200 (26/600) , and 5 mM β-ME). The six-histidine tag was cleaved by thrombin digestion overnight. The cleaved protein was reapplied to Ni-NTA column to remove protein with uncleaved six-histidine tag (SEC buffer above). This was followed by an additional final size-exclusion chromatography polishing step (SEC buffer above). The protein was stored at 4 °C in 300 mM NaCl, 50 mM Tris (pH 8.0), and 5 mM β-ME prior to use.HIV-1 RT was expressed in 2, and 50 mM Tris, pH 8.0. The mixture was allowed to cross-link overnight at room temperature. The complex was purified by anion-exchange chromatography with a linear gradient (75 mM to 1 M NaCl). This was followed by a size-exclusion chromatography step to remove any higher-molecular-weight aggregates. The purity and homogeneity of the final complex were assessed by SDS-PAGE  beta-octyl glucoside (β-OG) and 6 mM MgClSamples of 30 µM apo-miniRTIC, 60 µM miniRTIC–NVP, and 60 µM miniRTIC–EFZ were applied to glow-discharged Quantifoil grids (R 0.6/1 100 Holey Gold Supports + 2 nm C Grids: Au 200 mesh) and subsequently vitrified using an FEI Vitrobot .2 across all three datasets. In total 12,186, 12,675, and 12,402 micrographs were collected at defocus values ranging from −1.0 to −2.5 μm for the apo-miniRTIC, miniRTIC–NVP, and miniRTIC–EFZ, respectively  with a Gatan K2 Summit direct detection camera and energy filter in counting mode with 200-ms exposure per frame. The dose rate was 8.0 electrons per pixel per second and forty frames per micrograph were collected at a magnification of ×165,000 (corresponding to 0.82 Å per pixel at the specimen level). The total dose was 100–107 electrons per Å34. Cryo-EM data were processed using Relion 3.0 and cryoSPARC37. In total, 4,315,173, 2,656,613, and 3,072,304 particle projections were semiautomatically picked in RELION 3.0 from the apo-miniRTIC, the miniRTIC–NVP, and the miniRTIC–EFZ datasets, respectively. The particle projections were extracted, downscaled four times, imported, and sorted through subsequent rounds of reference-free 2D classification in cryoSPARC. In total, 1,587,090, 1,155,315, and 1,129,740 particle projections belonging to classes with well-defined protein and RNA features were selected and re-extracted with two-time binning for further processing. A 3D ab initio model was generated using cryoSPARC based on the selected 2D classes, used for 3D classification in RELION 3.0. In total, 1,344,402, 1,155,315, and 1,123,557 particle projections were selected and re-extracted without binning for subsequent 3D refinement from the apo-miniRTIC, the miniRTIC–NVP, and the miniRTIC–EFZ datasets, respectively. The 3D reconstructions were refined to a resolution of 2.8, 3.1, and 2.9 Å, respectively, and sharpened in RELION 3.0  was used as the starting model. Ligand structures were downloaded from the RCSB PDB Ligand Expo or fetched using Coot’s monomer library. Initial docking of the EFZ and NVP ligands was performed by aligning the ligands to prior RT–NNRTI structures and then fit into the density using Coot. The β-OG ligand was docked into its respective density and initially fit using Coot. Restraints for all ligands were generated by eLBOW in Phenix prior to real-space refinement. Final models and maps were validated using Phenix comprehensive validation, including MolProbity40 and EMRinger41 analysis. Nucleic acid helicity was analyzed with 3DNA42  by rigid docking in Chimera. Regions of major structural differences in RT were de novo modeled in CootApo-miniRTIC modeling: p66 subunit (chain A) modeled residues 4–62, 71–133, 142–557, p51 subunit (chain B) modeled residues 6–218, 231–356, 363–427, vRNA (chain C) modeled residues 182–203 and tRNA (chain D) modeled residues 55–76. p66 subunit truncated side-chain residues 22, 27–29, 31–32, 34–36, 38–40, 42–43, 46, 49–50, 53, 71, 73, 78–79, 89–92, 131, 142–143, 151, 169, 173–174, 177, 195, 197, 220, 248, 250, 286–287, 289, 297, 311, 334, 344, 357, 396, 404, 413, 432, 449, 512, 516. p51 truncated side-chain residues 11, 13, 22, 65–68, 79, 86, 113, 138, 169, 173, 174, 194, 203, 212, 214–215, 238, 240–241, 250, 275, 297–298, 315, 334, 404, 424.miniRTIC–EFZ modeling: p66 subunit modeled residues 4–63, 70–134, 141–558, p51 subunit modeled residues 5–216, 231–357, 362–428, vRNA modeled residues 182–203 and tRNA modeled residues 55–76. p66 subunit truncated side-chain residues 28, 32, 36, 40, 43, 50, 53, 70, 79, 86, 92, 117, 143, 177, 191, 194, 218, 220, 286–287, 290, 324, 334, 428, 448–449, 471, 557–558. p51 subunit truncated side-chain residues 5–6, 11, 22, 66–67, 86, 162, 166, 174, 214, 240, 250, 297–298, 315, 334, 356, 388, 428.miniRTIC–NVP modeling: p66 subunit modeled residues 2–64, 70–134, 141–557, p51 subunit modeled residues 6–214, 233–426, vRNA modeled residues 182–203 and tRNA modeled residues 55–76. p66 subunit truncated side-chain residues 29, 36, 40, 43, 53, 64, 70, 92, 110, 194, 255, 286–287, 297, 308, 324, 344, 428, 461, 514. p51 truncated side-chain residues 6, 11, 66–67, 69, 86, 89, 113, 177, 194–195, 250, 297, 298, 357, 358.Due to chain breaks in modeling, r.m.s.d. calculations comparing apo-miniRTIC, 6WAZ, and 6HAK for p66 used residues 4–21, 35–50, 77–89, 93–126, 142–190, 198–215, 253–430, 470–545 . r.m.s.d. calculations comparing apo-miniRTIC, 6WAZ, and 6HAK for p51 used residues 6–93, 95–166, 175–210, 231–240, 250–284, 293–350, 363–425 . r.m.s.d calculations comparing apo-miniRTIC, miniRTIC–NVP, and miniRTIC–EFZ for p66 used residues 4–21, 35–50, 77–89, 93–126, 142–190, 198–215, 253–430, 470–545 . r.m.s.d. calculations comparing apo-miniRTIC, miniRTIC–NVP, and miniRTIC–EFZ for p51 using residues 6–16, 20–211, 236–300, 320–355, 365–416 .Contacts were first identified with a < 5-Å cutoff distance between RT and the vRNA–tRNA. After identification, these contacts were inspected and curated  was preincubated in 50 mM Tris-HCl, pH 8.0, 50 mM KCl, and 6 mM MgCl2 in a 37 °C water bath for 5 min. For drug conditions, nevirapine (50 nM) and efavirenz (50 nM) were separately incubated with miniRTIC (50 nM) under the same conditions. Free vRNA–tRNA (50 nM) and RT (250 nM) was also preincubated under the same conditions. Incorporation reactions were initiated by adding a mixture of α-32P-dCTP (40 nM) and dCTP (50 μM). Reactions were quenched at various time points between 5 s and 4 h with the addition of EDTA and SDS loading buffer. The reaction sets for each condition  were run on a 12% SDS-PAGE gel, dried, and exposed for 18 h on a phosphoimager screen (Molecular Dynamics) and each gel was individually scanned with a Storm 860 (Molecular Dynamics)13. Bands were quantified using ImageQuant. The intensity was normalized to the highest intensity for the individual time-course assays after background subtraction (set to 1). The miniRTIC and free vRNA–tRNA with RT were repeated five times. Nevirapine and efavirenz conditions were each repeated three times. Plotting and curve fitting was performed using GraphPad Prism8.The cross-linked miniRTIC, RT, and vRNA–tRNA used in 13. Reactions were preincubated at 37 °C for 5 min in 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 6 mM MgCl2, and 4 mM β-ME at vRNA–tRNA concentration of 200 nM and RT at a concentration of 3 µM. For drug conditions, preincubation occurred under the same conditions and with the addition of 3 µM efavirenz or 3 µM nevirapine. Reactions were initiated by the addition of a dNTP mixture concentration of 100 µM. Reactions were performed in triplicate and quenched at 5-, 10-, and 30 min with EDTA and 2X formamide dye. For the unextended control sample, preincubation conditions were the same, followed by quenching and then the addition of 100 µM dNTP mixture. Samples were denatured and heated for 5 min at 95 °C and loaded on an 8.5% polyacrylamide gel that was prerun for 2 h at 100 W. Samples were run for 3 h at 120 W before imaging for Cy3 fluorescence with a Typhoon Trio (Amersham Biosciences). Unextended primer bands were quantified using GE ImageQuant. Percent primer extension was normalized to control and plotting/curve fitting was performed using GraphPad Prism813.vRNA–tRNA complexes were purified as described above using a tRNA primer with a 5′ cyanine-3 dye (Cy3) labelFurther information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting summary


Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells  were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of  Gram-negative bacterial pathogens exhibit high-level resistance to most classes of antibiotics due to the presence of an impermeable outer membrane ,2. PolymE)-4-methylbenzylidene)hydrazineyl)pyrimidin-2-amine  has been a valuable research tool in microbiology for high-resolution structural studies of bacteria and their components ,15. TEM NCL195, a novel pyrimidine compound ,25 Figu, was synE. coli Xen14  and bioluminescent P. aeruginosa Xen41  were purchased from PerkinElmer Inc. . E. coli Xen14 was grown on horse blood agar (HBA) containing 30 µg/mL kanamycin and P. aeruginosa Xen41 was grown in HBA containing 60 µg/mL tetracycline overnight at 37 °C in normal air for selection. A single colony was taken from the overnight growth, suspended in 10 mL Luria-Bertani (LB) broth, Miller  in a 50 mL flask and incubated at 37 °C under continuous agitation in an orbital shaker at 150 rpm. The overnight bacterial culture was diluted 1:30 in 40 mL LB broth in 50 mL flask of LB broth and then incubated again at 37 °C under continuous agitation until A600nm = 0.1 or 0.5.Bioluminescent g for 5 min at 4 °C to avoid cell damage. The cells were initially resuspended in either cacodylate buffer (pH 7.0) or phosphate-buffered saline  and centrifuged twice for 5 min at 2900× g. Thereafter, cell pellets were fixed overnight in fixative containing 3.0% formaldehyde, 0.035% glutaraldehyde, 4% sucrose in cacodylate buffer (Procedure 1); fixative containing 4.0% formaldehyde, 1.25% glutaraldehyde, 4% sucrose in PBS buffer (Procedure 2); fixative containing 4.0% formaldehyde, 1.25% glutaraldehyde in cacodylate buffer without sucrose supplementation (Procedure 3); fixative containing 4.0% formaldehyde, 1.25% glutaraldehyde, 4% sucrose and 0.01 M CaCl2 in cacodylate buffer (Procedures 4 and 5), as detailed in Xen14 was prepared in five different ways  to minimXen41 cells were prepared essentially as described for Xen14, then processed for TEM using Procedure 4  with eit2O) and Reynolds lead citrate for 10 min each, with three washes in distilled water in-between each stain. Sections were then viewed on a Tecnai G2 Spirit  Transmission Electron Microscope operated at 100 KV at Adelaide Microscopy, The University of Adelaide.Sections of Xen14 and Xen41 embedded in resin were cut to 1 μm using a glass knife, stained with 1% toluidine blue containing 1% borax and viewed under a light microscope at 400× magnification to identify stained bacteria. Ultrathin sections were then cut to 90 nm with an ultramicrotome EM-UC6 (Leica) using a diamond knife (Diatome) and placed on 200-mesh copper EM grids (Proscitech). Sections were sequentially stained with uranyl acetate  [3) were cut and infiltrated with 2.3 M sucrose in phosphate buffer overnight at 4 °C with gentle rocking. Blocks were stored in 2.3 M sucrose at 4 °C prior to sectioning. Blocks were transferred to aluminum sectioning pins (Leica) and quickly plunge-frozen in liquid nitrogen. Thin cryo-sections (80 nm) were cut at −100 °C with an EM-UC6/FC7 cryo-ultramicrotome (Leica) using a cryo-diamond knife (Diatome). Cryo-sections were removed from the knife with 2.3 M sucrose using a wire loop and transferred to formvar/carbon-coated, plasma-cleaned 200-mesh copper EM grids. Grids were stored in an airtight container on sucrose droplets at 4 °C. To stain, grids were floated face down on 2% gelatin for 30 min at 37 °C before washing in PBS (3 × 2 min) and staining with 2% uranyloxalate acetate pH7  and methyl cellulose–uranyl acetate (pH 4) on ice (10 min). Grids were looped out, drained and allowed to dry. Samples were imaged with a Tecnai G2 Spirit electron microscope (FEI Company) operated at 100 kV at Adelaide Microscopy, The University of Adelaide.Xen14 and Xen41 cells were prepared as described above before being fixed in 1 mL cacodylate buffer containing 4.0% formaldehyde, 1.25% glutaraldehyde, 0.01 M CaCltioning) ,27. SampA600nm = 0.1 (early logarithmic phase) and 0.5 (mid logarithmic phase) and then treated with colistin at 0.5 µg/mL for 1 h. Subsequently, Xen14 cells grown to A600nm = 0.1 were chosen for further analysis and were incubated with 0.5 µg/mL colistin for 2 h and 4 h to determine optimal treatment time.To determine the optimal conditions to observe NCL195-colistin interaction on Gram-negative membranes, Xen14 cells were initially grown until A600nm = 0.1. Aliquots were then treated as follows: (i) no treatment, (ii) NCL195 alone (2 µg/mL); (iii) colistin alone (1 µg/mL); (iv) colistin (1 µg/mL) + NCL195 (2 µg/mL) combination. Aliquots of Xen14 and Xen41 were then incubated for 1 h at 37 °C under continuous agitation in a shaker at 150 rpm. During the treatment time, each sample was manually mixed every 10 min to ensure adequate antibiotic contact with the cells. Following treatment, Xen14 cells were washed in buffer and then fixed overnight according to Procedures 4 or 5  no treatment, (ii) NCL195 alone (2 µg/mL); (iii), colistin alone (0.125 µg/mL); (iv), colistin alone (0.25 µg/mL); (v), colistin (0.25 µg/mL) + NCL195 (2 µg/mL) combination. For Xen 41, cells were washed twice in cacodylate buffer and resuspended in LB broth to s 4 or 5 , whereass 4 or 5 . For crys 4 or 5  and storE. coli and P. aeruginosa after exposure to NCL195 alone, colistin alone or NCL195-colistin combination. For this study, we examined the morphological changes to bacterial cells exposed to the test drugs using bioluminescent derivatives of E. coli and P. aeruginosa used routinely in our real-time in vivo assessments of drug efficacy. We initially used Xen14 cells to optimize the best TEM protocol for observing NCL195-colistin interaction on Gram-negative membranes. We found that several factors affected the morphology of the bacterial cells., et al. [Fixative: Fixative containing 3.0% formaldehyde, 0.035% glutaraldehyde, 4% sucrose and 0.075% ruthenium red, 0.075% L-lysine acetate (Procedure 1) caused shrinkage of the bacterial cell as well as detachment and perturbation of the cell membrane A,B. Ther, et al.  describeBuffer: We also found that the type of buffer used resulted in altered cell membrane morphology. PBS buffer caused detachment of the cell membrane (Embedding method: Following from the optimized fixative and buffer conditions above (Procedure 2), we observed that a TEM protocol using cacodylate buffer with fixative containing 4.0% formaldehyde, 1.25% glutaraldehyde, 0.075% ruthenium red, 0.075% L-lysine acetate, and 4% sucrose followed by embedding in LR-White resin (Procedure 4) provided the best delineation of the outer membrane, cell wall and inner membrane, with no wavy, detached or shrunk membranes A. This pIn this work, we sought to determine the most effective technique to accurately visualize drug interactions on the bacterial membrane as part of our on-going research aimed at gaining a better understanding of the complex interactions between membrane-active drugs and the consequent morphological changes occurring on the bacterial surface. To accomplish this, we compared two sample preparation methods for TEM  to visualize the cell membrane of P. aeruginosa Xen41. However, due to the production of exopolysaccharide by P. aeruginosa [Having optimized the best TEM protocol for visualizing Xen14 morphology (Procedure 4), this was then applied to ruginosa , which mA600nm = 0.1 or A600nm = 0.5). For this initial analysis, the Xen14 cells were treated with colistin at 0.5 µg/mL for 1 h. Significant morphological changes were observed following 1 h incubation in colistin at A600nm = 0.1. Compared to untreated cells , the majority of the tubular and fimbria-like radiant appendages were broken and had disappeared, although layered membrane structure could still be observed  produced clearly visualized membrane damage in cells processed under cryo conditions E, wherea2 0.01 M, 4% sucrose and 0.075% ruthenium red, 0.075% L-lysine acetate is the optimal mixture for the stability of bacterial cell membrane. We also suggest using LR-White resin due to its ease of use during TEM processing. Additionally, we show the cryo-ultramicrotomy technique provides higher resolution, artifact reduction, clearer visualization of bacterial cytoskeleton and better preservation of bacterial structural integrity compared to conventional TEM processing methods. To our knowledge, this study is the first to use Tokuyasu cryo-ultramicrotomy to examine the effects of multiple drug-interactions on the bacterial cell surface. Cryo-ultramicrotomy can also be employed in conjunction with other imaging techniques such as that described for correlative light and electron microscopy [In this study, we describe optimized TEM conditions for visualizing changes to Gram-negative bacterial morphology induced by treatment with a combination of NCL195, a novel pyrimidine, and colistin. We show that cacodylate buffer works better than PBS buffer, and that fixative containing 4.0% formaldehyde, 1.25% glutaraldehyde, CaClcroscopy . We sugg


Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate  for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDHLx) and DERATma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERATma and MDHLx.One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from  Lysinibacillus xylanilyticus (MDHLx) efficiently converts methanol and ethanol to formaldehyde and acetaldehyde, respectively [Thermotoga maritima  has been introduced as an intermediate enzyme to condense formaldehyde and acetaldehyde to 3-HPA for PDO biosynthesis in engineered Escherichia coli [One- or two-carbon C1 or C2) compounds have been utilized as attractive substrates for biotechnological production of valuable compounds because they are inexpensive and can be used as bio-renewable platform feedstock  or C2 co,4. Howevectively . Alcoholectively . To dateectively ,22,23,24hia coli . In thishia coli . MoreoveTma concentration, and formaldehyde and acetaldehyde concentrations as substrates for 3-HPA production. Moreover, we designed and performed a one-pot cascade reaction for 3-HPA biosynthesis from methanol and ethanol using two enzymes, MDHLx and DERATma  column. The purified DERATma enzyme (approximately 29.4 kDa), which converts formaldehyde and acetaldehyde to 3-HPA  of reported recombinant DERAs from araensis ; Hyperthutylicus ; Pyrobacrophilum , Yersiniinia sp. , Paeniballus sp. , and Haljaponica ; Thermotmaritima ; Acidulim boonei , and Rhohropolis ; E. coli[E. coli , Haemophfluenzae , and Psesyringae , were 4.syringae ; H. inflfluenzae ; Yersiniinia sp.  and Paenllus sp. ; H. japojaponica ; R. erythropolis ; H. butyutylicus  and A. b. boonei ; T. kodaaraensis , were 25Tma toward formaldehyde and acetaldehyde as the substrates were measured  of DERATma for formaldehyde was 2.8-fold higher than that for acetaldehyde, suggesting that DERATma prefers formaldehyde (C1) than acetaldehyde (C2). Zeng et al. investigated the Km of DERATma toward formaldehyde and acetaldehyde, which were 2.54 and <1 mM (at pH 7.0 and 30 °C), respectively [Km for formaldehyde was similar to that reported previously, but the Km value for acetaldehyde was 7-fold higher than that reported previously (at pH 7.0 and 40 °C). Moreover, the reported Km values for acetaldehyde in other aldol reactions with sugar substrates for DERA isolated from Rattus norvegicus [Lactiplantibacillus plantarum [E. coli [Salmonella enterica [Tma, respectively.Substrate specificity and kinetic parameters of DERAmeasured . The kcaectively . Howeverrvegicus , Lactipllantarum , E. coli[E. coli , and SalTma concentration. The optimal enzyme concentration for 3-HPA production was investigated using 0.28, 0.55, 0.83, 1.11, 1.39, 1.66, 1.94, 2.22, and 2.50 U/mL enzyme toward 10 mM formaldehyde with 20 mM acetaldehyde. The 3-HPA production increased when using <1.66 U/mL enzyme; however, product concentration reached a plateau when using >1.66 U/mL enzyme  [Tma and MDHLx A164F showed a maximal activity at pH 7.4 and 45 °C  . Interesnd 45 °C b,c, whicectively . Thus, wLx has higher catalytic efficiency toward ethanol than toward methanol, which can cause interference between the two substrates at the enzyme active site [To obtain the optimal concentrations of methanol and ethanol as substrates, we investigated the enzyme cascade reactions for 3-HPA production using 0.05–1 M methanol in the presence of 1 M ethanol and 0.16–1 M ethanol in the presence of 1 M methanol . The conive site , indicatTma and MDHLx A164F were pH 7.4, 45 °C, 1.66 U/mL of DERATma, 0.026 U/mL of MDHLx A164F, 0.8 M of methanol, and 0.15 M of ethanol with 5 mM of Mg2+ and 3 mM of NAD+. Under these optimal conditions, time-course reactions for 3-HPA biosynthesis were conducted  of 3-HPA from methanol and ethanol for 8 h with 2.04 mg/L/h productivity. Although the productivity was quite low, producing valuable products from methanol and ethanol as cheap materials without carbon loss and by-product formation is important for value-added material biosynthesis via eco-friendly processes. In addition, MDH and DERA with high activity should be developed using rational approach-based protein engineering or directed evolution in the future. Till date, 3-HPA has been produced as an intermediate by glucose or glycerol fermentation using wild-type strain cells and metabolically engineered cells such as  mM of 3-E. coli ,25,41, Lllus sp. ,46,47,48eumoniae ,51,52,53eumoniae ; 2,3-buteumoniae .Thus, 3-HPA has not yet been reported as the main product. However, 3-HPA can be designed as the main product, using enzymatic conversion. Furthermore, methanol and ethanol are considered some of the most useful substrates for obtaining complex chemical compounds. Therefore, these C1 or C2 chemicals have been used to produce valuable products such as polyhydroxyalkanoates, amino acids, organic acids, and other chemical compounds using diverse biocatalysts [+ were obtained from Sigma-Aldrich . PrimeSTAR® Max DNA polymerase for cloning the DERA gene was purchased from Takara . T4 DNA ligase, T4 kinase, and DpnI were obtained from NEB . PCR product purification and gel extraction kits were purchased from Promega . QIA prep Spin mini-prep kit for plasmid preparation was purchased from Qiagen . Ni2+-NTA column for protein purification was purchased from GE Healthcare . Oligonucleotides and plasmid sequencing services were provided by Macrogen facility . All materials for DNA techniques and SDS–PAGE were purchased from Bio-Rad . All DNA and protein techniques were carried out following the standard molecular biological protocols. 3-HPA was provided by Korea Research Institute of Bioscience (KRIBB) and used for standard curve using HPLC [Ethanol, methanol, formaldehyde, acetaldehyde, and NADing HPLC .E. coli C2566 (New England Biolabs) and pET-28a(+) carrying kanamycin resistance  were used as host cells and expression vector, respectively. The DERA gene (NCBI accession No. AE000512.1) from Thermotoga maritima, comprising 747 nucleotides (248 amino acids), was synthesized, and codon optimization was performed to enhance expression yield in E. coli by Cosmogenetech facility . The synthesized gene was cloned into the expression vector pET-28a(+) with His6-tag at the N-terminal domain. The DERA gene was amplified from the synthesized gene fragment using PCR and then cleaned up using a PCR purification kit (Promega). The primer sequences used for gene cloning are summarized in E. coli C2566 and then plated on Luria Bertani (LB) agar supplemented with 30 μg/mL kanamycin to select the kanamycin-resistant colony. The selected colony was isolated using the plasmid purification kit (Qiagen) and confirmed by DNA sequencing at the Macrogen facility .Lx was applied to a one-pot reaction with DERATma [Lx variants was added to the PCR mixture containing 10 pmol of each primer . These transformants (10–100 μL) were plated on LB agar with 30 μg/mL kanamycin, incubated at 37 °C overnight (14–16 h), and then inoculated in 3 mL LB broth. Recombinant E. coli cells harboring DERATma or MDHLx were cultivated in a 2-L flask containing 250 mL LB broth supplemented with 30 μg/mL kanamycin at 37 °C and 200 rpm. To induce DERATma expression, isopropyl-β-d-thiogalactopyranoside  was added to the LB broth when the optical density of recombinant cells at 600 nm reached 0.6–0.8 and continuously cultivated at 30 °C with shaking at 200 rpm for overnight. In case of MDHLx and its variants, the temperature was reduced to 20 °C after IPTG induction and they were continuously cultivated overnight to induce enzyme expression [Each plasmid, such as the DERApression .E. coli cells were harvested by centrifugation at 8660× g for 20 min at 4 °C and then stored at −80 °C. Then, they were resuspended in lysis buffer  and disrupted on ice by ultrasonication for 10 min. The disrupted cells were centrifuged at 31,660× g for 30 min at 4 °C to remove unbroken cells and cell debris. The supernatant (crude extract) was loaded to a Ni2+–NTA column, and the bound protein was eluted with the same buffer containing 250 mM imidazole at 4 °C. The purified enzyme was desalted with a 10 kDa Amicon ultra centrifugal filtration tube (Sigma-Aldrich), dialyzed with 20 mM Kpi buffer (pH 7.0), and confirmed using SDS–PAGE. The protein concentration was measured using the Bradford method.Recombinant O-benzylhydroxylamine hydrochloride dissolved in 33:15:2 (v/v/v) pyridine: methanol: water were mixed and incubated at 25 °C for 2 h. The samples were diluted with 30 µL methanol, and 3-HPA was quantified using HPLC with 3-HPA as the standard. The kinetic parameters of DERATma were measured with different substrate concentrations. The catk (min−1) and MK (mM) values were determined using non-linear regression with Michaelis–Menten equation using GraphPad Prism 8 software .One unit (U) enzyme activity was defined as the amount of enzyme required to produce 1 μmol 3-HPA per minute at 40 °C and pH 7.0. The enzyme specific activity was defined as the product amount produced per enzyme amount per unit reaction time. Unless otherwise stated, the reaction was conducted in 20 mM Kpi buffer (pH 7.0) containing 0.5 mg/mL enzyme at 40 °C for 10 min. To determine aldol condensation, the reaction was performed using 20 mM formaldehyde and 20 mM acetaldehyde. Then, 15 µL diluted samples and 50 µL 130 mM Tma activity, pH values were varied from 5.5 to 9.0 using 20 mM MES buffer (pH 5.5–6.5), 20 mM Kpi buffer (pH 6.5–8.0), and 20 mM Tris buffer (pH 8.0–9.0) at 30 °C. The effect of temperature on DERATma activity was monitored as a function of time by incubating at 25, 30, 35, 40, 45, 50, 55, and 60 °C, and pH 7.0.To evaluate the effect of pH on DERATma concentration for 3-HPA production from formaldehyde and acetaldehyde, 0.28–2.50 U/mL DERATma was incubated with 10 mM formaldehyde and 10 mM acetaldehyde. To determine the optimal substrate concentrations, the reactions were performed with 1.66 U/mL DERATma and 0–30 mM formaldehyde or acetaldehyde. Time-course reactions for 3-HPA production from formaldehyde and acetaldehyde were conducted with 1.66 U/mL DERATma for 180 min with 15 mM formaldehyde and 10 mM acetaldehyde.To determine the optimal DERA2+, 3 mM NAD+, 1 M methanol, and 1 M ethanol in 20 mM Kpi buffer (pH 7.4). The effect of temperature on enzyme stability was monitored as a function of time by incubating at 25, 30, 35, 40, 45, 50, 55, 60, and 65 °C in 20 mM Kpi buffer (pH 7.4) for 120 min. To evaluate the effect of pH on aldol activity, pH values were varied from 6.0 to 9.0 using 20 mM Kpi buffer (pH 6.0–8.0) and 20 mM Tris–HCl buffer (pH 8.0–9.0) for 120 min at 45 °C. To determine the optimum DERATma and MDHLx ratio, 0–2.22 U/mL DERATma was used, while 0.01 U/mL MDHLx was used and 0–0.03 U/mL MDHLx were used, while 1.39 U/mL DERATma was used at 45 °C for 120 min. To determine the optimal substrate concentrations, the reactions were performed in 20 mM Kpi buffer (pH 7.4) containing 1.39 U/mL DERATma, 0.026 U/mL MDHLx, 5 mM Mg2+, and 3 mM NAD+ with 0–1 M methanol or ethanol for 120 min at 45 °C. Time-course reactions for one-pot 3-HPA production from methanol and ethanol were conducted in 20 mM Kpi buffer (pH 7.4) containing 1.39 U/mL DERATma, 0.026 U/mL MDHLx, 5 mM Mg2+, 3 mM NAD+, 800 mM methanol, and 100 mM ethanol at 45 °C for 480 min.To optimize the reaction conditions for 3-HPA production, the reactions were performed with 5 mM Mg® 5 µm C18 110 Å, LC column of 250 × 4.6 mm dimensions  after derivatization as follows: 15 µL supernatant or reaction solution was mixed with O-benzylhydroxylaminehydrochloride solution (50 µL of 130 mM stock solution in pyridine:methanol:water 33:15:2). After being incubated at 25 °C for 2 h, the samples were diluted with 30 µL methanol and centrifuged at 8660× g for 3 min. The column was monitored by increasing the absorbance at 215 nm at 30 °C with a gradient of mobile phase A  and mobile phase B  as follows: mobile phase B was increased from 10% to 100% for 30 min, decreased to 10% for 30–32 min, and stabilized for 32–50 min at 1 mL/min flow rate.Formaldehyde, acetaldehyde, and 3-HPA were quantified using HPLC equipped with a GeminiTma, which also catalyzes bioconversion of the cheap C1 and C2 chemicals, such as methanol and ethanol, to 3-HPA via the MDHLx A164F cascade reaction. We optimized the reaction conditions, including pH, temperature, and enzyme and substrate concentrations, for DERATma activity and cascade reaction with MDHLx A164F to improve enzymatic 3-HPA bioconversion. This is the first study on one-pot 3-HPA production from methanol and ethanol using two biocatalysts. In the future, the results of this study could be useful for industrial value-added product production from bio-renewable platform feedstocks via eco-friendly processes using biocatalysts.In this work, we identified the biosynthetic pathway of 3-HPA from formaldehyde and acetaldehyde using DERA


The E4106.30×30K missense variant displays higher constitutive activity in G protein signaling as compared to wild-type H1R, whereas the opposite was observed for R1273.52×52H, R13934.57×57H, and R4096.29×29H. The E4106.30×30K missense variant displays a higher affinity for the endogenous agonist histamine than wild-type H1R, whereas antagonist affinity was not affected. These data support the hypothesis that the E4106.30×30K mutation shifts the equilibrium towards active conformations. The study of these selected missense variants gives additional insight into the structural basis of H1R activation and, moreover, highlights that missense variants can result in pharmacologically different behavior as compared to wild-type receptors and should consequently be considered in the drug discovery process.The Exome Aggregation Consortium has collected the protein-encoding DNA sequences of almost 61,000 unrelated humans. Analysis of this dataset for G protein-coupled receptor (GPCR) proteins (available at GPCRdb) revealed a total of 463 naturally occurring genetic missense variations in the histamine receptor family. In this research, we have analyzed the distribution of these missense variations in the four histamine receptor subtypes concerning structural segments and sites important for GPCR function. Four missense variants R127 G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins encoded by the human genome and play a dominant role in regulating human physiology in response to extracellular stimuli . Consequ−3) and 4.8 commons (MAF ≥ 1 × 10−3) MVs per GPCR subtype   of nearly 61,000 unrelated individuals in the exome aggregation consortium (ExAC) database  revealedry 2021)  5]. The. The−3) 1, H2, H3, and H4 receptor  . In . In 5]. 1R, H2R, H3R, and H4R, respectively  of H2R, and four common MVs in TM3 (V883.26×26G), TM4(A1384.48×48V), TM5 (H2065.70×70R) and ICL3 (S284C) of H4R  were predicted to have a functional impact based on their SIFT (0–0.05) and/or PolyPhen (>0.1) scores, whereas more than two-thirds of the MVs in the H3R and H4R  were predicted to be functionally deleterious mepyramine saturation binding , whereas R4096.29×29H has the same affinity as WT hH1R.Initially, the binding characteristics of the four selected natural hH binding . Next, mepyramine (specific activity 20.7 Ci/mmol), MicroScint-O scintillation liquid, and GF/C filter plates were bought from PerkinElmer . Doxepin, mepyramine maleate, levocetirizine·2HCl and histamine·2HCl were obtained from Sigma-Aldrich . NanoGlo® was bought from Promega . Fetal bovine serum (FBS) was obtained from Bodinco , and penicillin/streptomycin was purchased from GE Healthcare . FastDigestTM restriction enzymes, Dulbecco’s modified Eagles medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), trypsin-EDTA, Hanks’ balanced salt solution (HBSS), PierceTM BCA protein assay kit, GeneJET gel extraction kit and GeneJET plasmid Miniprep kit were bought from Thermo Fisher Scientific . 25 kDa linear polyethylenimine (PEI) was purchased from Polysciences . The Nluc-hH1R/pcDNA3.1 construct was previously reported mepyramine (0–20 nM) for 1 hour at 25 °C with gentle agitation in the absence or presence of 10 µM mianserin to detect total and nonspecific binding, respectively. For competition binding assay, cell homogenates were incubated with 7 nM [3H]mepyramine in combination with increasing concentrations of unlabeled histamine, doxepin, mepyramine, or levocetirizine, for 1 hour at 25 °C with gentle agitation. Incubations were stopped by rapid filtration with ice-cold wash buffer  over a 0.5% polyethyleneimine-coated 96-well GF/C filter plate using a 96-well FilterMate-harvester . The GF/C filter plates were dried, and filter-bound radioactivity was quantified after the addition of 25 μL/well MicroScint-O using a Wallac 1450 MicroBeta Trilux counter (PerkinElmer). The binding affinity (Kd) of [3H]mepyramine and the total number of receptors (Bmax) were determined using the “One site—Total and nonspecific binding” model in GraphPad Prism 8.4.3. The protein content of the cell homogenates was measured by the BCA kit. Competition binding curves were fitted using the “one-site—Fit logIC50” model in GraphPad Prism 8.4.3, and binding affinities (Ki) of unlabeled ligands were subsequently calculated using the Cheng–Prusoff equation:L] and dK are the concentration and binding affinity of [3H]mepyramine, respectively.HEK293T cells were collected two days after transfection with 1 µg of Nluc-hHescribed . For sat1R variants and 2.5 µg NFAT-Luc reporter gene plasmids per dish and transferred  into poly-L-lysine-coated white 96-well plates after 24 h, as previously described [2, 0.78 µM Na2HPO4, 38.9 mM Tris (pH 7.8), 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 µM dithiothreitol). Luminescence was measured (1 s/well) after another 30 min incubation at 37 °C in a Mithras LB940 multimode microplate reader . Concentration–response curves were fitted using the “log (agonist) vs. response (three parameters)” model in GraphPad Prism 8.4.3.HEK293T cells were transiently co-transfected with 5 ng Nluc-hHescribed . The celGraphPad Prism version 8.4.3  was used for nonlinear regression and statistics.


OI of 35%. Blended with other benzoxazines, it significantly improves their thermal stability and fire resistance. It emphasizes its potential as flame retardant agent.This work explores the strategy of incorporating a highly substituted reactive flame retardant into a benzoxazine moiety. For this purpose, a DOPO-based flame retardant received a chain extension via reaction with ethylene carbonate. It was then reacted with phloretic acid to obtain a diphenol end-capped molecule, and further reacted with furfurylamine and paraformaldehyde to obtain a benzoxazine monomer via a Mannich-like ring closure reaction. This four-step synthesis yielded a partly bio-based halogen-free flame retardant benzoxazine monomer (DOPO-PA-fa). The successful synthesis was proven via NMR, IR and MS analysis. The polymerization behavior was monitored by DSC and rheological analysis both showing the polymerization starts at 200 °C to yield pDOPO-PA-fa. pDOPO-PA-fa has a significant thermal stability with a residual mass of 30% at 800 °C under ambient atmosphere. Furthermore, it reached a V-0 rating against small flames and an  For instance, furfurylamine . DO. DO26]. p-benzoquinone, dimethylacetamide, ethylene carbonate, 2-propanol, phloretic acid, p-toluene sulfonic acid monohydrate, paraformaldehyde, furfurylamine, DOPO, potassium iodide, chloroform and ethyl acetate were supplied from Sigma–Aldrich  and DOPO from TCI . All chemicals were used without further purification.2-Ethoxyethanol, The synthesis of DOPO-HQ was adapted by Wang et al. . DOPO : δ (ppm) = . δ = 9.46 ; 9.17 ; 8.27–8.20 ; 7.74 ; 7.55–7.62 ; 7.50 ; 7.45 ; 7.32–7.26 ; 7.19 ; 6.89 ; 6.64  ppm.31P NMR  δ = 22.11 (s) ppm.The synthesis of DOPO-OH was adapted by Wang et al. . Dimethy1H NMR : δ (ppm) = . δ = 8.29–8.21 ; 7.76 ; 7.71–7.65 ; 7.52 ; 7.46 ; 7.36 ; 7.33–7.29 ; 7.20 ; 7.03 ; 4.87 ; 4.46 ; 4.03–3.91 ; 3.72–3.67 , 3.59–3.52 ; 2.98–2.93  ppm.31P NMR  δ = 22.11 (s) ppm.p-toluene sulfonic acid monohydrate  was added. The melt was stirred for 20 h. The glassy product was recrystallized from ethyl acetate to obtain a white powder in a 67% yield.The solventless method was adapted from previous reports . Phloret1H NMR : δ (ppm) = . δ = 9.16 ; 8.21–8.13 ; 7.74–7.69 ; 7.58–7.50 ; 7.45 ; 7.39 ; 7.28–7.21 ; 7.03–6.96 ; 6.94–6.91 ; 6.67–6.63 ; 4.35 ; 4.25–4.16 ; 3.87–3.80 ; 3.71–3.66 ; 3.49–3.42 ; 3.39–3.35 ; 2.75 ; 2.59 ; 2.55–2.51 ; 2.24–2.02  ppm31P NMR  δ = 21.70 (s) ppm.m/z, [M+Na]+ calculated for C40H37NaO10P+, 731.20165; found, 731.20080.MS (ESI) Furfurylamine  was mixed with paraformaldehyde  in chloroform (70 mL) and heated to 65 °C for 1 h before the addition of DOPO-PA , paraformaldehyde  and chloroform (100 mL). The reaction mixture was heated at 65 °C for 48 h. The reaction solution was added to a separation funnel and the organic layer was washed twice with aqueous hydrochloric acid (0.5 M), deionized water, saturated sodium carbonate and brine. The organic layer was pre-dried with magnesium sulfate and filtered, before the chloroform was removed under reduced pressure and the oil was further dried for 14 h at 50 °C. The product was obtained as yellow oil in a yield of 71%.1H NMR : δ (ppm) = . δ = 8.21–8.14 ; 7.71 ; 7.60 ; 7.56–7.50 ; 7.47–7.42 ; 7.38 ; 7.28–7.21 ; 7.04–6.87 ; 6.87 ; 6.76 ; 6.68–6.62 ; 6.44–6.38 ; 6.32–6.25 ; 4.77 ; 4.41–4.31 ; 4.26–4.16 ; 3.88–3.82 ; 3.80 ; 3.87–3.80 ; 3.71–3.66 ; 3.50–3.44 ; 3.39–3.35 ; 2.75 ; 2.62 ; 2.55–2.51 ; 2.24–2.02  ppm.31P NMR  δ = 19.80 (s) ppm.m/z, [M+H]+ calculated for C54H53N2O12P+, 951.32524; found, 951,32428.MS (ESI) −1 in a range between 500–4000 cm−1.An Equinox 55 ATR spectrometer  was used to conduct the Fourier transform infrared spectra in attenuated total reflection (ATR) mode using a diamond crystal. The sample were recorded with 32 scans and a resolution of 4 cm1H-NMR spectra were recorded on an AVANCE NEO 600 MHz-Spectrometer  with DMSO-d6 as solvent at 20 °C. 13C NMR and 31P NMR spectra were recorded using the same spectrometer with a frequency of 150 MHz and 243 MHz, respectively. The chemical shifts were reported in parts per million (ppm) and referred to residual protons in the DMSO-d6 at 2.50 ppm.2 atmosphere. All samples were measured 3 times and the average was taken for evaluation.DSC measurements were performed using a Discovery DSC  with 10 K/min in a range from 0 °C to 300 °C on a specimen scale of 1–2 mg under N3. Tg was determined by the maximum of tan(δ).DMA experiments were performed using a DMA Q800 . Thermo-mechanical properties were determined in a temperature range from 0 °C to 250 °C with a heating rate of 2 K/min with a frequency of 1 Hz in a single cantilever bending mode and test specimen of 40 × 10 × 4 mmTGA measurements were carried out with a Q5000  with a temperature range from 25 °C to 800 °C and a heating rate of 10 K/min under ambient and nitrogen atmosphere; 2, 5, 20 and 40 K/min were additional heating rates for samples under nitrogen atmosphere.3 and ground edges.UL94 vertical burning tests were performed according to DIN EN 60695-11-10 with specimen dimensions of 125 × 13 × 4 mmLOI experiments were performed according to EN ISO 4589-2 with specimen type 1, dimensions of 100 × 10 × 4 mm and ground edges with an LOI apparatus .v/v.Mass spectrometry (MS) analysis was conducted on a Bruker Impact II  mass spectrometer using electrospray ionization with 3 µL/min in DCM/Methanol 1/10 Rheological measurements were recorded using an Anton Paar Physica MCR 302 rheometer equipped with a CTD 450 temperature control device. Small quantities of the samples were loaded in a parallel plate–plate geometry (ϕ = 25 mm). The polymerization measurements were recorded in the oscillation mode at a frequency of 1 Hz and a controlled strain of 0.1%. Heating ramps of 2 K/min were applied to reach a temperature of 225 °C. The sample deformation was ramped linearly from 1% to 0.2% to remain within the instrument’s limitation and to maintain a linear viscoelastic behavior as the moduli  increase by several orders of magnitude upon curing. The gap between the plates was maintained at 0.5 mm during all the experiments.3. The melt was degassed for a second time before polymerizing with the following program 2 h at 170 °C and 2 h at 180 °C. BF-a was heated to 140 °C and p PEG400-PA-fa to 100 °C before transfer to the metal mold. After curing and demolding the specimens were cut according to DMA, LOI and UL94 test dimensions.The curing was performed with different parameters for the benzoxazines. DOPO-PA-fa was heated up to 105 °C, degassed and poured into a metal mold with the dimension 170 × 130 × 4 mmThe following paragraphs describe the synthesis and analysis of the bisbenzoxazine DOPO-PA-fa . The dirp-TSA  induce a spatial conformation.−1 to 1710 cm−1 and 1723 cm−1 was observed proving the formation of ester linkages. It is further confirmed by the disappearance of the C-O-H stretching signal of DOPO-OH at 1030 cm−1, indicating the full consumption of the aliphatic hydroxyl groups.The ester formation was confirmed by FTIR . A shiftDOPO-PA was reacted in chloroform with furfurylamine and paraformaldehyde to yield DOPO-PA-fa .−1 was assigned to the ring vibration of benzoxazine rings. The signal at 731 cm−1 corresponds to a mono substituted furan ring. Furthermore, the O-H stretching band at 3333 cm−1 was reduced significantly, but it was still present, presumably due to an incomplete benzoxazine formation.The benzoxazine ring formation was observed by FTIR . The sig1H NMR  can induce a preferential spatial conformation. Therefore, each type of end-chain oxazine ring methylene peak (30 and 48 or 31 and 49) deviate in the chemical shift, as they are not equivalent. A small residual phenolic hydroxyl group signal at 9.17 ppm indicates that the benzoxazine formation was incomplete, in line with FTIR characterizations. The signal of the O-CH2-N and the aromatic signal at 7.71 ppm were set in relation to each other to determine the degree of benzoxazine ring formation. The values of the integration were 2.6 and 1.00, indicating that 65% of the benzoxazine rings are closed.The benzoxazine ring formation was also confirmed by 1H NMR . The sigThe polymerization of the novel bio-based DOPO-PA-fa was determined by DSC analysis and compared with a conventional bisphenol F and aniline based benzoxazine monomer (BF-a) and a bisbenzoxazine, based on a flexible PEG400 linker (PEG400-PA-fa), as described earlier  14]. Th. Th14]. Tonset) of DOPO-PA-fa at 208 °C is similar to the Tonset of BF-a (216 °C) but higher to the Tonset of PEG400-PA-fa (177 °C). The Tonset of DOPO-PA-fa is in the range of previously reported benzoxazine containing DOPO [Tonset may, presumably, be due to the residual phenolic impurities observed by FTIR and NMR.The DSC analysis of DOPO-PA-fa reveals an exothermic signal in the range of 180–260 °C, assigned to the benzoxazine ring-opening polymerization . The teming DOPO ,27. The The polymerization enthalpies of BF-a and DOPO-PA-fa are in the same range: 112 and 127 kJ/mol, respectively  of the polymer pDOPO-PA-fa was measured at 150 °C (Tg of pBF-a (175 °C). It can be explained by the lower number of benzoxazine groups per grams of pDOPO-PA-fa compared with pBF-a. However, the rigid structure shows a beneficial effect on Tg when comparing with pPEG400-PA-fa (Tg = 26 °C). The storage modulus of pDOPO-PA-fa (2.7 GPa) at 20 °C is higher as for pPEG400-PA-fa (E0 = 1.2 GPa) as it already reached its Tg  for pDOPO-PA-fa and pBF-a. pPEG400-PA-fa had a slightly reduced initial stability with T5% = 291 °C. The evaluated temperature at 20 wt% mass loss showed that pDOPO-PA-fa and pPEG400-PA-fa show a comparable degradation temperature at 390 °C, whereas pBF-a is slightly more stable with T20% = 400 °C. While pPEG400-PA-fa achieved a char yield of 32%, both pDOPO-PA-fa and pBF–a achieved a char yield of 44%. The incorporation of DOPO in the linker structure of the bisbenzoxazine has a beneficial effect on the thermal stability by significantly increasing the char yield in comparison with pPEG400-PA-fa.The three polymers pBF-a, pDOPO-PA-fa and pPEG400-PA-fa all have a similar degradation profile under inert atmosphere a. The inIn thermo-oxidative conditions, the polymers showed only slight differences for the initial degradation stability with 309 °C, 317 °C and 328 °C for pPEG400-PA-fa, pDOPO-PA-fa and pBF-a, respectively. However, at temperatures above 500 °C pDOPO-PA-fa was more stable and reached a char yield of 30% at 800 °C in contrast to no residual mass formation for pBF-a and pPEG400-PA-fa. This effect shows the advantage of the DOPO presence in the molecular structure as it shows a significant effect on the char yield formation at 800 °C under ambient atmosphere.Ea) as an important kinetic parameter was derived from thermograms obtained under different heating regimes. Ea is directly related to the energetic needs for bond dissociation processes and indicates the tendency of material degradation during heating. The Flynn–Wall–Ozawa (FWO) method was used to calculate aE for pDOPO-PA-fa, pBF-a and pPEG400-PA-fa [Ea  in comparison to pPEG400-PA-fa (Ea = 136 kJ/mol). The reference pBF-a (Ea = 190 kJ/mol) needs more energy as it is slightly more stable than pDOPO-PA-fa. However, at a conversion of 10% the needed Ea for pDOPO-PA-fa (Ea = 200 kJ/mol) exceeds those of pBF-a (Ea = 172 kJ/mol).For all studied compounds, a higher UL94 and LOI tests were conducted to investigate the reaction of each polymer against small flames . AccordiOI) with 21.5%. pBF-a achieved an OI of 26% and the pDOPO-PA-fa an OI of 35%. The significant improvement is according to the literature as a DOPO-, aniline- and phenol-based monomer with a phosphorous loading of 7.8 wt% achieved an OI of 40% [As expected from the UL94 tests pPEG400-PA-fa achieved the lowest oxygen index . This approach is promising as it shows a similar effectivity as the neat DOPO-HQ without being limited in regards to the loading.Finally, the efficiency of DOPO-PA-fa to act as a flame retarding agent for other polymers was tested. For that purpose, it was blended with BF-a or PEG400-PA-fa (7 wt% of DOPO-PA-fa leading a phosphorous loading of 0.21 wt%) and compared to blends of BF-a and PEG400-PA-fa with the reactive additive DOPO-HQ resulting the same phosphorous loading of 0.21 wt%. Polymerized blends of DOPO-PA-fa and BF-a achieved an This study describes the incorporation of a DOPO spacer group into bio-based benzoxazine monomer. DOPO-HQ was reacted with ethylene carbonate to promote its reactivity toward a solvent-free esterification with PA, to obtain a diphenol end-capped DOPO molecule. This precursor was reacted with furfurylamine and paraformaldehyde, leading to the benzoxazine monomer DOPO-PA-fa. The successful 4-step synthesis pathway was verified by NMR, IR and MS analysis.Tg (150 °C) is lower than for rigid pBF-a (175 °C) but significantly higher than for the flexible pPEG400-PA-fa (20 °C).The polymerization behavior and thermo-mechanical properties of the corresponding polymer were analyzed by DSC, DMA and rheological analysis and compared to BF-a and PEG400-PA-fa. Results showed that for DOPO-PA-fa, the polymerization starts at around 200 °C, alike to BF-a. Its 2 atmosphere is already comparable to those of pBF-a, under ambient atmosphere it shows an increased stability with a char yield of 30% at 800 °C, whereas those for pBF-a and pPEG400-PA-fa are 0%. During UL94 test, pDOPO-PA-fa reached a V-0 rating and an OI of 35%. Thus, DOPO-PA-fa proved to be an interesting, partly bio-based benzoxazine monomer, with promising properties as an FR monomer for future applications.The thermal stability and reaction tests against small flames showed the improved stability of pDOPO-PA-fa. While the thermal stability for the partly bio-based pDOPO-PA-fa under N


Centaurea solstitialis) is an invasive annual plant that has infested multi-million ha in the western United States, causing multi-billion dollars of losses and management costs. The rosette weevil (Ceratapion basicorne) has recently been approved for biological control of this weed. However, this weevil reproduces only once a year, which hinders mass-rearing for release. Here, we tested whether insect hormones can break the reproductive diapause of female weevils. We found that applying two insect hormones, 20E and methoprene, can consistently terminate diapause of female weevils to enable them to lay eggs. Thus, topically applying insect hormones could be used to induce females to lay eggs at any time of year, which would permit rearing more than one generation per year. This could greatly increase the number of weevils produced per year in a mass-rearing program to accelerate the release of rosette weevils to help suppress yellow starthistle in the United States. These insects would revert to having one generation per year after release in synchrony with their host plant.Yellow starthistle (Ceratapion basicorne (a univoltine weevil), for yellow starthistle  as a model system to study the use of two insect hormones, 20-hydroxyecdysone (20E) and methoprene, to terminate reproductive diapause. Methoprene  terminated reproductive diapause of female weevils, whereas doses of 0.0, 0.01 and 0.1 μg did not. The combination of methoprene and 20E had a stronger effect and induced an increase in eggs  compared with a methoprene only group (1.00 ± 0.13 eggs/day), and a control group (0.21 ± 0.04 eggs/day). Thus, topical application of these hormones should enable us to rear the weevil out of its normal season and produce more than one generation per year, which will increase productivity of mass-rearing it for field release. Once released in the field, the insect would continue as a univoltine agent that is well-synchronized with its host plant.Classical biological control is an important method for controlling invasive alien weeds. Univoltine insects can be highly effective biological control agents of annual weeds because they are well synchronized with their host plant. However, having only one generation per year makes it difficult and slow to multiply them in the laboratory for initial field releases. If it were possible to terminate reproductive diapause early, then we could rear multiple generations per year, which would greatly increase annual production. We used a recently approved biocontrol agent,  Classical biological control, the use of host-specific natural enemies, is an important strategy for controlling invasive alien plants . Once neCentaurea solstitialis L., Asteraceae) is an invasive alien annual weed that has infested about 6 million ha in the Western United States , (2) acetone + methoprene [AM], and 20E + methoprene [2M]. A 20E alone application was not tested because of the small number of females available and because 20E alone failed to terminate reproductive diapause in Colorado potato beetles . Femalesribed in .To test whether the 2M treatment can induce egg laying on underperforming and unresponsive females used in Experiment 3, we applied 20E (1 μg) followed in 2 days by methoprene (1 μg) to underperforming and unresponsive females using the same methods as in Experiment 3. We define “underperforming females” as female weevils that oviposited several days and stopped oviposition (=a short oviposition period) or that oviposited less than 1 egg per day more than two consecutive days. “Unresponsive females” are defined as females that did not lay any eggs after one of the treatments used in Experiment 3. A total of 18 weevils were used with the same environmental conditions as Experiment 3. We recorded the number of feeding holes and eggs in each female every two days. Experiment 4 was conducted from 19 November to 21 December 2020.a is the inflection point and n is the Hill coefficient, which controls the curvature) was fit to the data using nonlinear regression.Because the normality of residuals was significantly different on the Shapiro–Wilk test, we used the Kruskal–Wallis test as a non-parametric test to examine whether different methoprene concentrations affected the number of feeding holes and eggs in Experiment 1 and whether the absence/presence of 20E and/or methoprene altered the number of feeding holes and eggs in Experiment 3. The two-parameter Hill equation  and experiments 3 vs. 4 . The unpaired Z = 4.404, p < 0.0001, 0.1 μg: Z = 3.100, p < 0.05, Z = 0.840, p > 0.999) or between 0.01 μg and 0.1 μg of methoprene , or between 0.1 μg vs. 1 μg of methoprene . However, the number of feeding holes was higher for females treated with 1 μg of methoprene than 0.01 μg of methoprene . Linear regression analysis showed no increase in feeding rate over time for the control treatment  = 1.61, p = 0.21), but it did increase for the other three treatments  = 10.25, p = 0.0021, Y = 1.63 (±1.05 SE) + 0.396 (±0.123) * X; trt = 0.1: F = 4.77, p = 0.032, Y = 4.80 (±1.41) + 0.361 (±0.166) * X; trt = 1.0: F = 14.27, p > 0.0003, Y = 5.44 (±1.83) + 0.812 (±0.215) * X,a = 2.06, n = 0.290).Among the four treatments, 1 μg of methoprene tended to induce the highest number of feeding holes per day  − 0.304 (±0.384) * X  = 0.62, p = 0.43), and for 1.0 μg methoprene was Y = 0.55 (±2.64 SE) + 2.911 (±0.468) * X  = 24.30, p < 0.0001), and for the oviposition period (Day 8-14) for the control was Y = 4.54 (±3.90) − 0.060 (±0.348) * X  = 0.029, p = 0.87), and for 1.0 μg methoprene was Y = 42.14 (±8.27) − 1.863 (±0.740) * X  = 6.34, p = 0.016). The females ate more after treatment with 1 μg of methoprene than they did in Experiment 1, when treated with 0.0, 0.1 and 0.01 μg of methoprene  than the feeding holes made by females in the control group from Day 8 to 14  − 0.006 (±0.695) * X  = 0.0073, p < 0.93). Likewise, this was significantly higher for these same females than in Experiment 1 , in which there was no oviposition , whereas none of the control females oviposited . The proportion of eggs that were properly placed inside the leaf blade or midrib  was 82.65%, 78.48% and 11.11% for the 2M, AM and AA groups, respectively : 2M vs. AM , 2M vs. AA , and AM vs. AA . Eggs were also found on both the leaf surface  and midrib surface . Even if these eggs were fertile, they were not viable because they dessicated before embryos could complete development and eclose. Hatching rate of eggs that were placed inside leaves was 56.8%, 45.9% and 0% for the 2M, AM and AA groups, respectively : 2M vs. AM , 2M vs. AA , AM vs. AA . The adult mortality was 25% for AA and 2M groups, and 12.5% for the AM group .The 2M (20E + methoprene) treatment showed the highest number of feeding holes from Day 8 to 24 , followed by AM  and AA  groups (n = 170) from the potted yellow starthistle plants. These adults were held at summer photoperiod for three months (at 19–24 °C) on cut yellow starthistle leaves, but did not oviposit, indicating that they were in reproductive diapause.After Experiment 3, six female weevils treated with hormones  oviposited viable eggs on potted plants for additional two months. The egg hatching rate was not explicitly examined on potted plants because it was difficult to check the 1st instar larvae inside the plant without damaging them. However, we reared out newly emerged weevils : Pre 2M vs. Pre AM , Pre 2M vs. Pre AA , Pre AM vs. Pre AA .The feeding holes were similar among the three retreated groups from Day 8 to 24  developed vitellogenic oocytes after topical doses of 0.1 μg vs. 19% for 0.01 μg [T = 3.38, df = 57, p < 0.01). A possible explanation for these differences could be the increased light intensity used in Experiment 2. Alternatively, these females were two weeks older and had been at warm temperature two weeks longer than in Experiment 1. In Experiment 2, feeding rate increased linearly during the preoviposition period (Days 1–7) and then gradually decreased during the first seven days of the oviposition period. A similar decrease in feeding rate was observed in naturally ovipositing females [C. basicorne, which enabled them to lay eggs [Our results demonstrated that doses of 0.01, 0.1 and 1.0 μg of methoprene progressively increased feeding by adult females. However, only the dose of 1 μg of methoprene stimulated oviposition. The fact that the egg production only occurred in 60% of females treated with 1 μg of methoprene in Experiment 1 suggests that 1 μg of methoprene is near the threshold for terminating diapause of female asicorne . The dosr 0.5 μg . One hun females . The artlay eggs ,31,36,50C. basicorne. Oviposition started on the same day for the AM and 2M treatments in Experiment 3, which suggests that the effect of 20E was to produce more eggs rather than reduce the time to initiate oviposition of C. basicorne. The higher egg production by the AM group compared with the AA group is consistent with the theory that exogenous JH treatment stimulates egg maturation; however, exogenous JH also inhibits CA activity and increases JH esterase activity, which degrades the exogenous JH [We did not test 20E alone because it was previously demonstrated that 20E alone failed to terminate reproductive diapause of Colorado potato beetles , and we enous JH ,31. Thusenous JH ,31. ThisT = 10.97, df = 80, p < 0.001; Exp. 3 vs. 2: T = 5.02, df = 19, p < 0.001). However, it is notable that in all three experiments, the oviposition rate was lower than that observed by non-diapausing females reported in [T = 3.43, df = 470, p < 0.001; vs. Exp. 2: T = 5.76, df = 509, p < 0.001; vs. Exp. 3: T = 5.65, df = 532, p < 0.001). Weevils were originated from Turkey, rather than Greece, and measured oviposition at 25/25 °C (12 h photoperiod) [The oviposition rate of females in Experiment 3 exposed to the AM treatment  was higher than in Experiments 1 (0.50 ± 0.17) and 2 (0.81 ± 0.28) (Exp. 3 vs. 1: orted in : (1.8 ± operiod) , rather operiod) ,52.C. basicorne, because it increased oviposition rate by 51% above that of methoprene alone. Our findings can be used in two ways in mass-rearing biocontrol agents. First, to increase the number of biocontrol agents to release by artificially terminating reproductive diapause when necessary, which would permit producing more than one generation per year in the laboratory. Adults produced out of season could be stored at cold temperature (5 °C) until needed for release in the spring [We conclude that 1 μg of methoprene alone was able to induce females to oviposit viable eggs, but that the combination of 20E (1 μg) followed by methoprene (1 μg) was the best treatment for artificially terminating reproductive diapause of female e spring . Second,