Morus macroura, which possesses enriched TPC (4.238 ± 0.26 mg GAE/g DW) and TFC (1.073 ± 0.18 mg QE/g DW), as well as strong FRSA (82.39%). These green synthesized NPs were evaluated for their anti-diabetic, anti-glycation, and biocompatibility activities. Furthermore, their anti-cancerous activity against HepG2 cell lines was assessed in terms of cell viability, production of reactive oxygen/nitrogen species, mitochondrial membrane potential, and apoptotic caspase-3/7 expression and activity. Synthesized NPs were characterized by techniques including ultraviolet-visible spectroscopy, SEM, EDX, FTIR, and XRD. UV-C mediated monometallic and bimetallic NPs showed well-defined characteristic shapes with a more disperse particle distribution, definite crystalline structures, and reduced sizes as compared to their respective controls. In the case of clinical activities, the highest anti-diabetic activity (67.77 ± 3.29% against α-amylase and 35.83 ± 2.40% against α-glucosidase) and anti-glycation activity (37.68 ± 3.34% against pentosidine-like AGEs and 67.87 ± 2.99% against vesperlysine-like AGEs) was shown by UV-C mediated AgNPs. The highest biocompatibility (IC50 = 14.23 ± 1.68 µg/mL against brine shrimp and 2.48 ± 0.32% hemolysis of human red blood cells) was shown by UV-C mediated ZnONPs. In the case of anti-cancerous activities, the lowest viability (23.45 ± 1.40%) with enhanced ROS/NOS production led to a significant disruption of mitochondrial membrane potential and greater caspase-3/7 gene expression and activity by UV-C mediated bimetallic Ag–ZnONPs (0.1/0.5). The present work highlights the positive effects of UV-C light on physico-chemical physiognomies as well as the clinical activities of NPs.A nano-revolution based on the green synthesis of nanomaterials could affect all areas of human life, and nanotechnology represents a propitious platform for various biomedical applications. During the synthesis of nanoparticles, various factors can control their physiognomies and clinical activities. Light is one of the major physical factors that can play an important role in tuning/refining the properties of nanoparticles. In this study, biocompatible monometallic (AgNPs and ZnONPs) and bimetallic Ag–ZnONPs (0.1/0.1 and 0.1/0.5) were synthesized under UV-C light irradiation from the leaf extract of  Nanoparticles (NPs) are nanoscale materials which have dimensions of 100 nm or less. At present, the interest of scientists in NP research is intense due to their enhanced chemical stability, thermal conductivity, nonlinear optical performance, and catalytic activity . Prior tMorus macroura leaf extract for the synthesis of AgNPs, ZnONPs, and Ag–ZnO bimetallic NPs has not been reported before. M. macroura is a tropical plant which exhibits many medicinal properties, including antimicrobial, wound healing, and anti-ulcer activity . C. C52]. CControl and UV-C mediated bimetallic Ag–ZnONPs (0.1/0.1) were 2.75 ± 0.21% hemolytic and 2.97 ± 0.30% hemolytic towards hRBCs, respectively. On the other hand, control and UV-C mediated bimetallic Ag–ZnONPs (0.1/0.5) were slightly more hemolytic, with values of 3.43 ± 0.24% and 3.60 ± 0.44%, respectively. Zare et al. also confirmed the slightly hemolytic nature of Ag–ZnO nanocomposites at lower concentrations; however, they showed greater hemolysis as compared to their monometallic counterparts . Here weOne way to control blood glucose level is to delay the breakdown of carbohydrates in small intestine by blocking the action of α-glucosidase and α-amylase . In our Control bimetallic Ag–ZnONPs (0.1/0.1) displayed better inhibitory activity (24.93 ± 1.74% α-glucosidase and 49 ± 3.98% α-amylase inhibition) as compared to control bimetallic Ag–ZnONPs (0.1/0.5) (23.27 ± 1.80% α-glucosidase and 40.6 ± 2.26% α-amylase inhibition). However, their UV-C mediated counterparts showed slightly less inhibition. Bakur et al.  reportedOur study highlights the plausible role of UV-C light in enhancing the biological activity of NPs by refining their morphology, as UV-C mediated AgNPs showed the most significant anti-diabetic activity among all NPs.Oxidative stress may lead to the build-up of advanced glycation end products (AGEs) . TherefoWe found out that UV-C mediated AgNPs inhibited AGE production most significantly amongst all NPs, therefore showing the positive role of UV-C light in tailoring the morphology as well as the subsequent anti-glycation activity of NPs.The anti-cancerous activity of NPs was evaluated in terms of % cell viability of HepG2 cells. Our results clearly demonstrate the anti-cancerous potential of green synthesized NPs as compared to non-treated cells which were 100% viable A. Cells Control AgNPs reduced the cell viability of HepG2 cells up to 36.25 ± 1.42% cell viability, while UV-C mediated AgNPs showed 31.8 ± 1.31% cell viability. Recently, a 40% death rate was reported for MCF-7 cells when treated with AgNPs . ControlIn this study, the production of ROS/RNS was correlated with cell viability, as oxidative burst results in reduced cell viability and proliferation . In NTC,2+ ions from ZnONPs resulted in mitochondrial dysfunction in PC12 cells [Control AgNPs also induced the oxidation of HepG2 cells, resulting in 2831 ± 801.12 relative DHR123 fluorescence units while UV-C mediated AgNPs resulted in the production of 2605.07 ± 190.43 relative DHR123 fluorescence ROS units. Chairuangkitti et al.  reported12 cells . AmongstA loss of MMP with an increase in intracellular ROS levels leads to cell death . Figure As compared to their monometallic counterparts, bimetallic NPs significantly reduced MMP in HepG2 cells, particularly UV-C mediated bimetallic Ag–ZnONPs (0.1/0.1), with an MMP of 1777.17 ± 40.12 RFU. In contrast, control bimetallic Ag–ZnONPs (0.1/0.1) resulted in an MMP of 1963.3 ± 71.21 RFU. Similarly, UV-C mediated bimetallic Ag–ZnONPs (0.1/0.5) showed an MMP of 1989.20 ± 80.76 RFU, whereas the control bimetallic Ag–ZnONPs (0.1/0.5) led to an MMP of 2140.76 ± 68.21 RFU. These findings show that UV-C mediated green synthesized NPs can alter mitochondrial dynamics in cancerous cells, especially UV-C mediated bimetallic NPs which show enhanced properties.Eleutherococcus senticosus AgNPs against lung cancer cells by activating the expression of the caspase-3 gene [Albizia adianthifolia mediated AgNPs increased the activity of effector caspase-3/7 proteins by 1.7-fold [The induction of caspase proteins increases ROS generation and mitochondrial dysfunction, causing the apoptosis of cancerous cells . Exposure-3 gene . In agre1.7-fold .Similarly, control ZnONPs enhanced caspase-3 gene expression by 154.7 ± 11.07 log 2-fold change, while UV-C mediated ZnONPs further enhanced gene expression by 174.7 ± 17.03 log 2-fold change. Control ZnONPs induced 221.5 ± 15.23 RFU/mg protein caspase-3/7 activity, while UV-C mediated ZnONPs further enhanced this by 250 ± 25.12 RFU/mg protein A,B. ZnONUV-C mediated bimetallic Ag–ZnONPs (0.1/0.1) significantly increased caspase-3 gene expression with a 395.95 ± 49.47 log 2-fold change, as compared to control bimetallic Ag–ZnONPs (0.1/0.1), which showed a 248.13 ± 23.32 log 2-fold change. Likewise, control bimetallic Ag–ZnONP (0.1/0.1) elevated caspase-3/7 activity by 288.5 ± 25.12 RFU/mg protein, and its UV-C counterpart further elevated this up to 333 ± 20.57 RFU/mg protein. UV-C mediated bimetallic Ag–ZnONPs (0.1/0.5) also resulted in similar caspase-3 gene expression with a 323.16 ± 53.23 log 2-fold change, while the control resulted in a 273.26 ± 54.23 log 2-fold change. An increase in the ZnO ratio in bimetallic Ag–ZnONPs (0.1/0.5) significantly increased caspase-3/7 activity (324.4 ± 31.12 RFU/mg protein). UV-C bimetallic Ag–ZnONPs (0.1/0.5) further heightened caspase-3/7 activity to 362.5 ± 23.45 RFU/mg protein. From these findings, it can be concluded that bimetallic NPs enhanced caspase-3 gene expression and caspase-3/7 activity considerably more than monometallic NPs. Moreover, UV-C mediated NPs further enhanced caspase-3 gene expression and caspase-3/7 activity.2, P53, and caspase expression, through which the intrinsic or extrinsic pathway is initiated. These events lead to DNA fragmentation, cell bleeding, and ultimately cell death [1 permeabilize the mitochondrial outer membrane, which discharges all soluble proteins and Bcl2 proteins to form the apoptosome [Nanoparticles play a role in bringing about programmed cell death by regulating Bclll death . The extll death . These rll death . The intoptosome . The apooptosome .Morus macroura leaves were collected from Botanical Garden of Kinnaird College for Women, Lahore, Pakistan, and verified by Dr. Samina Hassan . The 10 g leaves were washed first with tap water 3 to 4 times and then with distilled water thoroughly to remove dust particles, and then the leaves were shade dried. Leaves were grinded in powder form with the help of an electrical grinder and soaked in 400 mL deionized water. The mixture was heated right after 24 h at 40 ± 5 °C overnight and stirred constantly. When the remaining volume reached 100 mL the mixture was filtered with Whatman’s filter paper and stored at 4 °C for NP synthesis.For the preparation of aqueous leaf extract, fresh M. macroura leaf extract as described by Kamtekar et al. [−1) was taken in a test-tube, to which 5 mL distilled water and 0.5 mL Folin–Ciocalteu’s reagent were added and shaken well. After 5 min incubation at RT, 1.5 mL of 20% sodium carbonate was added and the total volume was increased up to 5 mL using distilled water. A deep blue color was observed. After 2 h of incubation at room temperature (RT), the calibration curve was plotted by measuring absorbance of known gallic acid concentrations at 750 nm using a spectrophotometer . The results for TPC were measured from a gallic acid calibration curve  and expressed as mg GAE/g DW. TFC was calculated by using formula:C = concentration of extract, V = volume of extract, and m = mass of extract. The TPC was expressed as mg/g of gallic acid equivalents in milligrams per gram (mg GAE/g) of dry extract. TPC was assessed in triplicate.The Folin–Ciocalteu method was used to evaluate the total phenolic content (TPC) of r et al. . Firstly−1) was placed in a test-tube, to which 0.2 mL of 1 M potassium acetate, 0.2 mL of 10% (w/v) AlCl3 solution, and 5.6 mL distilled water were added, mixed well, and incubated at RT for 30 min. The calibration curve was plotted by measuring the absorbance of known quercetin concentrations at 415 nm using a spectrophotometer . The results for TFC were measured from a quercetin calibration curve  of quercetin (25–200 µg/mL) and expressed as quercetin equivalent (QE) per gram DW. TFC was calculated by using the formula:C = concentration of extract, V = volume of extract, and m = mass of extract. All the experimental work was performed in triplicate.The aluminum chloride colorimetric method was adopted to measure total flavonoid contents (TFC), as reported by Aryal et al. . BrieflyM. macroura leaf extract was measured by using DPPH  as described by Anjum et al. [c = absorbance of plant extract and DPPH and As = absorbance of DPPH solution (standard).The antioxidant activity of m et al. . In shorv/v  to identify an appropriate concentration of silver nitrate for the optimal synthesis of AgNPs. The ratio of 1:10 was chosen as there was an instant color change from greenish yellow to dark brown, and characteristic peaks of AgNPs were observed when absorbance was measured between 380 and 460 nm using a UV-visible spectrophotometer. After mixing of the plant extract and precursor salt solution, it was constantly stirred at room temperature with and without UV-C light (200–280 nm) exposure. After 2 h, the NPs solution was centrifuged at 13,000× g rpm for 10 min. Dark brown pellets were obtained consisting of silver nanoparticles. The supernatant was discarded and then the pellets were washed with deionized water thrice. After washing, AgNPs were dried at room temperature and collected for characterization and biological applications.UV-mediated green synthesis of monometallic AgNPs was carried out as reported by Anjum et al. . For thiM. macroura were mixed, while 2 M NaOH was added dropwise until the pH was 12. The mixture was constantly stirred using a magnetic stirrer for 2 h at RT under the illumination of a UV-C (200–280 nm) lamp and without the lamp as a control. The color change from colorless to yellowish white indicated the initiation of ZnONP synthesis. After 2 h, the ZnONP solution was micro-centrifuged at 6000× g rpm for 15 min, the supernatant was discarded, and the pellets were washed by re-suspending in distilled water. This step was performed thrice. Pellets of ZnONPs were dried at 40 °C overnight in an oven. Finally, the dried ZnONPs were crushed to fine powder using a mortar and pestle.Control ZnONPs were synthesized via green route following the protocol of Anjum et al.  with ming rpm for 15 min and pellets were washed with deionized water thrice. In the case of Ag–ZnO 0.1/0.5 bimetallic NPs, pellets were of greyish white color. Meanwhile, Ag–ZnO 0.1/0.1 bimetallic NPs were of a greyish black shade. Lastly, the green synthesized bimetallic NPs were air dried.Bimetallic Ag–ZnONPs were prepared by following the method mentioned by Sumbal et al.  after miThe progress of green synthesis of NPs was monitored by using UV-visible spectroscopy after a 30 min interval during a 2 h reaction. The UV–Vis spectra of the AgNP, ZnONP, and bimetallic Ag–ZnONP reaction mixtures were recorded using a spectrophotometer  in the ranges of 380–460 nm, 300–800 nm, and at both wavelengths, respectively.−1 wavenumber range. The resolution of the instrument was about 4 cm−1. The FTIR data in this study are reported as the mean of triplicates.Fourier-transform infrared (FT-IR) analysis of NPs was conducted as reported by Tungmunnithum et al. . A refleThe morphology of NPs was evaluated by SEM using the SIGMA model , which operated at an accelerating voltage of 10 kV. Small amounts of samples were dropped on a carbon-coated copper grid. The film was dried for 5 min under a mercury lamp, and SEM images of monometallic and bimetallic NPs at different magnifications were collected. EDX analysis was performed by an EDX detector attached to an SEM for the elemental analysis of ZnONPs.The crystalline nature of NPs was investigated using X-ray diffraction (XRD). Briefly, monometallic and bimetallic NPs were coated on an XRD grid and measurements were taken in the scanning mode using an X-ray diffractometer  which operated at 40 kV with a current of 30 mA and Cu/kα radiation in the range of 20–80° in 2θ angles. Moreover, the sizes of all types of synthesized NPs were calculated using the Debye–Scherrer equation (4)D=kλβArtemia salina (brine shrimp) was evaluated in a 96-well plate (300 µL) for 24 h. The larvae of brine shrimps were used for toxicological study by following protocol reported by Ahmed et al. [A. salina were placed in a specifically designed plastic tray with 2 compartments containing sterile sea water (38 g/L) supplemented with 6 mg/L dried yeast. The eggs were hatched by incubation for a period of 24–48 h, with a constant supply of oxygen. Proper illumination was ensured to maintain the temperature (30–32 °C) and light necessary for hatching. Then, 10 mature (phototropic) nauplii were picked up with a Pasteur pipette and placed into the wells, to which AgNPs, ZnONPs, and bimetallic Ag–ZnONPs  were separately added and the final volume was adjusted to 300 µL. The negative control was 1% DMSO in sea water, while doxorubicin (1–10 µg/mL) served as a positive control. After 24 h of incubation the live shrimps were quantified and the median lethal concentration (LC50) was measured by using table curve 2D v5.01 of the test extracts with ≥50% mortality.The lethality of green synthesized AgNPs, ZnONPs, and bimetallic Ag–ZnONPs (20 mg/mL stock in water) against d et al. . Brieflyg rpm. Furthermore, 200 µL of pelleted erythrocytes were shaken in 9.8 mL of PBS (pH: 7.2). In a 1.5 mL Eppendorf tube, 100 µL of AgNPs/ZnONPs/Ag–ZnONPs and erythrocytes were taken separately, incubated at 35 °C for 1 h, and centrifuged for 10 min at 10,000× g rpm. In a 96-well plate, 100 µL of supernatant was added and the hemoglobin released was measured at 540 nm using a BioTek ELX800 Absorbance Microplate Reader . Triton X-100 and DMSO served as a positive and negative control, respectively. The results were measured as % hemolysis using the following formula:The biocompatibility of AgNPs, ZnONPs, and bimetallic Ag–ZnONPs was evaluated against freshly isolated hRBCs. A blood sample from a healthy donor  was withdrawn using a sterile syringe at the fingertip. Procedures dealing with human subjects were carried out in consideration of the ethical standards of international and national research committees and the 1964 Helsinki Declaration and its later amendments. Written informed consent was obtained from the healthy donor participating in this study. Blood was collected in tubes containing EDTA to prevent blood clotting . RBCs weα-Glucosidase was purified and immobilized by using rat intestinal acetone powder  and CNBr-activated sepharose 4B , respectively, according to the previously described protocol of Hano et al. . As suchd-maltopentaoside  and phosphate buffer  by constant mixing. The reaction mixture was incubated for 30 min with and without samples at RT. Then the reaction was stopped by using 1 M sodium carbonate solution followed by measuring enzyme activity by taking absorbance at 405 nm by Synergy II reader . The % inhibition of α-amylase was measured by calculating the difference in absorbance with and without the sample.Inhibition of α-amylase activity by NPs was measured by using α-amylase from porcine pancreas  according to the protocol established by Velioglu et al. . Brieflyw/v). The mixture was incubated for 5 days in the dark at 37 °C, and the fluorescent AGE formation was measured using a VersaFluor fluorometer  set with 410 nm emission and 330 nm excitation wavelengths, respectively. The AGE inhibition was expressed as the % inhibition relative to the control.The anti-aging activity of monometallic and bimetallic NPs was evaluated by assessing the inhibition of production of advanced glycation end-products (AGEs) based on the protocol reported by Shah et al. . The tesHuman hepato-cellular carcinoma cells (HepG2)  were cultured in Dulbecco’s Modified Eagle Medium for anti-cancerous studies. MTT -2,5-diphenyltetrazolium bromide) dye was utilized to assess the cytotoxicity of AgNPs, ZnONPs, and bimetallic Ag–ZnONPs in vitro. The reduction of tetrazolium dye MTT by living cells into an insoluble purple colored product formazan was recorded using a spectrophotometer as a measure of cell viability.4 cells/well; 200 µL per well), for 24 h. Then, 10 µL of MTT dye (5 mg/mL) was added per well and incubated for 3 h. Then, 10% acidified sodium dodecyl sulfate was added to dissolve insoluble formazan. Lastly, after the overnight incubation of cells, plates were analyzed at 570 nm using a microplate reader . Non-treated cells (NTC) acted as a control. The experiment was performed in triplicate. Cell viability was measured in terms of the percentage with respect to the control using the equation below:Thus, 200 µg/mL of green synthesized AgNPs, ZnONPs, and bimetallic Ag–ZnONPs were added to a 96-well plate which was pre-seeded with HepG2 cells  were washed twice with phosphate-buffer saline (PBS), resuspended in PBS containing 0.4 μM DHR-123, and incubated for 10 min at 30 °C in the dark. Lastly, the fluorescence signal  was recorded on a VersaFluor Fluorimeter .The intracellular ROS/RNS level was assessed by using dihydrorhodamine-123 (DHR-123) fluorescent dye  as reported by Nazir et al. . Pre-seem) of HepG2 cells treated with green synthesized monometallic and bimetallic NPs was taken using 3,3′-dihexyloxacarbocyanine iodide (Sigma), which works by staining mitochondria according to their MMP. Cells were incubated in culture media supplied with 25 nM 3,3′-dihexyloxacarbocyanine iodide at 37 °C for 40 min. The MMP was expressed as relative fluorescent units (RFU). The experiment was performed thrice.The MMP  and Quant-iT RNA Assay Kit , respectively. A first-strand cDNA synthesis kit  was used to perform reverse transcription. The PikoReal real-time PCR system  was used to perform quantitative PCR with the DyNAmo Color Flash SYBR Green qPCR Kit (Thermo Fisher). A 10 µL PCR reaction mixture was prepared which contained 0.5 µL diluted cDNAs, 2 × SYBR Green mix, and 1 µM of each primer pairs. The PCR reaction was carried out as follows: 7 min at 95 °C, 40 cycles of 10 s at 95 °C, 10 s at 60 °C, and 30 s at 72 °C. Data were analyzed using Pikoreal software. A total of 3 biological replicates and 2 technical repetitions were performed for each sample. The caspase-3 primers used were: 5′-TGTTTGTGTGCTTCTGAGCC-3′ (Forward primer) and 5′-CACGCCATGTCATCATCAAC-3′ (Reverse primer) (amplicon size: 210 bp).w/v) sucrose, 100 mM HEPES (pH 7.2), 5 mM DTT, 1% (v/v) NP40, and 0.1% (w/v) CHAPS. Incubation of the homogenate was carried out on ice for 15 min, and it was centrifuged twice at 13,000× g for 10 min at 4 °C to pellet out cell debris. The supernatant was filtered through a 0.22 mm filter. Ab Apo-ONE Homogeneous Caspase-3/7 Assay kit  was used to measure in vitro caspase-3/7 activity, following the instructions of manufacturer.Caspase-3/7 activity was measured by using frozen cells to obtain cytosolic protein extracts. A glass chilled mortar and pestle were used to ground the samples in 500 mL ice-cold extraction buffer containing 10% . The data are expressed as the mean ± SE.Morus macroura leaf extract under the influence of UV-C light for the first time. The phytochemical profile of M. macroura showed that the plant is enriched with phenolic and flavonoid contents, showing increased antioxidant activity. FTIR spectra showed the involvement of polyphenolic and carboxylic acid as well as aromatic compounds in the bio-reduction and stabilization of monometallic and bimetallic NPs. Characterization techniques such as SEM-EDX and XRD helped to draw the conclusion that UV-C light had a more profound effect on modeling the morphological features of green synthesized NPs. A reduction in size and more defined shapes were observed in the case of UV-C mediated green synthesis of NPs. The physico-chemical properties of NPs also determine their applications and influence their efficacy. In our study, UV-C mediated AgNPs showed exceptionally strong anti-diabetic and anti-aging activities. All NPs were moderately hemolytic towards hRBCs and moderately toxic towards brine shrimp, hence showing their bio-safe nature. Here, the role of UV-C irradiation in the green synthesis of monometallic and bimetallic NPs and ultimately in enhancing their in vitro biological activities has been described for the first time. Interestingly, the differences observed in biological activity between the control and UV-irradiated NPs could be attributable to differences in cellular absorption. Future research will be needed to test this theory. Once the process is further optimized and scaled up, these biocompatible NPs could be used as strong contenders in anti-cancerous and anti-diabetic therapeutics as well as in cosmetics.In this research, monometallic AgNPs, ZnONPs, and bimetallic Ag–ZnONPs were green synthesized from


Halorubrum sp. LN27 and has a head-tailed morphotype with a contractile tail, typical of myoviruses. The average head diameter is 64 ± 2.0 nm, and uncontracted tails are 15 ± 1.0 × 65 ± 2.0 nm. The latent period is about 10 h. The maturing time of VOLN27B in cells of Halorubrum sp. LN27 was nearly 8 h. The adsorption time of VOLN27B on cells of Halorubrum sp. LN27 was less than 1 min. Virus particles are unstable at pH values less than 5 or when the NaCl concentration is below 12% (w/v). VOLN27B and Halorubrum sp. LN27 were recovered from the same hypersaline environment and provide a new virus-host system in haloarchaea.A novel halovirus, VOLN27B, was isolated from a drill core sample taken at a depth of approximately 430 m, from a layer formed during the Cretaceous period . VOLN27B infects the halophilic archaeon  Halobacteria), which are often the dominant cellular microorganisms and can grow to high cell densities (>107 cells per milliliter). Viruses infecting haloarchaea are typically observed to be in much higher concentration than their host cell population, and direct examination of natural brines shows a number of different morphological types of virus-like particles (VLP), including spherical, spindle-shaped, and head-tail [Thumleimavirales, Kirjokansivirales, and Methanobavirales .Hypersaline environments, near salt saturation, are commonly found to harbor members of the extremely halophilic archaea . Haloarchaeal viruses Hardycor1, Hardycor2, and Serpecor1 are able to infect Halorubrum coriense strain Ch2, while ChaoS9 and HFTV1 can infect Halobacterium salinarum and Haloferax strains, respectively. HFTV1 can also infect Halorubrum spp. although it is eight times less effective than Haloferax strains [Since 1974, more than 70 head-tailed viruses infecting haloarchaea have been reported, and their morphotypes include those with contractile tails (myovirus), noncontractile tails (siphovirus), and very short tails (podovirus) , 7, 9–11 strains .Halspiviridae), pleomorphic (family Pleolipoviridae), tailless icosahedral (family Spherolipoviridae), and tailed icosahedral  [To date, more than 100 viruses have been isolated from hypersaline environments, a majority of them (~90) infect extremely halophilic euryarchaea in the class Halobacteria , 10. Allvirales) . Archaeavirales) . The divvirales) .Halorubrum sp. LN27 from a drill-core of salt mine (~430 m underground) which formed during the Cretaceous period . Here, we determined the primary viral characteristics, including particle morphology, stability, virus life cycle, and nucleic acid type. This provides the first description of a novel virus-host system recovered from an extremely hypersaline environment that can be applied to studies of virus-haloarchaea interactions and coevolution.In this study, we isolated a new infectious head-tailed virus, namely, VOLN27B, infecting w/v) after washing with distilled water for six times to entirely scour off the residual NaOH. Insoluble substances were removed by suction filtration with sterile filter paper . The filtrate was concentrated from about 22 L to 500 mL through tangential flow filtration (TFF) using Vivaflow 50  with a molecular weight cut-off (MWCO) at 10 kDa. The concentrated salt solution was stored in a refrigerator at 4°C until halovirus screening.A drill core was collected at the Dingyuan Salt Mine , Anhui province, China, in May 2016 . The enthttps://www.jcm.riken.jp/) (pH 7.5). The medium consisted of Casamino acids (BD-Difco) 5.0 g, yeast extract (BD-Difco) 5.0 g, sodium glutamate 1.0 g, trisodium citrate 3.0 g, MgSO4·7H2O 20.0 g, KCl 2.0 g, NaCl 200.0 g, FeCl2·4H2O 36.0 mg, and MnCl2·4H2O 0.36 mg per liter. Solid and soft agar (top layer) media were obtained by adding 15 g or 5 g of Bacto agar (Difco Laboratories) per liter, respectively.The haloarchaea used as host strains to isolate haloviruses are listed in Table Halorubrum that have been previously described [μL volume of the salt solution was mixed with 900 μL of cell suspension  and incubated at 37°C for 30 min; then, the entire mixture was added to 5 mL soft agar , vortexed briefly, and poured on to a base layer agar plate (1.5% agar) of the same medium. Plates were incubated at 37°C for three days and observed for plaque formation. Several clear plaques were formed on the lawn of Halorubrum sp. strain LN27. Three consecutive plaque purifications were performed to obtain a pure culture. Ultimately, the halovirus infecting cells of strain LN27 was named VOLN27B.To screen for haloviruses present in the concentrated salt solution obtained from the drill core sample, plaque assays were performed on ten haloarchaeal strains Table . Three wescribed , and theHalorubrum sp. strain LN27 using the plaque formation assay. To obtain a virus stock, titration plates with near confluent plaques were flooded with NaCl  and followed by collecting the NaCl solution after incubation for about 12 h. The fluid collected from the plate was defined as primary virus stock (PVS). Virus titer in this PVS was determined to be approximately 1011 pfu/mL via plaque formation assay.Plaques on the top soft agar were picked with a sterile toothpick into JCM 168 liquid medium, homogenized by votexing, and retitrated on The influence of time and temperature on plaque formation was examined by titration of virus on lawns of strain LN27 and incubation of plates for (1) 3, 5, 6, and 7 days at 37°C and (2) incubation at 30°C, 38°C, 42°C, and 50°C for 3 days.Halorubrum, two species of Halopenitus, two species of Haloferax, and one species from the following genera: Halalkalicoccus, Halococcus, Haloarchaeobius, Halostagnicola, Haloterrigena, Haloparvum, Natrinema, Natronomonas, and Natrialba . Multiple sequence alignment and phylogenetic analysis were conducted by using the MEGA X  [Strain LN27 was isolated from the Dingyuan Salt Mine. Based on the sequence similarity of 16S rRNA gene, strain LN27 was classified as sp. LN27 . To inven et al. . Referenjoining) .v/v) in buffered 20% (w/v) NaCl solution adjusted to pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 and incubation at room temperature for 12 h. The buffers used were pH 4.0 and 5.0, 2-morpholinoethanesulfonic acid; pH 6.0, 7.0 and 8.0, 1,4-piperazine bis-ethanesulfonic acid; pH 9.0, 2-cyclohexylamino ethanesulfonic acid; and pH 10.0, N-cyclohexyl-3-aminopropanesulfonic acid. All buffers were at 1 mol/L.PVS was used for testing virus stability under different conditions, after which the virus titer was determined by plaque assay. Temperature stability was tested at (1) 4°C for 1, 2, 3, and 5 months; (2) 37, 50, 60, 70, 80, and 90°C for 10 min; and (3) frozen at -20°C, -40°C, and -70°C overnight followed by a thawing. Stability to pH was tested by diluting PVS 1 : 100 (v/v) in 1, 2, 4, 8, 9, 10, 11, and 12% (w/v) NaCl solution and incubation at room temperature for 12 h. Chloroform sensitivity was tested by mixing PVS with an equal volume of chloroform , separation of the phases by centrifugation  at 4°C for 3 min, and removing the aqueous phase.Salt stability was tested at by diluting PVS 1 : 100  and Halorubrum sp. strain LN60 (not host) were infected with halovirus VOLN27B (m.o.i 1 : 10) at postinfection (p.i.) times of 1, 5, 10, 15, 20, 25, and 30 min. The cells were removed from the sample through centrifugation  at 4°C for 10 min. Then, cell-free supernatants were used for plaque formation assay.To investigate the adsorption time, exponentially growing cells of 600 = 0.7 ~ 0.8; ≈2.0 × 108 cfu/mL). (2) VOLN27B stock  was added into above cell suspension to an m.o.i = 20. (3) After an adsorption period (30 min), virus infected cells were harvested by centrifugation  and washed gently with JCM 168 broth twice to remove unbound virus. Then, cells were resuspended with 100 mL JCM 168 broth; (4) the cell suspension was incubated at 37°C with shaking (180 rpm). The OD600 was measured in 1, 3, or 6 h intervals for the infected and uninfected cultures, respectively. (5) Viable cells in both infected and uninfected cultures were monitored using plate counts. The virus titer of the culture supernatants was determined by plaque formation assay.A one-step growth curve was conducted in a 250 mL flask containing 100 mL liquid JCM 168 medium as follows: (1) Cells of strain LN27 were inoculated into the liquid medium and cultivated at 37°C to exponential phase , infected with halovirus VOLN27B , and unabsorbed virus removed by washing cells three times with 100 mL sterilized 20% NaCl solution. After resuspension in 100 mL JCM 168 broth, cells were incubated at 37°C with shaking (180 rpm). At hourly intervals, 100 μL culture was removed, the cells collected by centrifugation , washed three times with 20% NaCl solution, lysed by the addition of 500 μL ddH2O, and the virus titer of the lysate determined by plaque titration.Cells of μg) was digested with DNase I (5 U) , Exonuclease III (20 U) , and Mung Bean Nuclease (4 U)  at 37°C for 1 h, and the resulting product was checked by DNA electrophoresis. An equal amount of undigested plasmid pBR322  was used as a circular dsDNA standard, and the EcoRI  cleaved pBR322 was used as a linear dsDNA standard. These untreated and EcoRI leaved pBR322 plasmids were also used as controls in treatments nucleases DNAse I, Exonuclease III, and Mung Bean nuclease.To determine the genome type of halovirus VOLN27B, the genome of VOLN27B was isolated from the virus stock according to the method described by Summer  using WiHalorubrum sp. strain LN27 was grown in liquid cultures to midexponential phase  then infected with virus halovirus VOLN27B (m.o.i = 20) and incubated at 37°C with shaking (180 rpm). At 36 h p.i., the cells were removed by centrifugation at 8,000 rpm for 15 min at 4°C, and virus in the supernatant concentrated 10-fold using tangential flow filtration (TFF) with a molecular weight cut-off (MWCO) of 10 kDa. The concentrated virus (109 pfu/mL) was precipitated using 10% (w/v) polyethylene glycol 6000 (PEG 6000) overnight at 4°C. Precipitated virus was collected by centrifugation , resuspended in 3 mL 20% (w/v) NaCl solution, loaded on to a linear 5% to 20% (w/v) sucrose gradient, and centrifuged at 28,000 rpm for 8 h at 4°C . The virus zone was collected and stored at 4°C until use. The titer was ~1011 pfu/mL.w/v) uranyl acetate (pH 4.5) for 30 s, and the dried copper grid was subjected to observation through transmission electron microscope (TEM). Electron micrographs were taken using JEM-1400 Transmission Electron Microscope (80 kV) .To determine the morphology of halovirus VOLN27B, concentrated and purified virus particles were stained with 3% . With an extension of cultivation period, the diameter of plaques increased from 2-3 mm to 7-8 mm  Figures . The arele Table . Plaquesain LN27 . This hansparent . Regardlo 7-8 mm .Halorubrum saccharovorum strain JCM 8865  and the alignment used to reconstruct a phylogenetic tree  of NaCl concentration . A significant production of free viruses in supernatants began at 10 h p.i. and peaked at approximately 21 h p.i. (Turbidity of cell suspension infected by VOLN27B (m.o.i = 20) begun to drop at 18 h p.i (hour postinfection) . Althoug1 h p.i. . The shaHalorubrum sp. LN27 were infected by VOLN27B, no infectious viruses were detected before 8 h p.i  is an inland athalassohaline hypersaline environment formed approximately 650 m.y.a (millions of years ago) without any marine transgressions . Halorubapproach . Later, approach . Numerouapproach , 13. Durns Table  as the hns Table . The dians Table .Viruses can be classified into two different categories based on their lifestyles: temperate and lytic . HalovirHalobacterium and Haloferax, while HF2 can infect strains from different species  in genus Halorubrum [Halorubrum and different genera have been used , which was very similar to the head-tailed halovirus phiN infecting Halobacterium salinarum [Halorubrum sp. strain LN27 certainly provide a good virus-host system for probing the interaction between virus and its host.Infectivity of VOLN27B decreased along with the decreasing of salinity , and viralinarum . Haloviralinarum . In summ


Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1, which we show to be crucial to host growth. Finally, using simultaneous knock-down ‘paradox’ controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1, Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria–Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote–eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system,  For details of additional transcriptome sequence acquisition from P. bursaria CCAP 1660/12, which also included ‘single-cell’ transcriptome analyses, please refer to the electronic supplementary material, Methods.RNA was extracted from Raw reads were trimmed at Q5 in Trimmomatic (v. 0.32) . Reads wTetrahymena) and universal encodings. The longest peptide sequences were retained for each. The remaining ORFs were then annotated via a BLASTX (v. 2.2.31) search against a genome database consisting of: Arabidopsis thaliana, Aspergillus nidulans, Bacillus cereus ATCC 14579, Burkholderia pseudomallei K96243, Candidatus Korarchaeum cryptofilum OPF8, Chlamydomonas reinhardtii, Chlorella variabilis NC64A, Chlorella vulgaris C-169, Escherichia coli str. K-12 substr. MG1655, Homo sapiens, Methanococcus maripaludis S2, Oxytricha trifallax, Paramecium biaurelia, P. caudatum, P. multimicronucleatum, P. primaurelia, P. sexaurelia, P. tetraurelia, Saccharomyces cerevisiae S288C, Streptomyces coelicolor A32, Sulfolobus islandicus M.14.25, Tetrahymena borealis, T. elliotti, T. malaccensis, T. thermophila macronucleus, T. thermophila micronucleus and Ustilago maydis.ORFs were called from Trinity assembled transcripts using Transdecoder, using both ciliate . Binned sequences were further annotated using SignalP (v. 4.0), TMHMM (v. 2.0) and BLAST2GO (v. 4). Each dataset was filtered to remove any sequences with a predicted peptide sequence shorter than 30 amino acids.Assembled transcripts were subsequently binned into either ‘host’, ‘endosymbiont’, ‘food’ or ‘other’ datasets, using a phylogeny-based machine-learning approach  and universally encoded predicted proteins (‘endosymbiont’ bin) were searched using BLASTp and a minimum expectation of 1×10meciumDB  for Para species  to assesProtein sequences were aligned using MAFFT  . Sequences and cloning sites for each plasmid construct are detailed in electronic supplementary material, table S1. All modified constructs were confirmed by Sanger sequencing (Eurofins Genomics).Sequences for plasmid constructs were synthesized . 4.5P. bursaria was fed with E. coli transformed with an L4440 plasmid construct with paired IPTG-inducible T7 promoters, facilitating targeted gene knock-down through the delivery of complementary dsRNA. L4440 plasmid constructs were transformed into E. coli HT115 competent cells and grown overnight on LB agar (50 µgml−1 Ampicillin and 12.5 µgml−1 Tetracycline) at 37°C. Positive transformants were picked and grown overnight in LB (50 µgml−1 Ampicillin and 12.5 µgml−1 Tetracycline) at 37°C with shaking (180 r.p.m.). Overnight pre-cultures were back-diluted 1 : 25 into 50 ml of LB (50 µgml−1 Ampicillin and 12.5 µgml−1 Tetracycline) and incubated for a further 2 h under the same conditions, until an OD600 of between 0.4 and 0.6 was reached. E. coli cultures were then supplemented with 0.4 mM IPTG to induce template expression within the L4440 plasmid, and incubated for a further 3 h under the same conditions. E. coli cells were pelleted by centrifugation (3100 g for 2 min), washed with sterile NCL medium, and pelleted once more. E. coli cells were then re-suspended in NCL medium supplemented with 0.4 mM IPTG, 100 µgml−1 Ampicillin and 0.8 µgml−1 β-sitosterol, and adjusted to a final OD600 of 0.1.P. bursaria cells were pelleted by gentle centrifugation in a 96-well plate (10 min at 800 g), taking care not to disturb the cell pellet by leaving 50 µl of supernatant, and re-suspended 1 : 4 into 200 µl of induced E. coli culture medium . Feeding was conducted daily for up to 14 days using freshly prepared bacterized medium.. 4.6P. bursaria 186b for gene expression analysis after three days of RNAi feeding. P. bursaria cells (approx. 103 per culture) were pelleted by gentle centrifugation (800 g for 10 min), snap-frozen in liquid nitrogen, and stored at −80°C. RNA extraction was performed using TRIzol reagent (Invitrogen), following the manufacturer's protocol after re-suspending each pellet in 900 µl TRIzol reagent. RNA was precipitated using GlycoBlue Co-precipitant (Invitrogen) to aid RNA pellet visualization, and then cleared of residual DNA using the TURBO DNA-free Kit (Ambion), following the manufacturer's protocol for routine DNase treatment.RNA was extracted from ® III First-Strand Synthesis SuperMix (Invitrogen), following the manufacturer's protocol. Quantitative PCR (qPCR) was performed in a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). Reaction conditions were optimized using a gradient PCR, with a standard curve determined using 10-fold dilutions of P. bursaria cDNA: u2af1 , dcr1 , dcr2/3 , dcl1/2 , dcl3/4 , dcl5  and GAPDH , using StepOne software v. 2.3. Each 20 µl reaction contained 10 µl PowerUp SYBR Green Master Mix (Thermo Fisher Scientific), 500 nM of each primer and 1 µl (50 ng) cDNA. Primers pairs for each reaction are listed in electronic supplementary material, table S2. Each reaction was performed in duplicate for each of three biological replicates, alongside a ‘no-RT’ (i.e. non-reverse transcribed RNA) control to detect any genomic DNA contamination. Cycling conditions were as follows: UDG activation, 2 min at 50°C and DNA polymerase activation, 2 min at 95°C, followed by 40 cycles of 15 secs, 95°C and 1 min at 55–65°C (u2af1 (57°C), dcr1 (60°C), dcr2/3 (60°C), dcl1/2 (60°C), dcl3/4 (60°C), dcl5 (60°C) and GAPDH (60°C)). Each reaction was followed by melt-curve analysis, with a 60–95°C temperature gradient (0.3°C s−1), ensuring the presence of only a single amplicon, and ROX was used as a reference dye for calculation of CT values. CT values were then used to calculate the change in gene expression of the target gene in RNAi samples relative to control samples, using a derivation of the 2−ΔΔCT algorithm [RNA was reverse transcribed into single-stranded cDNA using the SuperScriptlgorithm .. 4.7P. bursaria  cultures using TRIzol reagent (Invitrogen), as detailed above. To isolate sRNA from total RNA, samples were size separated on a denaturing 15% TBE-urea polyacrylamide gel. Gels were prepared with a 15 ml mix with final concentrations of 15% acrylamide/Bis (19 : 1), 8 M urea, TBE , and the polymerization started by the addition of 150 µl 10% APS (Sigma-Aldrich) and 20 µl TEMED (Sigma-Aldrich). Gels were pre-equilibrated by running for 15 min  in TBE before RNA loading. The ladder mix consisted of 500 ng ssRNA ladder , and 5–10 ng of each 21 and 26-nt RNA oligo loaded per lane. The marker and samples were mixed with 2X RNA loading dye (NEB) and heat denatured at 90°C for 3 min before snap cooling on ice for 2 min prior to loading. Blank lanes were left between samples/replicates to prevent cross-contamination during band excision. Gels were then run for 50 min .Total RNA for sRNA sequencing was extracted from ® Gold Nucleic Acid Gel Stain. Bands of the desired size range (approx. 15–30-nt) were visualized under blue light, excised and placed into a 0.5 ml tube pierced at the bottom by a 21-gauge needle, resting within a 1.5 ml tube, and centrifuged (16 000 g for 1 min). Four hundred microlitres of RNA elution buffer (1 M Sodium acetate pH 5.5 and 1 mM EDTA) was added to the 1.5 ml tube containing centrifuged gel slurry, and the empty 0.5 ml tube discarded. Gel slurry was manually homogenized until dissolved using a 1 ml sterile plunger and incubated at RT for 2 h with shaking at 1400 r.p.m.Once run, gels were stained by shaking (60 r.p.m.) for 20 min at RT in a 40 ml TBE solution containing 4 µl SYBRg for 1 min). The filter insert containing acrylamide was discarded. One millilitre of 100% EtOH was added to each solution, alongside 15 µg of GlycoBlue™ Coprecipitant (Invitrogen) to aid sRNA pellet visualization, and stored overnight at −80°C to precipitate. Precipitated solutions were centrifuged at 4°C (12 000 g for 30 min), and the supernatant discarded. sRNA pellets were washed with 500 µl of cold 70% EtOH (12 000 g for 15 min at 4°C), and air dried in a sterile PCR hood for 10 min, before re-suspending in 15 µl of RNAse-free water and storage at −80°C.Solutions containing RNA elution buffer and gel slurry were transferred to a Costar Spin-X 0.22 µm filter column and centrifuged .sRNA concentrations were determined using an Agilent 2100 Bioanalyzer, following the Agilent Small RNA kit protocol, and all samples matched to 0.7 ngmlThe raw paired-end reads from the RNA-seq libraries were trimmed using Trim Galore in order to remove barcodes (4-nt from each 3′- and 5′-end) and sRNA adaptors, with additional settings of a phred-score quality threshold of 20 and minimum length of 16-nt. Results were subsequently checked with FastQC.https://github.com/guyleonard/paramecium) to produce a table of mapped read accessions and their respective read lengths. Size distributions of sRNA abundance for each sample were plotted using the R programming language packages; tidyverse, grid.extra and ggplot2 in R Studio (v. 1.3.1073).Trimmed reads were mapped against the ‘host’ or ‘endosymbiont’ dataset of assembled transcripts using the HISAT2 alignment program with default settings. Post-mapping, the BAM files were processed using SAMTOOLS and a set of custom scripts (Click here for additional data file.


Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein–protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G , an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains. Crkl is a ubiquitously expressed 39 kDa adapter protein, member of the proto-oncogene CRK family, that mediates and regulates the linking of several signaling proteins. It was originally discovered in cells from patients with chronic myelogenous leukemia, and its overexpression is correlated with the onset of a number of cancer diseases (recently reviewed in ). Crkl p10 helix, that typically mediate the binding and recognition of proline-rich sequences, in particular those characterized by the P-X-X-P consensus (X being any amino acid)  was fitted with Equation (1), taking into account the non-pseudo-first order conditions  (chevron plot) for the wild-type, and the three variants are reported in In an effort to analyze the mechanistic role of the residues D138, E140 and D141 in the binding reaction with C3G, we performed site-directed mutagenesis and generated the D138A, E140A and D141A variants of the N-SH3 domain. At first, to monitor the effect of these mutations on the stability of the domain, we performed (un)folding kinetic experiments in buffer Hepes 50 mM pH 7.0 at 25 °C. The dependence of the logarithm of the observed rate constants . Such a decrease in binding affinity may be at the basis of our impossibility to time-resolve the binding reaction with a stopped-flow apparatus, with the reaction possibly occurring in the dead time of the instrument.It is of particular interest to discuss the effect of D141A mutation. When employed in kinetic binding experiments, this variant did not return any measurable trace describing a change in FRET signal upon mixing, possibly due to an overall destabilization of the complex and/or sub-millisecond kinetics that could not be resolved by the stopped-flow. To further investigate this aspect and obtain a quantitative measure of the binding affinity of D141A variant with C3G, we resorted to conduct equilibrium binding experiments. Experiments were conducted by exciting samples containing a constant concentration (1 µM) of N-SH3 D141A at 280 nm and following the progressive quenching of two tryptophan residues (in position 164 and 165) fluorescence emission at increasing concentrations of dansyl-C3Gkoff. We challenged a preincubated complex of D141A NSH3 domain (2 µM) and dansyl-C3G277–296 (10 µM) versus a high excess of nondansylated C3G (50 µM), and we could not measure any change in fluorescence emission, suggesting the koff being too high to be measured at the stopped-flow. To test this and to slow down the diffusion of molecules, we repeated the experiment increasing the viscosity of the solution adding 20% w/v sucrose to the buffer Hepes 50 mM pH 7.0. As expected, the increase in solution viscosity allowed us to obtain a displacement trace  occuAchieving a deep understanding of the interaction occurring between Crkl protein and its ligands is of fundamental importance to gaining useful information about the molecular basis of several physiological pathways and human pathologies in which this protein is involved. The employment of rigorous kinetic characterization of the binding reaction at different experimental conditions, together with site-directed mutagenesis, allowed us to describe in detail the roles of electrostatic forces occurring between the N-SH3 domain and a peptide mimicking one of its physiological partners, C3G. Importantly, whilst the SH3 domains are generally thought to recognize their ligands via the P-X-X-P recognition motif, our data exemplify the existence of a negatively charged stretch in the SH3, which is critical in determining the affinity between the interacting molecules. In this view, our study complements and enriches the structural knowledge on this important protein system, by providing a mechanistic insight on the role of these residues, as probed by the effect of site-directed mutagenesis on the association and dissociation rate constant, respectively. Our data represent a first step for future structural and extensive mutational characterization of this protein system.The construct encoding the N-SH3 domain of Crkl was subcloned in a pET28b+ plasmid vector. The constructs encoding D138A, E140A and D141A were obtained through site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent technologies) according to the manufacturer’s instructions. All the mutations were confirmed by DNA sequencing.E. coli cells, strain BL21. Bacterial cells were grown in LB medium, with 30 μg/mL of kanamycin, at 37 °C until OD600 = 0.7−0.8 and then induced with 0.5 mM IPTG. The cultures were grown at 37 °C for three hours after induction, kept at 25 °C overnight and then collected by centrifugation. Purification was performed resuspending the pellet in 50 mM TrisHCl, 0.3 M NaCl, pH 7.5 buffer with the addition of antiprotease tablet , and then sonicated and centrifuged. The soluble fraction from bacterial lysate was loaded onto a nickel-charged His-Trap chelating HP  column equilibrated with 50 mM TrisHCl, 0.3 M NaCl and pH 7.5. Protein was then eluted with a gradient from 0 to 0.5 M imidazole by using an ÄKTA-prime system. Fractions containing the protein were collected, and the imidazole was removed using a HiTrap Desalting column , with the protein purified in the final buffer of TrisHCl 50 mM, NaCl 0.3 M, pH 7.5. The purity of the proteins was analyzed through SDS-page.The expression of all the His-tagged constructs was performed in Peptides mimicking the portion of C3G ranging from residue 277 to 296 (sequence VVDNSPPPALPPKKRQSAPS) in their dansylated and nondansylated variants were purchased from GenScript Biotech.Kinetic (un)folding experiments were performed on an Applied Photophysics Pi-star 180 stopped-flow apparatus, monitoring the change of fluorescence emission, exciting the sample at 280 nm and recording the fluorescence emission by using a 320 nm cutoff glass filter. In all experiments, performed at 25 °C in buffer 50 mM Hepes pH 7.5, refolding and unfolding were initiated by an 11-fold dilution of the denatured or the native protein with the appropriate buffer (0 M and 6 M Guanidine HCl). For each denaturant concentration, at least five individual traces were averaged, and the final protein concentration was 1.5 μM. The fluorescence time courses obtained was satisfactorily fitted by using a single-exponential equation. The chevron plots obtained were fitted using an equation describing a two-state folding mechanism.w/v sucrose was added to the buffer Hepes 50 mM pH 7.0. Displacement traces were fitted with a single-exponential equation.Kinetic binding experiments were performed on a single-mixing SX-18 stopped-flow instrument (Applied Photophysics), by mixing a constant concentration (0.5 μM) of C3G dansylated versus increasing concentrations of N-SH3 at 10 °C. Ionic strength dependence of wt N-SH3 was performed with concentrations ranging from 1 to 5 µM for 0 M NaCl condition and from 0.5 to 5 µM for 0.15 M, 0.3 M, 0.5 M and 1 M NaCl, buffer Hepes 50 mM pH 7.0. The excitation wavelength was 280 nm, and fluorescence was collected using a 455 nm cut-off filter. At least, five independent acquisitions were collected and averaged for each experiment. The resulting averages were all satisfactorily fitted with a single-exponential equation. Buffers used for pH dependence were: 50 mM Sodium Acetate pH 5.0, 50 mM Sodium Acetate, pH 5.5, Bis–Tris 50 mM pH 6.0, Bis–Tris 50 mM pH 6.5, Hepes 50 mM pH 7.0, Hepes 50 mM pH 7.5, Tris–HCl 50 mM pH 8.0, Tris–HCl 50 mM pH 8.5, Tris–HCl 50 mM pH 9.0, all with added 0.5 M NaCl. Concentrations of N-SH3 used for each experiment were: 4.0–8.0 µM at pH 5.0, 2.0–7.0 µM at pH 5.5, 1.0–5.0 µM at pH 6.0, 0.5–5.0 µM at pH 6.5, 1.5–6.0 µM at pH 7.0, 0.5–5.0 µM at pH 7.5, 1.5–6.0 µM at pH 8.0, 1.5–6.0 µM pH 8.5, 1.5–6.0 µM at pH 9.0. For the ionic strength dependence of D138A and E140A variants buffer used were 0.05 M NaCl, 0.1 M NaCl, 0.15 M NaCl, in 50 mM Hepes pH 7.5. Concentrations of N-SH3 D138A used for each experiment were: 2.0–4.5 µM at 0 M NaCl, 2.5–6.0 µM at 0.05 M, 0.1 M, 0.15 M NaCl. Concentrations of N-SH3 E140A used for each experiment were: 2.0–4.5 µM at 0 M NaCl, 3.0–5.5 µM at 0.05 M NaCl, 3.0–5.0 µM at 0.1 M NaCl, 3.0–5.5 µM at 0.15 M NaCl. Displacement kinetic experiments were performed by mixing a preincubated complex of N-SH3  and dansylated C3G versus a high excess of nondansylated C3G (see text for details). Experiments were performed in the same buffer conditions as the binding experiments, except for D141A variant, for which 20% Equilibrium experiment on D141A (fixed concentration at 1 μM) was carried out on a Fluoromax single-photon counting spectrofluorometer , by mixing the construct with increasing dansyl-C3G concentrations. Experiments were performed at 10 °C, using a quartz cuvette with a path length of 1 cm, in 50 mM Hepes pH 7.0 and measuring the change in fluorescence of the naturally present tryptophan residues in position 164 and 165 at increasing concentration of dansyl-C3G. The excitation wavelength was 280 nm, and fluorescence spectra were recorded between 300 and 400 nm.


Furthermore, 16-epi-pyroaconitine could antagonize the arrhythmogenic effect caused by the prototype compound aconitine; the antiarrhythmic effect of 16-epi-pyroaconitine was stronger than lidocaine and propafenone, which had the potential to be developed as antiarrhythmic drugs.The transformation pathways of diterpenoid alkaloids have been clarified in the boiling and steaming process. Aconitine, a famous diterpenoid alkaloid, is successively transformed into benzoylaconine and aconine during the processes of boiling and steaming, but the transformation pathway remains to be determined in the sand frying process. The present study aims at investigating the transformation pathways of aconitine in the process of sand frying, as well as assessing the cardiotoxicity and antiarrhythmic activity of aconitine and its converted products. The parameters of temperature and time for the structural transformation of aconitine were confirmed by HPLC. The converted products were further separated and identified by column chromatography, NMR, and HR-ESI-MS. Furthermore, by observing the lead II electrocardiogram (ECG) changes in rats under an equivalent dose, the cardiotoxicity of aconitine and its converted products were compared. Ultimately, the antiarrhythmic effect of the converted products was investigated by employing the model of aconitine-induced arrhythmia. Consequently, the structure of aconitine was converted when processed at 120°C–200°C for 1–40 min. Two diterpenoid alkaloids, a pair of epimers, namely, pyroaconitine and 16-epi-pyroaconitine, were further isolated from processed aconitine. 0.03 mg/kg aconitine induced arrhythmias in normal rats, while the converted products did not exhibit arrhythmias under an equal dose. In the antiarrhythmic assay, 16-epi-pyroaconitine could dose-dependently delay the onset time of VPB, reduce the incidence of VT, and increase the arrhythmia inhibition rate, demonstrating comparatively strong antiarrhythmic activity. Conclusively, compared with the prototype compound aconitine, the converted products exhibited lower cardiotoxicity. Further investigations on the cardiotoxicity indicated that pyroaconitine with  Aconitum plants belonging to the family of Ranunculaceae worldwide, mainly distributed in the temperate regions of the northern hemisphere, of which over 200 species are distributed in China, most of them are found in Sichuan, Yunnan, and Tibet Autonomous Region D 20+ 30.8° . Its molecular formula was deduced to be C32H43NO9 from a pseudomolecular ion at m/z 586.3010 [M + H]+  in its HR-ESI-MS. The NMR spectra , four methoxyl groups , a benzoyl group , 128.4 d (2C), 133.3 d), as well as a carbonyl group (δC 211.8 s). These data suggested that compound 1 was a pyro-type alkaloid D20 − 101.1 . The pseudomolecular ion at m/z 586.3008 [M + H]+ (cacld. 586.3011) in its HR-ESI-MS spectrum suggested a molecular formula of C32H43NO9. 1H and 13C NMR spectra of 2 , four methoxyl groups , a benzoyl group , 128.6 d (2C), 133.6 d), as well as a carbonyl group (δC 211.7 s).16-Epi-pyroaconitine (tra of 2  showed tJ = 5.16 Hz) at δH 5.41 was attributed to H-14β, implying the appearance of a benzoyl group at C-14 position [3 (δH 3.24) and C-1 (δC 83.6 d), 6-OCH3 (δH 3.24) and C-6 (δC 84.1 d), 16-OCH3 (δH 3.80) and C-16 (δC 86.1 d),18-OCH3 (δH 3.29) and C-18 (δC 76.8 t) in HMBC spectrum  and H-5 (δH 2.04), H-18 , H-19 , as well as C-13  and H-9 (δH 2.80), H-12 , H-14 (δH 5.41) in the HMBC of 2.The one-proton double signal  s and (142.3 ± 17.5) s, respectively. Compared with the control group, two positive drugs had a significant difference (P < 0.05).Lidocaine and propafenone delayed the onset time of VPB induced by aconitine, and the incubations of VPB were (180.9 ± 51.0) s, (263.9 ± 103.5) s, (430.1 ± 217.8) s, and (560.4 ± 288.5) s, respectively. Compared with the control group and propafenone, the onset time of VPB in all dose groups had a significant difference (P < 0.05). Moreover, compared with the positive drug lidocaine, there was an obvious difference in the 0.25 mg/kg and 0.30 mg/kg groups (P < 0.05).The effect of 16-epi-pyroaconitine on the latency of VPB was shown in VT is the result of further development of VPB, as well as an important factor in sudden cardiac death. The incidence of VT can be used to evaluate whether the experimental compound can effectively prevent the progress of VPB; the lower the occurrence is, the better the efficacy is.χ2 = 10.023, P=0.007), as illustrated in χ2 = 7.318, P=0.007; χ2 = 7.813, P=0.005).Complying with the data analysis requirements of chi-square test, the adjacent dose groups with similar incidence should be combined to analyze. For this reason, four dose groups of 16-epi-pyroaconitine were partially combined and subsequently divided into three subgroups, namely, 0.05 mg/kg, 0.15–0.25 mg/kg, and 0.30 mg/kg dose groups. The chi-square test showed that the incidence of VT in the aforementioned three groups was significantly different , indicating that 16-epi-pyroaconitine could dose-dependently increase the arrhythmia inhibition rate, as depicted in The effect of different dose groups of 16-epi-pyroaconitine on the arrhythmia inhibition rate in rats was analyzed. The results of chi-square test showed that there was a significant difference in the arrhythmia inhibition rate among the four groups  P < 0.05, The antiarrhythmic results demonstrated that 16-epi-pyroaconitine could dose-dependently delay the onset time of VPB, decline the occurrence of VT, and increase the arrhythmia inhibition rate. For example, the VPB incubation periods at doses of 0.25 mg/kg and 0.30 mg/kg were significantly longer than the positive drugs propafenone and lidocaine (in vivo experiments that, with the structural transformation of aconitine, the cardiotoxicity of the converted products was reduced. Comparatively, the cardiotoxicity of pyroaconitine was stronger than that of 16-epi-pyroaconitine, which exhibited relatively strong antiarrhythmic effects.In summary, this paper used the oil bath heating method to simulate the sand frying process and screened out the temperature and time parameters for the structural transformation of aconitine. In addition, it also demonstrated from the


N-carboxyanhydrides (NCA) monomers, which requires dry solvents, Schlenk line/gloveboxe, and protection of side-chain functional groups. Here we report a robust method for preparing unprotected NCA monomers in air and under moisture. The method employs epoxy compounds as ultra-fast scavengers of hydrogen chloride to allow assisted ring-closure and prevent NCA from acid-catalyzed decomposition under moist conditions. The broad scope and functional group tolerance of the method are demonstrated by the facile synthesis of over 30 different α/β-amino acid NCAs, including many otherwise inaccessible compounds with reactive functional groups, at high yield, high purity, and up to decagram scales. The utility of the method and the unprotected NCAs is demonstrated by the facile synthesis of two water-soluble polypeptides that are promising candidates for drug delivery and protein modification. Overall, our strategy holds great potential for facilitating the synthesis of NCA and expanding the industrial application of synthetic polypeptides.A great hurdle in the production of synthetic polypeptides lies in the access of  N-carboxyanhydrides (NCAs), but the methods for NCA synthesis usually require anhydrous and air-free conditions. Here, the authors report a method for the synthesis of NCAs under atmospheric conditions and room temperature, based on the use of epoxides as acid scavengers.Synthetic polypeptides are biomimetic materials that are commonly prepared by ring-opening polymerization of amino acid  N-carboxyanhydrides (NCA), are a family of biomimetic materials that can be applied as catalysts for heterogeneous reactions, self-assembling building blocks, surfactants for double emulsion, drugs carriers, tissue engineering scaffolds, antimicrobial agents, and alternatives to polyethylene glycol (PEG)15. For example, poly-l-alanine and poly-l-leucine are established industrial catalysts for asymmetric Juliá–Colonna epoxidation reactions16. Copaxone®, a random co-polypeptide prepared through the ROP of four different NCAs, is a blockbuster drug for multiple sclerosis which generated an annual global sales of 4 billion USD in 201218. Several additional polypeptide-based therapeutic candidates are being tested at different phases of clinical trials19. Despite these achievements, synthetic polypeptides are difficult and costly to produce. Although recent advances in ROP has allowed, sometimes even in unpurified solvent or at aqueous-organic interfaces, the rapid generation of polypeptides with predictable and high molar masses (Mn), low dispersity (Đ), and well-defined reactive end groups31, there are few new methods for simpler synthesis of NCA. The “Fuchs-Farthing’s method” for the generation of NCA, which involves the phosgenation and ring-closure of amino acids via (tri)phosgene, is usually carried out under strictly anhydrous and air-free conditions  are tolerated on NCA35, the introduction of more nucleophilic and/or proton-bearing functionalities such as alcohol, thiol, and carboxylic acid has been considerably difficult. Recently, Kramer and Deming achieved the notable breakthrough of purifying non-crystallizable NCAs via silica gel column chromatography in a glovebox36. In another outstanding study, Fuse reported efficient NCA synthesis in a mixture of organic and aqueous solvents with the use of a microflow reactor, followed by a sequence of flash dilution, extraction, and purification37. Despite all these advances, the scalable, open-vessel, and moisture-tolerant synthesis of challenging NCAs without the burden of protecting functional side groups remains an urgent and unmet need.Synthetic polypeptides, commonly prepared by the ring-opening polymerization (ROP) of amino acid Here, we report a robust method for preparing unprotected NCA monomers in air and under moisture. The method employs epoxy compounds as ultra-fast scavengers of hydrogen chloride to allow assisted ring-closure and prevent NCA from acid-catalyzed decomposition under moist conditions Fig. . The brol-glutamate NCA (Bn-GluNCA) was observed to undergo ROP and the reaction reached completion within 4 h in THF-d8 (2.0 M) when stoichiometric D2O was added  and without Schlenk line/glove box. The model monomer γ-benzyl n THF-d8 .0 M when40. We ruled out the use of TEA because (i) it can prematurely initiate the ROP of NCA when in excess, (ii) it cannot quench the nucleophilic chloride, which is known to attack the carbonyls of NCAs to form isocyanates41, and (iii) the removal of triethylammonium chloride salt adds extra burden to the workup procedure. On the other hand, we conducted the model reaction of α-pinene (1.0 M) and HCl (1.0 M) in THF-d8 at room temperature, which showed low conversion and slow rate , (+)-limonene, or α-pinene, particularly in the synthesis of challenging NCAsate Fig. . This is43. To our delight, the use of PO (1.0 M) led to complete, rapid conversion of HCl (1.0 M) in less than 3 min  and epichlorohydrin (ECH) could be ideal additives in NCA synthesis, due to their ability to remove both proton and chloride even at relatively low temperatures Fig. 43. To ouion Fig. , 6.2 and HCl, (ii) all hydrogen chloride could be rapidly removed by PO , triphosgene (0.5 equiv), and PO (4.0 equiv) in analytical grade THF  at room temperature in a sealed 350-mL heavy-wall flask. Unlike the conventional method, which would require heating the reaction mixture to 50–60 °C for 3–4 h under nitrogen, our protocol achieved complete conversion of the amino acid substrate within ~1.5 h without external heating. Thanks to the efficient and complete elimination of HCl, pure Bn-GluNCA  could be obtained at 84% yield following recrystallization without the need for anhydrous solvents or a glovebox . The NCA synthesis succeeded even with intentionally added H2O  to the reaction mixture as long as triphosgene and PO were kept slightly excessive. The high purity of Bn-GluNCA was confirmed by a combination of 1H and 13C NMR spectroscopy, high-resolution mass spectrometry, and FT-IR spectroscopy , which we were previously unable to synthesize at 40% environmental humidity and above, regardless of how other reaction conditions were optimized. In the current study, however, we successfully obtained 2.7 g of pure EG3-GluNCA  residual water in the solvent could be efficiently consumed by the excessive triphosgene, generating CO PO Fig. , and (iiN-substituted NCAs (NNCA), we surprisingly ran into roadblocks in our initial attempts to prepare sarcosine NCA (SarNCA) and ProNCA. Analysis of the crude reaction mixtures revealed the formation of byproducts from the aminolysis of PO by the secondary amines  and l-threonine NCA (ThrNCA)46, had been attempted but without convincing characterization results. In contrast, we speculated that hydroxy-bearing NCAs should be well-tolerated by our method, based on the fact that neither 1-chloro-2-propanol nor 2-chloro-1-propanol, the ring-opening products of PO and HCl, exhibited any detrimental effect on product formation. Indeed, SerNCA  mediated by benzylamine (BnNH2) in dry DMF provided one-step access to poly-l-glutamic acid (PLG), a therapeutically relevant biodegradable carrier polymer being evaluated in phase 3 clinical trials19, at >95% monomer conversions within 3 h in general  with highly corrosive HBr, leading to substantial time saving and yield increase compared to the conventional multistep procedure (yield: 80% vs. 40%). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry of a 20-mer PLG offered solid evidence to confirm the proposing polymer structure, which included the benzyl amide terminus . The integration of Glu/Bn in the 1H NMR spectra of the two PLG polymers agreed with the SEC results on Mn  as characterized by SEC  could serve as an inimer. Copolymerization of CysNCA (3r) and EG3-GluNCA (3k) by BnNH2/4-pyridine (DMAP) in dry DMF afforded P(EG3-Glu)-branch-PCys in a single step, which branches off at the thioester linkages -branch-PCys exhibited a characteristic α-helical conformation with a helicity of ~73% -branch-PCys found its Mn and Đ to be 33 kg/mol and 1.66, respectively -branch-PCys gave a sulfur content of 2.4 wt%, and thus a molar EG3-Glu/Cys ratio of ~4.8/1, which agreed well with the 1H NMR spectroscopy  and Bn-GluNCA  by BnNH2 (1.0 equiv)/DMAP (1.0 equiv) in dry DMF afforded PBLG-branch-Pcys in a similar fashion 54 and β-amino acid NCA (βNCA)55. Similar to that of NCA, the synthesis of OCA generally involves stringent anhydrous conditions, but also requires activated charcoal to remove impurities. Here, by implementing PO as the acid scavenger, the representative l-phenyllactic acid OCA  for the preparation of PLG with a much shorter procedure and spared efforts in protection and deprotection  with other NCAs allowed facile production of branched polypeptides, further underscoring the synthetic utility of unprotected NCAs, which were attainable only with the epoxy-assisted NCA synthesis. We have previously shown that the linear helical P(EG3-Glu) is a promising non-fouling polypeptide for surface coating and modification of protein drugs59. We envision the broader application of P(EG3-Glu)-branch-PCys as a high-performance biomedical material owing to its unique topology and the intrinsically embedded reactive thiol and thioester functionalities, which is amenable to further bioconjugation, surface anchoring, and responsive degradation. It should be pointed out that the results of the copolymerization could be modulated by simply altering the feeding ratio, allowing fine-tuning of Mn and microstructure of the polymer products. Moreover, the branching degree of the copolymer can be potentially manipulated via intra/inter-molecular S-to-N acyl shift during the course of polymerization. Detailed kinetic studies, mechanistic investigations, and optimization of the homopolymerization of GluNCA at higher M/I ratios, copolymerization of CysNCA with other NCAs, along with the related application testing are currently underway in our laboratory. The generality of the method was further demonstrated by the synthesis of several OCAs and βNCAs in high purity , THF (150 mL), methyloxirane  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the vessel was sealed immediately. The amino acid gradually disappeared in ~30 min with noticeable heat release. The reaction was stirred at room temperature for ~1.5 h in total and cooled down to ~4 °C in an ice bath. For safety reasons, the excessive triphosgene was quenched by adding 70 mL cold water at ~ 4 °C with 1–3 min stirring. The mixture was extracted with ethyl acetate  at room temperature. The combined organic phase was washed with brine and dried with anhydrous Na2SO4. After the removal of the solvent by rotatory evaporation in a vacuum under 45 °C, the crude product was purified by crystallization in hexane/THF below 10 °C (preferably in a cold room) without N2 protection. The pure product 3a was obtained as a white needle crystal . The NCA was stored at −10 °C for at least 5 months.To a pressure vessel with a heavy wall (air humidity: 70%), γ-benzyl 1H NMR  δ 7.42 – 7.31 , 6.70 , 5.13 , 4.38 , 2.59 , 2.32 – 2.21 , 2.18 – 2.06 .13C NMR  δ 172.5, 169.6, 152.2, 135.3, 128.8, 128.7, 128.4, 67.2, 57.0, 29.8, 26.9.HRMS : [M + H]+ calculated for C13H14NO5+: 264.0866; found: 264.0865.FT-IR (cm−1) 1854, 1781, 1728.l-glutamate 1k , THF (50 mL), methyloxirane  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the vessel was sealed immediately. The reaction was stirred at room temperature for 2 h. (This NCA cannot be washed with water!!!) After the removal of the solvent by rotatory evaporation in a vacuum under 50 °C, the crude product was purified by flash column chromatography . The pure product 3k was obtained as a colorless oil . The NCA was stored at −30 °C for 1 month.To a pressure vessel with a heavy wall (air humidity: 66%), γ-(2-(2-(2-methoxyethoxy)ethoxy)ethyl 1H NMR  δ 9.09 , 4.46 , 4.20 – 4.07 , 3.60 , 3.57 – 3.47 , 3.46 – 3.39 , 3.24 , 2.47 , 2.10 – 1.96 , 1.99 – 1.83 .13C NMR  δ 171.8, 171.4, 151.9, 71.3, 69.8, 69.7, 69.6, 68.2, 63.5, 58.1, 56.2, 29.1, 26.5.HRMS : [M + H]+ calculated for C13H22NO8+: 320.1340; found: 320.1339.FT-IR (cm−1) 1855, 1782.l-cysteine 1r , THF (200 mL), methyloxirane  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the vessel was sealed immediately. The reaction was stirred at room temperature for 7.5 h and filtered directly. (This NCA cannot be washed with water!!!) The filtrate was concentrated by rotatory evaporation in a vacuum under 40 °C and during the course, the crude product precipitated out as a crystal with ~1–2 mL oil left in the flask. The wet crude product was then added a mixture of PE/EA , stirred, sit for 1 min, and the liquid phase was decanted. The washing was repeated three times at room temperature with no need of N2 protection. The pure product 3r was obtained as a white crystal . The NCA was stored at −20 °C for 1 month. For longer storage, the NCA can be stored in a glove box at −30 °C.To a pressure vessel with a heavy wall (air humidity: 50%), 1H NMR  δ 8.00 , 4.56 , 2.98 – 2.82 , 2.05 .13C NMR  δ 170.1, 152.9, 60.5, 26.5.HRMS : [M + H]+ calculated for C4H6NO3S+: 148.0063; found: 148.0064.FT-IR (cm−1) 1863, 1792.l-glutamic acid 1t , THF (150 mL), ECH  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the vessel was sealed immediately. The reaction was stirred at room temperature for 18 h and filtered directly. After the removal of the solvent by rotatory evaporation in a vacuum under 45 °C, the crude product was purified by crystallization in PE/THF below 10 °C (preferably in a cold room) without N2 protection. The pure product 3t was obtained as a white powder . The NCA was stored at −20 °C for 2 months.To a pressure vessel with a heavy wall (air humidity: 40%), 1H NMR1H NMR  δ 12.32 , 9.09 , 4.45 , 2.36 , 1.99 , 1.85 .13C NMR  δ 173.4, 171.5, 152.0, 56.3, 29.1, 26.6.HRMS : [M + H]+ calculated for C6H8NO5+: 174.0397; found: 174.0395.FT-IR (cm−1) 1858, 1791, 1741.l-glutamic acid NCA  in anhydrous DMF (500 μL), the initiator benzylamine  was added. The reaction was stirred at room temperature for 16 h. After extensive dialysis and lyophilization, the polymer was obtained as a white powder . The experiments were independently repeated twice. The obtained Mn was 3.6 ± 0.1 kg/mol, and Đ was in the range of 1.06‒1.08 determined by aqueous SEC. The dn/dc value of PLG in PBS was measured as 0.1184 ± 0.002859 mL/g  in dry DMF (0.5 mL), the initiator benzylamine  was added. The reaction was stirred at room temperature for 24 h. Then, 54 μL (58 mM) of the solution of poly(l-glutamic acid) was added to the solution of EG3-GluNCA  in dry DMF (300 μL). The reaction was stirred in the glove box at room temperature for 24 h. After extensive dialysis and lyophilization, the polymer was obtained as a white fiber solid.To the solution of l-proline 2b , CH3CN (15 mL), methyloxirane  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the flask was kept slightly open to air to allow CO2 escape. The reaction was stirred at ~4 °C in an ice bath for 2.5 h. For safety reasons, the excessive triphosgene was quenched by adding 0.5 mL cold water at ~4 °C with 1–3 min stirring. Then brine (1 mL) was added to facilitate phase separation and subsequent extraction. The aqueous phase was extracted with ethyl acetate . The combined organic phase was directly dried with anhydrous Na2SO4. After the removal of the solvent by rotatory evaporation in a vacuum under 40 °C, the crude product was purified by flash column chromatography (from PE/EA = 5:1 to 1:1 to pure EA) as soon as possible. The pure product 4b was obtained as light yellow oil, which became a solid after frozen . The NCA was stored at −20 °C for 1 month. To a 100 mL flask (air humidity: 74%), Boc-1H NMR  δ 4.32 , 3.73 , 3.30 , 2.28 , 2.24 – 2.14 , 2.09 , 1.92 13C NMR  δ 168.9, 154.9, 63.1, 46.5, 27.6, 26.9.HRMS : [M + H]+ calculated for C6H8NO3+: 142.0499; found: 142.0499.FT-IR (cm−1) 1848, 1763.l-hydroxyproline 2c , CH3CN (50 mL), methyloxirane  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the flask was kept slightly open to air to allow CO2 escape.  The reaction was stirred at ~ 4 °C in an ice bath for 2.5 h. For safety reasons, the excessive triphosgene was quenched by adding 0.5 mL cold water at ~4 °C with 1–3 min stirring. Then 1 mL brine was added to separate the monophase. The organic phase was directly dried with anhydrous Na2SO4. After the removal of the solvent by rotatory evaporation in a vacuum under 35 °C (Do not rotavapor too long), the crude product was purified by flash column chromatography (from PE/EA = 5:1 to 1:1 to pure EA). The rude product 4c was obtained as a colorless oil at room temperature with ~5% solvent residue . After being stored in a −20 °C freezer, the oil became a chunk-like solid. Purer HypNCA was attainable via careful recrystallization of the crude product in mixed THF/hexane. The NCA was stored at −20 °C for 1 month. .To a 100 mL flask (air humidity: 72%), Boc-1H NMR  δ 5.31 , 4.72 , 4.54 , 3.71 , 3.06 , 2.23 , 1.95 .13C NMR  δ 170.3, 154.7, 72.6, 62.3, 55.0, 35.5.HRMS : [M + H]+ calculated for C6H8NO4+: 158.0448; found: 158.0447.FT-IR (cm−1) 3419, 1851, 1760.l-phenyllactic acid 5a , THF (15 mL), methyloxirane  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the vessel was sealed immediately. The reaction was stirred at room temperature for 24 h in total. After the removal of the solvent by rotatory evaporation in a vacuum under 45 °C, the crude product was purified by crystallization in hexane/THF below 10 °C (preferably in a cold room) without N2 protection. The pure product compound 7a was obtained as a white needle crystal . The NCA was stored at −10 °C for 1 month.To a pressure vessel with a heavy wall (air humidity: 55%), 1H NMR  δ 7.41 – 7.29 , 7.26 – 7.19 , 5.30 , 3.38 , 3.25 .13C NMR  δ 166.5, 147.9, 131.6, 129.8, 129.3, 128.5, 80.0, 36.5.HRMS : [M]+ calculated for C10H8O4+: 192.04171; found: 192.04173.−1) 3451, 1883, 1792.FT-IR , THF (30 mL), ECH  were added sequentially under magnetic stirring. Triphosgene  was finally added in one portion and the vessel was sealed immediately. The reaction was stirred at room temperature for 2 h. After the removal of the solvent by rotatory evaporation in a vacuum under 45 °C, the crude product was purified by crystallization in PE/THF below 10 °C (preferably in a cold room) without N2 protection. The pure product 8c was obtained as a white crystal . The NCA was stored at −20 °C for 2 months.To a pressure vessel with a heavy wall (air humidity: 80%), 1H NMR  δ 8.97 , 4.32 , 3.70 , 3.23 , 2.90 .13C NMR  δ 170.9, 165.1, 148.8, 52.9, 48.1, 30.6.HRMS : [M + H]+ calculated for C6H8NO5+: 174.0397; found: 174.0396.FT-IR (cm−1) 1807, 1766.Supplementary InformationPeer Review File