Petroselinum crispum (PcPAL) resulted in specifically tailored PcPAL variants, contributing to a rational design template for PAL-activity enhancements towards the differently substituted substrate analogues. Within this study we tested the general applicability of this rational design model in case of PALs, of different sources, such as from Arabidopsis thaliana (AtPAL) and Rhodosporidium toruloides (RtPAL). With some exceptions, the results support that the positions of substrate specificity modulating residues are conserved among PALs, thus the mutation with beneficial effect for PAL-activity enhancement can be predicted using the established rational design model. Accordingly, the study supports that tailoring PALs of different origins and different substrate scope, can be performed through a general method. Moreover, the fact that AtPAL variants I461V, L133A and L257V, all outperformed in terms of catalytic efficiency the corresponding, previously reported, highly efficient PcPAL variants, of identical catalytic site, suggests that not only catalytic site differences influence the PAL-activity, thus for the selection of the optimal PAL-biocatalysts for a targeted process, screening of PALs from different origins, should be included.Unnatural substituted amino acids play an important role as chiral building blocks, especially for pharmaceutical industry, where the synthesis of chiral biologically active molecules still represents an open challenge. Recently, modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from  Their substrate scope has been intensively studied within the last decade, with several PALs, such as those originary from Petroselinum crispum (PcPAL)6, Anabaena variabilis (AvPAL)8, Rhodotorula glutinis (RgPAL)10, Arabidopsis thaliana (AtPAL)11, Planctomyces brasiliensis (PbPAL)12, Kangiella koreensis (KkPAL)13, Pseudozyma antarctica (PzaPAL)14, shown to possess broad substrate scope. However, all these studies also revealed significant differences in their catalytic efficiencies towards specific substrates14. As example, PbPAL and the recently explored AL-11 PAL15 transformed substrates with electron-donor substituents, previously shown to be poor substrates for other PALs, such as PcPAL, AtPAL and AvPAL. Comparison of the catalytic sites of PALs of different origins shows a highly conserved polar substrate binding region responsible for the fixation of substrate’s carboxyl- and NH2- group  activity, such as in case of RgPAL and RtPAL from Rhodotorula sp., with reported TAL/PAL activities20. Histidine ammonia-lyases (HALs) show a characteristic His residue at position corresponding to 138 of PcPAL21. Exploration and characterization of novel PALs from different origins is continuously expanding15 driven by the aim to find PALs of increased operational and/or thermostability, or of activity towards substrates hardly transformed by existing PALs.The current state of art of the PAL mediated biotransformations revealed several synthetically useful PALs of eukaryotic (plant and yeast) or bacterial originsoup Fig. , which ak18 Fig. . The difk18 Fig. , responsPAL Fig. . These lPcPAL22, AvPAL23, RgPAL24, PbPAL12, AL-1115, also focused to provide variants of expanded substrate scope or increased operational and thermal stability. Diversification at positions 137 and 138 has been performed at several PALs, such as PcPAL18, AvPAL23, RtPAL24 and PbPAL12, and provided variants of improved catalytic performance, with modifications especially at analogue positions of 137 of PcPAL, highlighting the substrate specificity modulator effect of this residue. Our recent mutational analysis of the hydrophobic binding pocket of PcPAL2, revealed other specificity modulator active site residues and depicted their specific interaction with the differently positioned ortho-, meta-, para- substituents of the non-natural substrates, thus providing excellent tool for the rational protein engineering of PcPAL. Considering the differences in the hydrophobic substrate binding pocket of PALs of different origins, that strongly influence their substrate scope, the general validity of our recently developed rational engineering strategy among PALs of diverse origins, hence of diverse substrate scope, should be assessed. This might provide a desirable general rational design strategy among PALs, allowing facile development of substrate-tailored PALs of various origins. Accordingly, we tested whether the mutational strategy developed for PcPAL applies to other PALs, such as AtPAL and RtPAL, of different sequence similarities to PcPAL, AtPAL possessing high, 81% sequence identity and identical catalytic site with PcPAL, while RtPAL shares lower, 38% sequence identity with PcPAL, and contains specific ‘TAL-activity provider’ His and Gln residues at positions analogue to 137 and 138 of PcPAL , while in comparison RtPAL shows ~ twofold increased kcat values, but lower specificity constants (kcat/KM) in the natural PAL-reaction (deamination of l-Phe) and also within the reverse ammonia addition route of trans-cinnamic acid11.Protein engineering efforts on PALs of different origins, such as PcPAL, revealed mutant variants, obtained by mutagenesis of residues L256, L134, F137 and I460, of enhanced catalytic activity to ortho-, meta-, para- substituted substrates, respectively2. Accordingly, the homologues of all four substrate specificity modulating residues were similarly replaced in AtPAL and RtPAL  and four RtPAL variants . Notable, that residue H137 of RtPAL, homologue of F137 of PcPAL, is most probably involved in H-bonding with Q138, homologue of L138 of PcPAL. Since H137V RtPAL variant showed reduced activity within the activity screens, we presumed that the presence of residue Q138 within a complete hydrophobic active site environment is non-favourable. Thus, the mutational strategy was adapted, and within the ‘PcPAL-like’ RtPAL variants, besides the specific mutation of residues involved in aromatic substituent accommodation, additional mutations H137F and Q138L have also been included. Thus, RtPAL variant H137F/Q138L resembles the catalytic site of wt-PcPAL, variants H137F/Q138L/L266V and H137F/Q138L/I472V being homologues to L256V and I460V PcPAL, respectively, and variant H137V/Q138L RtPAL to F137V PcPAL variant. Despite our efforts, including different PCR protocols for mutagenesis, RtPAL variant L134A/H137F/Q138L resembling the catalytic site of L134A PcPAL could not be obtained, thus only L134A RtPAL was employed within the activity tests. The slight variations in thermal unfolding temperatures (Tm) of the purified enzyme variants indicated that mutations did not affect the protein folding  and electron-withdrawing  groups at all positions  of their aromatic ring , based on initial velocity measurements using purified enzymes, were also assessed. During enzyme kinetic measurements for the ammonia additions, factors such as (i) the high ammonia concentration of the reaction buffer leading to increased background absorbance, (ii) the high extinction coefficient of the cinnamic acid derivatives, hindering the use of large substrate concentrations, (iii) the overlapping absorbance spectra of the cinnamic acid and phenylalanine counterparts, provided high standard deviations, low reproducibility and/or incomplete Michaelis–Menten curves.The activities of the obtained ing Fig. . Both reortho-substituted substrates 1,2a–d, mutations L257V, L133A in case of AtPAL and mutation L266V in case of RtPAL provided similar enhancement of the conversion-based enzyme activity, relatively to their wild-type variants, as the one reported for L256V PcPAL2  and AtPAL (94% for 2a after 1 h reaction time and 48% for 1a after 6 h reaction time), thus the increased catalytic efficiency of variants L256V PcPAL and L257V AtPAL is less reflected within the conversion-based enzyme activities. However, the 2.3- and 2.1-fold increased kcat values, comparatively to the wt-variant’s  of the corresponding L256V and L257V variants. AtPALs provided stationary conversions of ~ 86% for 2b and ~ 50% for rac-1b within significantly shorter reaction times of 3 h and 30 min, respectively, in comparison with similar conversions obtained only after 24 h reaction times using PcPALs. Interestingly, while in case of 1a the KM value was not significantly altered upon mutations analogue to L256V, in case of 1b the mutation resulted in highly decreased substrate affinity  for all three PALs of different origin, supporting a more relaxed accommodation of 1b within the modified active site. Wild-type RtPAL in case of both substrates 2a, 2b provided only moderate conversion of 30% (6 h) and 35% (24 h), respectively, thus the beneficial effect of mutation L266V was clearly visible also within the conversion-based enzyme activity, with 94% (6 h) and 82% (24 h) conversion for 2a and 2b, respectively. In case of o-OCH3- substituted substrates 1c, 2c, the beneficial effect of mutations analogue with L134A from PcPAL was also observed in case of AtPAL, where the corresponding L133A variant provided high conversions of 95% and 3.1-fold increased kcat values. In case of L134A, but also wild-type RtPAL, very low/no conversions of < 1–3% were detected, while enzyme kinetics also revealed low initial velocities and substrate affinities. Interestingly, in this case the ‘PcPAL-like’ RtPAL L266V variant, with mutations H137F/Q138L/L266V, provided increased conversions of 19% for 1c and 17% for 2c after 16 h reaction time. Indeed, in this particular case, due to the bend caused by the oxygen atom of the o-OCH3 substituent, the methyl group positions between residues 134 and 266 , than their corresponding PcPAL homologues  or no conversion was detected when using wild-type PcPAL and RtPAL variants, in accordance with the reported steric clashes between the p-substituent and active site residues6. Interestingly, wt-AtPAL afforded close to maximum conversion of all para-substituted phenylalanines, except for p-OCH3-phenylalanine, where similarly to Pc/RtPALs, low conversion of 14% and kcat value of 0.007 s−1 were obtained within the ammonia elimination of rac-1k and no conversion within the ammonia addition to 2k.In case of AtPAL variants I461V and F136V, similarly as in case of PcPAL, provided important conversion and activity enhancements for all substrates 1i–2l and 2i–l. Accordingly, while in case of p-Br- and p-CF3-substituted substrates the mutation-induced increase in the conversions is less significant, due to the well-performing wild-type variant, the 2.9-fold and 3.4-fold increased kcat values of variant I461V for 1i and 1j support the beneficial effect of the mutation. In case of substrates 1k, 1l and 2k, 2l, p-substituted with the electron-donating -OCH3 and -CH3 groups, the superior catalytic efficiency of I461V variant to wt-AtPAL is also resembled within the highly increased conversion values. While mutation F136V of AtPAL also induced significant increase in the conversions of all substrates, in case of substrates 1i, 1j and 2i, 2j even surpassing the conversions registered with I461V variant, however the enantiomeric excess (ee) of the l-phenylalanines 1i, 1j and 1 k produced within the ammonia additions, were of lower value  in comparison with the highly enantiopure forms (ee > 99%) produced by wt- and I461V variant. This is in accordance with the results from PcPAL, where mutation F137V also decreased the enantioselectivity of the enzyme with ee values of 97% and 82% being obtained for l-1i and l-1j, respectively.Related to the effect of the mutational strategy, we observed that in case of RtPAL, in general, proved to be inefficient for the transformation of para-substituted amino acids, while the destabilization effect of mutation residue I472V, as described in case of meta-substituted substrates, resulted in no detectable activity. Instead, the mutation H137V, provided minor to moderate conversion increase of 7.9–23.1%, in case of p-Br- and p-CF3-substituted substrates 1i, 1j and 2i, 2j, where the beneficial effect of the mutation is also supported by the significantly increased kcat values. The lower catalytic efficiency of H137V RtPAL, reflected in significantly lower conversion values, in comparison to its homologue variants F136V AtPAL and F137V PcPAL, might result from the presence of polar Q138 residue in the proximity of the hydrophobic, mutated V137 residue  and RtPAL  the general applicability of the rational design strategy among PALs is supported. Notable, that in case of RtPAL, besides the modification of the substrate specificity-modulator residues, replacement of residue Q138, in proximity of position 137, to hydrophobic residues, further enhanced the catalytic properties of H137V RtPAL, supporting that the mutational strategy is adaptable for further additional mutations based on simple rational considerations, allowing facile development of substrate-tailored PALs of various origins. Despite the identical catalytic site residues of AtPAL and PcPAL, in several cases AtPAL variants in comparison with the corresponding PcPAL variants, showed higher catalytic efficiencies/conversions . The site-directed mutagenesis was performed following the protocol described by Naismith and Liu25, using as template the pET-19b vector harbouring the gene encoding PALs from Arabidopsis thaliana and Rhodosporidium toruloides, respectively. Using as homology model the active site of PcPAL6 (PDB ID 1W2726), several residues from the hydrophobic binding pocket were selected for point mutations, namely L133/L134 (to A), F136/H137 (to V), L257/L266 (to V) and I461/I472 (to V). New MIO-enzyme libraries were created at these positions and screened for activity towards the substrates. The primers used within the mutagenesis are listed in Table The codon optimized genes encoding PALs from wild-type RtPAL and its mutant variants  was performed according to our optimized protocol via immobilized affinity chromatography (IMAC)27. In case of AtPAL , precultures were prepared at 37 °C, 200 rpm, overnight in 50 mL LB (Luria Bertani) medium supplemented with carbenicillin (50 μg/mL) and chloramphenicol (30 μg/mL) from glycerol stocks of E.coli Rosetta (DE3)plysS cells harbouring the pET-19b vector carrying the wt- or mutant atpal gene. 2% (v/v) from the starter culture was used to inoculate 2 × 500 mL LB medium in 2 L flasks. The OD600 was monitored and when a value of 0.45 was reached, the temperature was lowered from 37 to 25 °C, and the shaking continued till an OD600 value of 0.6–0.8, when PAL expression was induced via IPTG . The cell growth continued at 25 °C, 200 rpm for another 6 h, when cells were harvested by centrifugation at 4000 rpm (1751×g), 4 °C for 20 min. The supernatant was discarded and the cell pellet was stored at − 20 °C until further use or processed immediately using the optimized protein isolation protocol as described for PcPAL27.Expression, isolation and purification of m) was determined by fitting the experimental data using a polynomial function, in which the maximum slope is indicated by the peak of its first derivative (F350/F330). All measurements were performed in triplicate . PAL variants were diluted with 20 mM Tris, 120 mM NaCl pH 8.8 buffer to a final concentration of 1 mg/mL. 10 μL of each sample were loaded into UV capillaries (NanoTemper Technologies) and unfolding of PAL enzymes was detected during heating in a linear thermal ramp of 1.5 °C/min between 20 and 95 °C, with an excitation power of 70%. Data analysis was performed using NT Melting Control software and melting temperature  pLysS cells harboring the pET-19b vector carrying the wt or mutant atpal or rtpal gene, followed by incubation at 37 °C and shaking at 200 rpm. 2% (v/v) of the overnight culture was used to inoculate 50 mL LB medium. Cultures were grown at 37 °C, 200 rpm until OD600 reached 0.6–0.8 (approx. 3 h), when enzyme production was induced via the addition of 0.5 mM IPTG , and the cell growth was maintained at 20 °C, 200 rpm, overnight (approx. 17 h). The final OD600 was measured for each mutant variant and wild-type PAL. The culture volumes required for the biotransformation screenings were harvested by centrifugation in 1.5 mL polypropylene tubes for 10 min at 13,300 rpm . The required volume of bacterial culture, providing the amount of whole-cell pellet needed was calculated considering the volume of the reactions, the whole-cell biocatalysts concentration (with fixed cell density OD600 of ~ 2)22 and the final OD600 value of the induced cells. The harvested cells were washed with 500 μL PBS buffer   and stored at − 20 °C until further use.The overnight precultures were prepared in 20 mL LB (Luria Bertani) medium supplemented with carbenicillin (50 μg/mL) and chloramphenicol (30 μg/mL) in 100 mL Erlenmeyer flasks, being inoculated with glycerol stocks of 600 of ~ 2, in 500 µL substrate solution (2 mM cinnamic acids 2a–l or 2 mM racemic amino acids rac-1a–l) prepared in 6 M NH4OH buffer pH 10 adjusted with CO2 (in case of ammonia addition) or 20 mM Tris.HCl, 120 mM NaCl buffer, pH 8.8 (in case of ammonia elimination). The reaction mixtures were incubated at 30 °C, 250 rpm. Reaction samples were taken after 3, 6, 16, and 24 h and quenched by adding an equal volume of MeOH, vortexed and centrifuged . The supernatant was filtered through a 0.22 μm nylon membrane filter prior to analysis by HPLC. In order to determine the conversions values, a Gemini NX-C18 column  was chosen, using as mobile phase: A: NH4OH buffer /B: MeOH, with a flow rate of 1.0 mL/min. The enantiomeric excess values were determined by chiral HPLC separations, using Crownpak CR-I (+) chiral column  and HClO4 (pH = 1.5)/acetonitrile as mobile phase at a flow rate of 0.4 mL/min. HPLC methods and response factors used for the conversion value determinations, as well as retention times of the enantiomers of rac-1a–l can be consulted in our previous reports6. All analytical scale biotransformations were performed in duplicates, while during the initial activity screens using a significantly sized reaction-subset the HPLC analysis have been performed for all samples within the duplicate set  was resuspended to an ODtrans-cinnamic acid analogues 2a–l at 290 nm (wavelength where the corresponding amino acids rac-1a–l showed no absorption), using substrate concentrations of 0.1–20 mM of 1a–l, 100 mM Tris.HCl, 120 mM NaCl (pH 8.8) as buffer and purified PAL variants at fixed enzyme concentration of 0.322 μM. Kinetic constants  were obtained from the Michaelis–Menten curves by non-linear fitting. Standard deviations for the determined kinetic parameters are given within Tables The initial enzyme activities were spectrophotometrically determined, using a Tecan Infinite Spark 10 M microplate reader and Corning 96-well Clear Flat Bottom UV-Transparent microplates. The kinetic measurements were performed in triplicate at 30 °C by monitoring the production of 2a–l were obtained by calculations based on the density functional theory, performed using the Gaussian 09 software28 by employing the B3LYP density functional and the 6-31G basis set. Geometry optimizations were carried out in a water solvated environment using the Polarizable Continuum Model (PCM)29.The ground state geometries of the monosubstituted cinnamic acid derivatives 30, using flexible-ligand and rigid-receptor docking. The search space was defined by embedding the binding site residues and the MIO prosthetic group. In both cases the receptor grid was defined as a cubic box with the dimension of 20 Å × 20 Å × 20 Å. The exhaustiveness search parameter of Vina was increased to 100.The molecular docking calculations were performed with the Autodock Vina softwarePcPAL was retrieved from Protein Data Bank entry 6F6T31, whereas in case of RtPAL, the AlphaFold32 predicted model was retrieved from the UniProt database (entry P11544)33. The assembled tetrameric structure was submitted for minimization using the YASARA web server34. Although crystal structures of RtPAL are available, PDB entries 1T6J and 1Y2M, both structures present the open conformation of the protein, missing the loop containing the Y110 residue, responsible for the catalytic site closure upon substrate binding.The crystal structure of Petroselinum crispum to other PALs with the aim to provide a general rational design strategy, highly desirable for developing substrate-tailored PALs of diverse substrate scope and origins. Accordingly, AtPAL and RtPAL, both well-characterized PAL representatives, that share different sequence identity  to PcPAL, with RtPAL known to possess dual PAL/TAL-activity, were selected for this purpose. As expected, wild-type RtPAL with low sequence identity to Pc/AtPAL, showed different substrate specificity towards the substrate library, revealing its higher catalytic efficiency towards meta-substituted substrates in both ammonia elimination and ammonia addition reaction routes, while the substrate specificities of wt-Pc/AtPAL have been found very similar. However, the enzyme activity tests of the generated focused AtPAL, RtPAL, PcPAL mutant library towards the mono-substituted substrates revealed that AtPAL variants, with some exceptions, surpassed in terms of conversion and catalytic efficiency the corresponding, previously reported PcPAL homologues . Since their active sites possess identical residues, the results highlight that besides active site residues, other structural elements also determine the different enzyme activities/substrate specificities of PALs. Furthermore, the activity of PAL variants tailored towards substrates of different  substitution pattern, revealed that the mutational approach is applicable among different PALs, resulting the expected catalytic efficiency increase towards the targeted non-natural substrates, with minor sequence alignment-based rational refinements further improving its efficacy. Accordingly, in case of RtPAL, besides the modification of the substrate specificity modulator residues L266V, L134A, F137V and I472V, replacement of residue Q138, in proximity of mutated position 137, to hydrophobic residues, further enhanced the catalytic properties of RtPAL variants. In this context, the study paves the way and contributes for the development of the general rational design strategy among the PAL (E.C. 4.3.1.24) and PAL/TAL families (E.C. 4.3.1.25).Within this study we tested the applicability of the mutational strategy developed for PAL from Supplementary Information.


Prunus genotypes were investigated. Microshoots were rooted in vitro on Murashige and Skoog (MS), ½ MS, Driver and Kuniyuki (DKW), or New Prunus Medium (NPM) media formulations in combination with 15 µM indole-3-butyric acid (IBA), 4-day 15 µM IBA pulse, 1 mM 30 s quick-dip, or IBA-free treatments. Shoots were observed pre- and post-acclimatization to determine rooting methods to maximize quality and minimize labor. A genotype-specific response to auxin application was observed with seven of eight genotypes achieving 100% survival when paired with the recommended IBA treatment. Peaches performed best when treated with 4-day IBA pulse or 30 s quick-dip. Rooting of P. cerasifera, it’s hybrid to P. persica, and P. munsoniana all benefitted from IBA application. Shoots rooted with 15 µM IBA were smaller and lower quality in most genotypes. DKW maximized size and quality in six genotypes. Better shoots and larger root systems during in vitro rooting produced better plants in the greenhouse with no detrimental effect of callus growth. Rooting techniques to maximize plant quality while reducing labor are specified.Difficult-to-root plants often perform poorly during acclimatization and in vitro rooting can increase the survival and quality of plants. The influence of auxin application and mineral nutrition on in vitro rooting and subsequent effects on plant quality in eight  Prunus, involves planting of orchards that are intended to be cultivated for decades and withstand biotic and abiotic stresses. Newly emerging plant pathogens cause breeders to generate interspecific hybrids to integrate novel alleles into improved germplasm. Interspecific hybrids, often sexually sterile, require vegetative propagation to provide material to tree nurseries. Traditional vegetative propagation of Prunus utilizes seasonally available soft and semi-hardwood stem cuttings which slows multiplication and may possibly spread disease. Alternatively, clean, pathogen-free clones can be rapidly multiplied through micropropagation. However, in vitro plant responses to culture medium have been observed to be genotype-dependent and nutrient salt composition needed to be reconsidered to maximize production [Fruit tree culture, including oduction ,2. CommePrunus has been observed in studies with media salt formulations and plant growth regulators (PGRs) ratios. For almond-peach and peach-almond hybrids, rooting efficiency were higher when treated with a strength of Murashige and Skoog (MS), Driver and Kuniyuki (DKW), and woody plant medium (WPM) relative to standard MS medium with optimal levels of IBA concentrations specific to individual cultivars [Prunus in Oasis® phenolic foam [Prunus. Optimal IBA concentrations ranging from 4–7µM were reported for in vitro rooting of 11 Prunus rootstocks but a considerable variation in rooting was observed [Prunus induce roots with a short duration of exposure to IBA and followed by transfer to PGR-free medium [Malus that showed a short period of auxin exposure followed by the removal of the PGR minimized the inhibition of root elongation and the formation of callus under prolonged IBA exposure [Malus, Pyrus, Camellia, and in other species of Prunus japonica Thunb. [Genotypic variation in rooting micropropagated ultivars ,4,5. A nlic foam . In peaclic foam . Large, observed . Alternae medium . This meexposure ,11. Howea Thunb. ,14,15,16Prunus genotypes important to ongoing breeding efforts. P. persica ‘Guardian®’, P. munsoniana 59/1, and P. cerasifera 14/4, 20-3, 20-4 serve as potential parental lines in novel rootstock development and P. persica × P. cerasifera 106.4, P. persica ‘GF-305’, P. persica × P. umbellata ‘MP-29’ were used for their utility in breeding and importance to disease management strategies. Methods were developed to maximize in vitro and greenhouse growth and determine possible correlations between early plant health and subsequent plant size following acclimatization. Additionally, techniques to maximize plant quality while accounting for the time required to apply IBA were considered.The present study examined the effect of various inorganic nutrient formulations in combination with methods of IBA application on in vitro rooting of eight ®’ and ‘GF-305’, had the highest percent rooting when treated with a longer duration of IBA in the 4-day pulse or 15 µM IBA treatments. ‘MP-29’, 106.4, and 20-3, exhibited the highest rooting percentage when treated with either the 4-day pulse (87–97%) or 30 s quick-dip. 59/1 followed this trend with lower rooting, averaging 70–77%. 14/4 and 20-4 performed uniformly across all IBA treatments, with 14/4 averaging 85–87.5% rooting and 20-4 averaging 65–75% rooting. The three myrobalan plums, 14/4, 20-3, and 20-4, and hybrid 106.4 had the lowest rooting in the absence of IBA, indicating the exogenous auxin’s importance. ‘MP-29’ had the same rooting percentage without IBA application and under 15 µM IBA, while 59/1 produced roots at a higher rate when no IBA was applied relative to the 15 µM IBA treatment. ‘GF-305’ rooted similarly when treated with 30 s quick-dip and IBA-free treatments (45–72%). ‘Guardian®’ rooted at 34–73% when 30 s quick-dip or when no IBA was administered  . The appPlant size segregated into three categorical groupings of small, medium, and large plants with corresponding scores of ≤8.7, between 8.7 and 10, and ≥10, respectively . IBA trePrunus propagation by stem cuttings, leaf area has been documented to influence rooting where a greater canopy results in better rooting [®’ and ‘GF-305’ performed best when treated with 4-day pulse. Hybrids ‘MP-29’ and 106.4, and plums 20-4, 20-3, and 59/1 were largest with 4-day pulse and were only distinct only from 15 µM IBA. 14/4 performed similarly across all treatments  were considered in recommended micropropagation practices for each genotype . If treaPyrus and Prunus [The largest plant (20-3) grew the best with MS medium, the smallest plant 59-1 grew best with NPM, and the other 6 genotypes grew best when grown on DKW medium. MS has the largest number of macronutrients. DKW also has almost as many macronutrients, ample mesonutrients, but much larger amounts of calcium and sulfate, than the other media and was most often the optimal media. NPM has much less macronutrients, similar to ½ MS, but the same mesonutrients as MS, and which was recommended for the genotype that produced the smallest plant size when treatment conditions were maximized. ½ MS provides the least amount of mesonutrients, calcium, magnesium, sulfur, and micronutrient manganese, with the low concentration of ammonium, potassium, and nitrate. In vitro plant quality in our trials requires adequate meso-nutrients as is consistent with published multifactor media optimization research in d Prunus ,29,30. OP. persica ‘GF-305’ and P. persica × P. umbellata ‘MP-29’ shoots were washed in dilute liquid detergent, followed by a 1 min dip in 70% ethanol and subsequent rinse in deionized water, followed by immersion in 10% bleach for 10 min. Shoot buds were peeled and placed in Baxter shell vials containing 20 mL Murashige and Skoog (MS) [−1 m−2 with a 16 h photoperiod.Aseptic shoot cultures were established following previously described methods . Brieflyoog (MS)  adjustedP. persica ‘Guardian®’ and ‘GF-305’ were maintained consistently on 40 mL of modified Quoirin and Lepoivre (QL) [−1 fructose, 1 mg L−1 ferulic acid, 2 mg L−1 ascorbic acid, 100 mg L−1 inositol, 2 mg L−1 glycine, 1 mg L−1 thiamine, 1 mg L−1 nicotinic acid, 1 mg L−1 pyridoxine, 2.2 µM 6-benzylaminopurine (BA), 0.025 µM indole-3-butyric acid (IBA), and 0.15 µM gibberellic acid (GA3) as described in Kalinina and Brown [Prunus genotypes: P. cerasifera 14/4, 20-3, 20-4, P. munsoniana 59/1, P. persica × P. cerasifera 106.4 and P. persica × P. umbellata ‘MP-29’ were maintained using New Prunus Medium (NPM) [−1 ferulic acid, 2 mg L−1 ascorbic acid, 100 mg L−1 inositol, 2 mg L−1 glycine, 1 mg L−1 thiamine, 1 mg L−1 nicotinic acid, 1 mg L−1 pyridoxine, with cycling between multiplication medium (2.2 µM BA) and resting medium (16 µM IAA). Media cycling was done to maintain healthy cultures and avoid prolonged exposure to BA. All media were autoclaved prior to use.Culture methods were selected to maintain plant quality during recurrent subculturing and were tailored independently to each genotype. vre (QL)  solidifind Brown . Plantle−1 sucrose, 1 mg L−1 ferulic acid, 2 mg L−1 ascorbic acid, 100 mg L−1 inositol, 2 mg L−1 glycine, 1 mg L−1 thiamine, 0.5 mg L−1 nicotinic acid, and 0.5 mg L−1 pyridoxine as described by Kalinina and Brown [Root induction was observed using a full-factorial arrangement of four media salt components and four IBA application techniques for the eight genotypes. Media containing salt formulations NPM , Murashind Brown . Media wnd Brown . Briefly®’ and ‘GF-305’. Seven ‘Guardian®’ seedling clones were observed as independent genotypes but pooled during analysis due to the similarities in responses. Run 2 contained the myrobalan plums 14/4, 20-3, 20-4, goose plum—59/1, and peach-plum hybrids ‘MP-29’ and 106.4. Each vessel contained 15 plants, five plants  of each of the three genotypes and was conducted in two technical replicates. Vessels were grown at an ambient temperature of 22 ± 2 °C under fluorescent lighting at 50 µmol s−1 m−2 with a 16 h photoperiod. After two weeks, the microporous vents integrated into the lid of the vessels were opened. Run 1 were grown for two additional weeks under a vented environment while the plums in run 2 were grown for one additional week. Plantlets were given a quality score based on the appearance of the shoot system using the following criteria: −1—chlorotic or necrotic tissue, no notable growth; 0—no notable growth, green tissue present; 1—green tissue present, notable leaf expansion, and growth. The presence or absence of callus was recorded, the number of roots was counted, and root quality was scored as follows: 0—no roots; 1—roots present, <5 mm in length; 2—roots present and elongated > 5 mm; 3—roots present, elongated, and with secondary roots present.The in vitro rooting experiment was conducted in two runs: run 1 contained the seedlings of ‘Guardian−1 m−2 at bench height and maintained at 75% ± 5 relative humidity by micro-mist irrigation emitters. The growing area was maintained at 25 ± 2 °C during peak sunlight and 20 ± 2 °C night temperature. Dry cells were hand watered as needed. Individual growth was quantified following the conclusion of the two-week growth period to include- stem height (mm) measured from the soil surface to the shoot tip, the number of leaves > 1 cm, and the length (mm) of the longest leaf. Root quality was scored after removing each plant from its cell and using the following scale: 0—no roots present; 1—roots present in the upper half of cell; 2—roots present throughout the cell; 3—roots present throughout the cell and soilless media plug remained intact by root adhesion after removal from the tray. A greenhouse vigor grade was assigned by observing the aerial tissue of each plant and using the following criteria: −1—chlorotic or necrotic tissue present, no notable growth; 0—green tissue present, no notable growth; 1—green tissue present, notable leaf expansion, and new leaves; 2—green tissue present, notable leaf expansion, new leaves, and elongated internodes. Ex vitro survival was calculated as a percent of plants that did and did not root in vitro and received a greenhouse vigor grade other than −1. Greenhouse growth was calculated as a percent of plants that received a greenhouse vigor grade of 1 or 2 for rooted or unrooted shoots from in vitro culture. Plant size was quantified by an index value using measurements in the following formula:Plantlets and unrooted shoots were gently removed from the agar matrix and then rinsed in tap water to remove residual media. Unrooted shoots were dipped in a 1 mM K-IBA solution for 5 s prior to transplanting. Treatment runs were transferred into polypropylene seedling trays composed of 63 cells measuring 1.5” × 1.5” × 3.5”  and filled with Fafard 3B soilless mix . The acclimatization phase was conducted from November to March in a greenhouse environment at 34.68° latitude with shade cloth and under a 3 mm polyethylene tent. Peak light intensity was approximately 150 µmol sPrunus genotypes were screened using a combination of treatment factors of four nutrient salt formulations, four IBA application treatments and replicated asynchronously. Treatment factor combinations were averaged to develop mean responses for each value. Data were analyzed using a 4 × 4 × 8 full factorial model with the inclusion of replicate as a blocking factor using JMP 16.1 . An analysis of variance (ANOVA) was used to determine if the primary model terms effects with factors considered significant. p < 0.01 was considered evidence of statistical significance. Performance groups of responses were created using 95% confidence intervals. Multivariate correlations were used to quantify relationships between laboratory and greenhouse responses.In vitro rooting was conducted using a randomized block design. Eight Prunus vary broadly by genotype, trends in recommended practices based on genotypic lineages exist. Genotypes with peach lineage performed best when treated with the 4-day pulse. Genotypes with myrobalan plum lineage were not sensitive to prolonged exposure to IBA but also responded well to a 30 s quick-dip. The 30 s quick-dip, an alternative rooting method to the 4-day pulse, requires less time and resulted in similar-sized plants in three of the eight genotypes screened. Little prior research has been done on quick-dip applications of exogenous PGRs under sterile conditions and improvements in this technique may warrant further investigation. The 4-day pulse treatment usually worked well but required the most technician time. Mineral nutrition during in vitro root induction was important and significantly affected final plant quality with DKW conferring the best quality in vitro with 6 of 8 genotypes screened. Replicate was significant in some ex vitro response potentially from the asynchronous replication and sunlight quality associated with the replicate. This study provides in vitro rooting methods that were useful for the different Prunus genotypes most used in our rootstock disease resistance breeding program.While propagation techniques within


Orange peel waste (OPW) is known to contain an abundant amount of polyphenols compounds such as flavonoids, well-reported for their antioxidant and anti-inflammatory properties. While OPW is generally regarded as a food waste, the opportunity to extract bioactive compounds from these “wastes” arises due to their abundance, allowing the investigation of their potential effects on endothelial cells. Hence, this study aims to use a green extraction method and pressurized hot water extraction (PHWE) to extract bioactive compounds from OPW. Liquid chromatography with UV detection (LC/UV) and liquid chromatography mass spectrometry (LC/MS) were subsequently used to identify the bioactive compounds present. Through the optimization of the extraction temperature for PHWE, our results demonstrated that extraction temperatures of 60 °C and 80 °C yield distinct bioactive compounds and resulted in better antioxidant capacity compared to other extraction temperatures or organic solvent extraction. Despite having similar antioxidant capacity, their effects on endothelial cells were distinct. Specifically, treatment of endothelial cells with 60 °C OPW extracts inhibited TNFα-induced vascular inflammation and endothelial dysfunction in vitro, suggesting that OPW possess vasoprotective effects likely mediated by anti-inflammatory effects. Citrus fruits are one of the most consumed and cultivated fruits globally, with increasing projection of production to meet global needs in the future . The incOne of the biggest groups of naturally occurring polyphenolic substances, flavonoids, are well-recognised to possess several biological activities, including antioxidant, anti-inflammatory, and vasodilatory properties ,8,9. GivTraditional extraction methods involve the use of organic solvents, such as methanol and ethanol, which are generally neither environmentally friendly nor sustainable. Furthermore, the use of organic solvents during the extraction process may increase costs of production, as the resultant organic wastes would require proper disposal ,21,22. ITherefore, the aim of the current study is to explore the use of PHWE, with water as an eco-friendlier alternative to organic solvents, in the extraction of bioactive compounds from OPW. The OPW extracts (OPWE) obtained using PHWE do not require an additional solvent removal step, reducing the overall complexity of the extraction method. Furthermore, as PHWE does require the presence of a simultaneously operating heating and cooling system, it may be a more energy-efficient compared to other methods ,24,25,26HPLC-grade water, formic acid, methanol, sand, 2,2′-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and standards used in HPLC were purchased from Sigma-Aldrich (Singapore). Fresh Australian navel OPW were donated by a local food vendor . OPW were lyophilised and blended using a food blender into powder form and sieved to obtain powder of particle size <0.3 mm. The <0.3 mm powder was used for the respective extraction methods.The PHWE system was assembled as previously described, consisting of a stainless-steel extraction column (250 mm × 10 mm i.d.), an isocratic LC10 series pump , and a 5890 Series II temperature-controlled oven  ,28. Stai2r, and relative standard deviation (RSD) were performed. The calibration curve was obtained and would be based on for the prediction of analyte concentration based on the LC/UV’s response to the standards used. The 2r values for all 5 standards used were at least 0.9990, indicating strong correlation between values obtained and actual values. RSD values (%) obtained for 6 consecutive injections of the respective analytes were ≤1.00%, indicating less variation between values and high repeatability of results. To normalise the peak areas to a constant sum, the measured peak intensity for each sample was normalised prior to obtaining an average reading for the three samples. For LC/UV, peaks were obtained based on the wavelengths at 254 nm. As for LC/MS, peak intensities obtained based on the molecular weights (m/z) and were normalised within each sample to the total peak intensity of the sample; normalisation was carried out to address concentration differences. The normalised peak area for each sample were further analysed using the principal component analysis (PCA) scores plot. The current approach was consistent with our earlier reports [The chemical profiles of the respective OPWE were determined using LC/UV/MS as described previously. Briefly, for separation of the compounds, a longer C18 reverse phase HPLC column  was used for LC/UV, while a shorter column  was used for LCMS. The gradient elution consisted of a mobile phase involving 0.1% formic acid in (A) water and (B) acetonitrile, respectively. Detection of bioactive compounds based on the standards involved was detected by the UV detector at the absorbance value of 280 nm. For LC/UV, the calibration curve, coefficient of determination  reports ,28,29.2O) were transferred into a 96-well plate. Subsequently, 100 µL of 0.1 mM DPPH solution was pipetted into those wells and mixed well. For the positive control, 10 mM ascorbic acid was used in place of the OPWE. The plate was wrapped with aluminium foil and incubated at 25 °C for 30 min. After incubation, the Multiskan GO microplate reader  was used to record the absorbance at 517 nm. Readings were processed as below:The DPPH assay was used to determine the antioxidant activity of the OPW extracted using different temperatures of PHWE and methanol extraction as described previously ,29. Brie•+ solution. The plate was incubated at 25 °C for 30 min in the Multiskan GO microplate reader , and absorbances were read at 734 nm. The CEAC values of the orange peel samples were calculated based on the equation obtained from the linear regression of the standard curve plotted.The vitamin C equivalent antioxidant capacity (CEAC) values of the OPWE were calculated and determined using ABTS assay as previously described . Briefly2. At 75–85% confluency, cells were seeded into cell-cultured plate under serum-starved condition (2% FBS) and incubated for 24 h. The HMEC-1 cells were treated with control (absence of TNF-α), OPWE alone (1 mg/mL), TNF-α (1 ng/mL), or TNF-α (1 ng/mL) co-incubated with OPWE (1 mg/mL) for 24 h. After treatment, the cells were harvested for cellular antioxidant, qPCR, or metabolomics experiments.Human Dermal Microvascular Endothelium (HMEC-1) cells were obtained (American Type Culture Collection (ATCC), USA) and cultured in MCBD-131 media , 5% L-glutamic acid (200 mM), 1% penicillin-streptomycin, and 0.001% recombinant human epidermal growth factor (EGF) (10 ng/mL)) using T75 flasks at 37 °C, 5% CO2 for 60 min. Subsequently, the cells were rinsed 3X with HBSS to remove excess DCFDA and counter-stained with 10 µM Hoechst 33,342 for further 15 min at 37 °C, 5% CO2. After incubation, the cells were rinsed 3X with HBSS, and the plate was read with the Varioskan LUX multimode microplate reader  with the following excitation and emission wavelength for DCFDA (485 nm/520 nm) and Hoechst 33,342 (350 nm/461 nm). Readings from DCFDA levels were normalised to Hoechst 33,342 and expressed as fold change to control levels.After 24 h of treatment, the wells were rinsed with Hanks’ Balanced Salt Solution (HBSS) to removed cell debris and culture medium. The cells were incubated with 2′,7′-Dichlorofluorescin diacetate (DCFDA)  (10 µM) at 37 °C, 5% COTM Total RNA Mini Kit , and the RNA quality and quantity were analysed as described earlier. Briefly, reverse transcription was carried out for the RNA samples in a single run with the T100TM Thermal cycler  to produce complementary DNA (cDNA) using the Bio-rad iScript cDNA Synthesis Kit. The final reaction volume was 20 µL, containing 0.5 µg of RNA. The comparative cycle threshold (2−ΔΔCt) method of qPCR was carried out according to earlier reports [Icam-1), vascular cell adhesion molecule 1 (Vcam-1), interleukin 1 Beta (Il1β), endothelin 1 (Edn1), prostaglandin-endoperoxide synthase 2 (Ptgs2), nitric oxide synthase 3 (Nos3), glutathione peroxidase 1 (Gpx1), superoxide dismutase 1 (Sod1), and nuclear factor erythroid 2-related factor 2 (Nfe2l2). qPCR was performed in 96-well plates with each well containing 10 µL volume reactions ) prepared in triplicates. Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh) was used as the reference housekeeping gene. Negative template controls were prepared by substituting cDNA with water or by having the reverse transcriptase in the cDNA synthesis substituted with RT negative controls.After 24 h of treatment, the wells were washed with phosphate-buffered saline (PBS), RNA was extracted using the Aurum reports ,31. The m/z) are normalised within each sample to the total peak intensity of the sample. Normalisation was performed to address the differences in concentration. Additionally, normalized data for each sample were analysed using the PCA and orthogonal projection to latent structure discriminant analysis (OPLS-DA) scores plot for the selected peaks. The fatty acids, lipids, and other small molecules were identified based on our earlier work and comparison with reference standards [Lipids were extracted using chloroform:methanol method, and LCMS was performed as previously reported with minor modifications ,33. The tandards ,33.p-value was <0.05.PCA and OPLS-DA plots were generated by the Soft Independent Modelling by Class Analogy (SIMCA) software. Concentration–response curves for DPPH inhibition were fitted using Graphpad Prism 6 software  to generate a sigmoidal curve. Nonlinear regression was performed to calculate the sensitivity of DPPH inhibition (IC50) by the OPWE. Group mean values were analysed by one-way ANOVA with post hoc analysis using Tukey’s test. All data are presented as mean ± SEM. Statistical significance was considered when While there are several factors, such as applied temperature, particle size, flowrate, and solvent system, known to affect the extraction efficiency of PHWE, the applied temperature was observed to affect the yield of each compound ,25. As sO-glucoside) at the higher temperatures. This could also explain why the yield difference between extraction at 100 °C and at 120 °C were comparable. While higher temperature would reduce the polarity of water, resulting in lower solubility of compounds, this was not the case for narirutin and p-coumaric acid, as their yields had increased with temperature; intermolecular forces such as hydrogen, van der Waals, and dipole–dipole bonds may have been reduced, resulting in lower activation energy required for the desorption of extractable compounds. Increased diffusivity due to increased water temperature, together with the reduction of water’s surface tension, may have also improved the desorption of the two compounds from the matrix of the OPW sample [The amount of hesperidin and narirutin determined were consistent with other studies at the same temperature, as these two flavones in orange peels were reported to be present in abundance in previous studies ,34. MostW sample ,36. FurtTo obtain the chromatographic chemical fingerprint and to reduce bias due to factors such as possible differences between sample replicates and instrument variability and sample peaks obtained, chromatographic peaks from LC/UV and LC/MS were normalized such that the value obtained for each compound at the respective temperature was expressed as a proportion of the total peak area at that condition. With reference to 50 concentration, which were significantly lower compared to PHWE extracts at 100 °C and 120 °C, indicating that the antioxidant activity is more potent at lower extraction temperature. Both 100 °C and methanolic extracts reported relatively similar IC50 concentrations, which are also significantly less potent that PHWE extracts at 60 °C and 80 °C (One well-reported biological activity of flavonoids is their potent antioxidant activity although it is unclear if the antioxidant activity of OPWE is affected by the different extraction method given the distinct chemical profile observed. Hence, we evaluated the antioxidant capacity of the OPWE from different PHWE temperatures and methanol extraction using two different antioxidant assays. Based on the DPPH and ATBS assays, OPWE using either PHWE or methanol extraction demonstrated antioxidant activity, which were consistent with earlier studies. However, in this study, we demonstrated that different PHWE temperatures exhibited different antioxidant efficacies. Specifically, the PHWE extracts at 60 °C and 80 °C reported comparable ICnd 80 °C A. Similand 80 °C B. Our rend 80 °C B. One poGpx1, in the TNF-α-induced endothelial cells (Sod1 and Nfe2l2 in the endothelial cells (Oxidative stress and inflammation are thought to be important causes of endothelial dysfunction, which is recognised as an important contributing factor to vascular complications in diabetes, reproductive diseases, and hypertension ,41,42,43al cells A. Co-treal cells A and notal cells D signifial cells B–F. Whilvcam-1 and icam-1, and inflammatory cytokine, Il1β, in the endothelial cells. In this study, TNF-α caused significant upregulation of the gene expression of vcam-1, icam-1, and Il1β in the endothelial cells (vcam-1, icam-1, and Il1β. Interestingly, only the 60 °C OPWE (vcam-1, icam-1, and Il1β in the endothelial cells. This suggests that only 60 °C OPWE but not 80 °C OPWE exhibited anti-inflammatory effects in the endothelial cells.To evaluate the effects of OPWE on TNF-α-induced endothelial dysfunction, we measured the gene expression of adhesion molecules, al cells . Neither °C OPWE A–C co-trEdn1 and Ptgs2 but had no effect on Nos3 expression, suggesting the upregulation of the vasoconstrictor Edn1 (Edn1 (Ptgs2 or Nos3 (Edn1, Ptgs2, and Nos3 in the endothelial cells (Endothelial cells are known to regulate vascular tone through the secretion of several vasoactive factors such as endothelin-1, prostanoids, and nitric oxide . Endothetor Edn1 . Co-trean1 (Edn1 A but not or Nos3 B,C. Howeal cells D–F. OverThe presence of oxidative stress and inflammation were reported to cause changes in the lipid profile, contributing to endothelial dysfunction. Therefore, to further characterize the potential vascular protective effects of 60 °C OPWE in the context of TNF-α-induced endothelial dysfunction, metabolite profiling of several lipid markers was carried out , Table 2OPW consists of a rich source of essential constituents that can be transformed into highly value-added bioproducts. Indeed, a plethora of studies have evaluated the potential to extract many different valuable bioactive compounds from OPW, including essential oils, pectin, and carotenoids. Specifically, other bioconversion technologies or additive manufacturing techniques have also been explored to convert OPW into biochemicals, biopolysaccharides, and bioenergy ,15,24,34


Macrotermes bellicosus at digesting lignocellulose is due to its gut bacterial symbionts. We report the metagenome-assembled genome sequence of Burkholderia cepacia UJ_SKK_1.2, reconstructed from metagenomes produced from Macrotermes bellicosus gut microbiota. The 7,460,271-bp genome obtained consists of 6,763 protein-coding sequences, with 6,719 functionally assigned genes and 59 RNA genes.The efficiency of the termite  Burkholderia species occupy diverse ecological niches , in February 2021 were cleaned 3 times (dipped in 70% ethanol for 3 min and rinsed with sterile water). To select only organisms with the characteristic of interest in this project (lignocellulose degradation), a termite gut was extracted in phosphate-buffered saline (PBS) and crushed; it was cultured on plated medium prepared from Kraft lignin, M9 salts, and agar-agar at a ratio of 2:1:2 at 37°C for 72 h and subcultured in four successions on the same medium. We thought that the colonies were pure isolates, but Gram staining revealed a mixture of cocci and rods appearing either in pairs, clusters, chains, or singly. To obtain cell pellets, the mixed organisms were grown overnight in nutrient broth at 37°C. Two sets of 2-mL portions were centrifuged at 14,000 × g for 3 min. The combined cell pellets were washed  and centrifuged at 14,000 × g for 3 min twice.N50 value of 270,795 bp, and a GC content of 67.7%. The BUSCO score was as follows: C:115 , F:0, M:9, n:124. The genome was annotated using PGAP v6.1  and Nextera index kit (Illumina), and the genomic DNA was fragmented using Illumina Nextera XT fragmentation enzyme. Combinatory dual indexes were added to each sample, followed by 12 cycles of PCR to construct libraries, which were purified using AMPure magnetic beads. The libraries were then eluted in Qiagen elution buffer (EB), quantified using a Qubit 4 fluorometer and Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit, and sequenced on an Illumina HiSeq X platform with 2 × 150-bp read lengths, producing 7.208 million raw reads. The raw metagenomic sequencing reads were trimmed and processed using Fastp v0.20.1 , with a GAP v6.1 , identifBurkholderia cepacia. Therefore, we named the organism Burkholderia cepacia strain UJ_SKK_1.2.The assembled contigs were processed through the CosmosID core genome single-nucleotide polymorphism (SNP) typing pipeline to evaluate the phylogenetic placement and SNP differences, using Parsnp  as the cJAMDXW000000000. The version described in this paper is JAMDXW010000000. The raw reads were deposited in the Sequence Read Archive under accession number SRR19200149.The genome data have been deposited at DDBJ/EMBL/GenBank under accession number


The mean age was 11.5 years (range: 9–15 years). There was a significant improvement in UCVA (p ≤ 0.001) and a decrease in the spherical equivalent measured with auto-refraction at 6 months (p ≤ 0.001). Total HOAs significantly increased after OK lens wear (p ≤ 0.001), with spherical aberration increasing approximately 3.9-fold (p = 0.05). Spherical aberration demonstrated statistically significant positive correlations with the change in spherical equivalent at 3 and 6 months . After wearing properly prescribed OK lens, all subjects had significantly improved UCVA and decreased myopic spherical equivalent, with increased total HOAs and positive spherical aberration at 1 month, and the changes were maintained at 6 months. Serially checked spherical aberration could evaluate the anti-myopia effect of the orthokeratology lens in children.We aimed to investigate the changes in higher-order aberrations (HOAs) after wearing orthokeratology (OK) lenses in myopic patients. The study included 15 eyes from ten myopic patients, whose refractive error was myopia less than −4.5 diopters (D) and astigmatism less than 1.5 D. Uncorrected visual acuity (UCVA) and best-corrected visual acuity (BCVA) were measured, and Zywave The orthokeratology (OK) lens is a contact lens used for correction of refractive errors such as myopia, hyperopia, and astigmatism by temporarily modifying the shape of the cornea ,2,3,4. TIn the present study, we attempted to investigate the changes in visual acuity, refraction, keratometric parameters, and HOAs, and to know whether the higher-order aberrations including spherical aberration were correlated with myopic improvement in children after 6 months of OK lens wear.Eleven myopic patients who commenced OK lens wear in the Bitsarang Eye Clinic between December 2014 and March 2015 were enrolled in this retrospective study. All subjects had less than 4.5 diopters (D) of myopia and astigmatism less than 1.5 D, with corrected visual acuity of 20/20. Excluding refractive error, no history of ocular disease, surgery, or trauma were noted among the study participants during medical record reviews. The study was approved by the hospital Ethics Committee and followed the tenets of the Declaration of Helsinki.® aberrometer , and corneal topography  was also performed on all subjects. All examinations were carried out at the initial visit and repeated at 1, 3, and 6 months following the commencement of OK lens wear.Uncorrected visual acuity (UCVA) and best-corrected visual acuity (BCVA) were measured on the logMAR scale. Keratometric readings (K) and spherical equivalent were obtained with an auto-refractometer . HOAs were measured with the Zywave® aberrometer. Zernike polynomials were used to quantitatively express the wavefront aberrations measured by the Zywave® aberrometer at 5 mm pupil size [Automated refractometry (KR-8800) was used to measure curvature (K) of the central 3 mm of the cornea. It acquired three consecutive K measurements automatically, and gave us an average of K for the analysis. For corneal topography, simulated keratometric readings (Sim K) and steep K and flat K of the central cornea with a radius of 5 mm were measured for the analysis. The pupil size was measured by the Zywavepil size ,15,16.Second order aberrations, third and fourth order HOAs, total HOAs (third and fourth order), and total aberrations were calculated for analysis. Defocus (Z200) and primary astigmatism (Z22) of second order aberrations, coma (Z31) and trefoil (Z330) of third order HOAs, and spherical aberration , secondary astigmatism (Z420+Z421) and quadrafoil (Z440+Z441) of fourth order HOAs were measured in root-mean-square  units ,15,16. Rp values < 0.05 were considered as statistically significant.SPSS software  was used for statistical analysis. Analysis of variance (ANOVA) and paired Wilcoxon signed-ranks test were performed to compare the changes in clinical parameters before and after the use of OK lenses. As Mauchly’s sphericity test was not satisfied, the Greenhouse–Geisser correction was performed. To investigate measurement differences at 1, 3, and 6 months compared with baseline, tests of contrasts were performed. Correlation analysis with Spearman’s correlation coefficient was performed to test for a relationship between the changes in aberrations and refractive power. Among 22 eyes of 11 subjects that commenced OK lens wear, 15 eyes from ten subjects were included in this study. In five eyes of five subjects, we failed to obtain reliable aberrometry measurements due to small pupils or poor cooperation. One subject did not present for re-examination during the follow-up period. The mean age of the subjects was 11.5 years (range: 9–15 years) and demographic characteristics are listed in ® Contact Lens  was used in 11 eyes and the LK® CH2 OK Lens  was used in four eyes. OK lens fitting was performed without difficulty and the lenses were well-tolerated in all subjects. A central bull’s eye pattern was confirmed in topographic examination at 1, 3, and 6 months after OK lens wear in all subjects.The Paragon CRTp ≤ 0.001). Mean spherical equivalent measured by auto-refractometry was −2.88 ± 0.68 D at baseline and decreased to −0.59 ± 0.36 D at 6 months (p ≤ 0.001), showing the greatest decrease at 3 months (p ≤ 0.001). Mean K measured by auto-refractometry decreased from 43.49 ± 1.03 D at baseline to 41.92 ± 1.34 D at 6 months (p ≤ 0.001), showing the greatest decrease at 3 months  was 0.96 ± 0.12 at baseline and improved to 0.01 ± 0.02 at 6 months . Astigmatism measured by the Sim K also decreased but was not statistically significant (p = 0.10). Both steep and flat K of the central 5 mm corneal radius significantly decreased , but astigmatism increased significantly at 1 month and was maintained at 6 months (p = 0.05). Moreover, the pupil size measured with the aberrometer did not show statistically significant changes at 1, 3, and 6 months . Total aberrations showed a significant decrease at all follow-up examinations  and the greatest decrease was at 3 months. Defocus (Z200) significantly decreased at all follow-ups . 0° astigmatism (Z220) showed no statistically significant change at 1 month (p = 0.35), but showed a significant decrease from the baseline at 3 months (p = 0.01) and the decrease was maintained at 6 months (p = 0.03). Astigmatism at 45° (Z221) had no statistically significant changes (p = 0.16). Vertical coma (Z311) significantly increased in the negative direction at 1 month (p ≤ 0.001) but decreased to an insignificant value at 3 months (p = 0.78), and the change was maintained at 6 months (p = 0.73). Horizontal coma (Z311) and trefoil (Z331) did not change significantly at any follow-up . SA (Z400) significantly increased at all follow-ups with the greatest increase at 6 months (p = 0.05). Secondary astigmatism (Z420+Z421) had no statistically significant change (p = 0.42). Quadrafoil (Z440+Z441) showed a significant change in the negative direction at 1 month (p = 0.005), but showed a positive effect change at 3 months (p = 0.01), then a negative change again at 6 months (p = 0.01), showing a significant change in negative direction overall (p = 0.03).p = 0.007 and p = 0.003, respectively). Other than SA, the remaining aberrations including total HOAs showed no statistically significant correlations with the correction of refractive error.SA (Z400) demonstrated statistically significant positive correlations with the change in spherical equivalent at 3 and 6 months , but 0° and 45° astigmatism were not measured separately . In our Horizontal coma (Z310) showed an effect change in the positive direction, but this was not statistically significant. Conversely, vertical coma Z311) had a significant change in the negative direction from −0.002 ± 0.153 µm at baseline to −0.463 ± 0.351 µm at 1 month. However, this changed to −0.086 ± 0.244 µm at 3 months, showing no significant difference from the baseline, and was maintained at 6 months. Gifford et al.  reported had a siOf the other HOAs, Stillitano et al. noted that trefoil (Z331) changed in the positive direction at 30 days and then stabilized, and quadrafoil (Z440+Z441) changed in the negative direction at 30 days but re-calibrated to a similar value as the baseline at 3 months . In our In this study, total HOAs significantly increased from 0.231 ± 0.089 µm at baseline to 0.654 ± 0.219 µm at 6 months after the commencement of OK lens wear. By contrast, total aberrations including lower-order aberrations decreased from 3.165 ± 0.914 µm at baseline to 1.623 ± 0.707 µm at 6 months. These changes in aberrations provide some empirical corroboration for the theoretical basis of OK to improve vision in myopic patients. However, no aberrations measured in this study showed significant correlation with the amount of correction in refractive error following the use of OK lenses, except for the changes in SA (Z400) at 3 and 6 months. Hiraoka et al. reported that decentration of an OK lens was related to an increase in coma (Z31) and SA (Z400) as well as a reduction in contrast sensitivity [Subjects had significant improvements in UCVA and myopic spherical equivalent with properly prescribed OK lenses, and showed increases in total HOAs and SA (Z400), with decreases in defocus (Z200) and 0° (Z220) astigmatism at 1 month. The changes were maintained at 6 months. Vertical coma (Z311) and quadrafoil (Z440+Z441) significantly increased in negative directions at 1 month, but decreased at 3 months. The changes observed in 45° astigmatism (Z221), horizontal coma (Z310), trefoil (Z331), and secondary astigmatism (Z420+Z421) were not statistically significant. Moreover, it was noteworthy that SA (Z400) demonstrated statistically significant positive correlations with the change in spherical equivalent at 3 and 6 months, which showed the feasibility of serially checked SAs to evaluate the anti-myopia effect of the orthokeratology lens in children. This study has several limitations. First, concerning measurement of wavefront and HOAs, the constants of Zernike polynomials vary with the size of the pupils, making pupil size an important factor ,23. HoweAfter wearing properly prescribed OK lens, all subjects had significantly improved UCVA and decreased myopic spherical equivalent, with increased total HOAs and positive spherical aberration at 1 month, and the changes were maintained at 6 months. Serially checked spherical aberration could evaluate the anti-myopia effect of the OK lens in children.


Atmospheric aerosols are important drivers of Arcticclimate changethrough aerosol–cloud–climate interactions. However,large uncertainties remain on the sources and processes controllingparticle numbers in both fine and coarse modes. Here, we applied areceptor model and an explainable machine learning technique to understandthe sources and drivers of particle numbers from 10 nm to 20 μmin Svalbard. Nucleation, biogenic, secondary, anthropogenic, mineraldust, sea salt and blowing snow aerosols and their major environmentaldrivers were identified. Our results show that the monthly variationsin particles are highly size/source dependent and regulated by meteorology.Secondary and nucleation aerosols are the largest contributors topotential cloud condensation nuclei  in the Arctic. Nonlinear responses totemperature were found for biogenic, local dust particles and potentialCCN, highlighting the importance of melting sea ice and snow. Theseresults indicate that the aerosol factors will respond to rapid Arcticwarming differently and in a nonlinear fashion. Responses of high-latitude particle numberconcentrationsto environmental drivers are nonlinear and source and process dependent. Onlyrecently, a study has investigated particle volume size distributions in the Arctic and linkedthem to coarse-mode anthropogenic, marine and mineral dust particles.5 However, the contributions from various sourcesto total particle number concentration (PN) are still not clear, leadingto high uncertainties in quantifying their impacts on the regionalclimate. Understanding PNSD/PVSD from nucleation mode to coarse modecan shed new light on aerosol sources and processes in the Arctic.The Arctic is a uniqueregion undergoing tremendous environmentalchanges at a much faster pace than the global average.k-Means cluster analysis is widely used to semi-quantitativelyinterpret PNSD/PVSD on the basis of occurrence frequency.8 However, this method could not apportion aerosols to specific sourcesbecause each cluster includes multiple sources.5 Positive matrix factorization (PMF) can quantitativelyseparate source-related factors for both number and volume/mass concentrations.12 While there are a few studies of source apportionment of Arcticaerosols in mass,12 no studies to date have quantifiedsource contributions to PN, which is crucial for cloud formation.Furthermore, previous studies suggested that annual cycles of Arcticaerosols with different diameters are governed by different processes.13 The limited knowledge on different processes controlling size-resolved/source-specificaerosols means that climate models are still unable to realisticallyreproduce processes in the rapidly changing Arctic.15 which is an explainable machine learning technique with PMF at the Gruvebadet Observatory in Svalbard in 2015. Environmentalfactors driving source-specific aerosol processes were then exploredusing the SHapley Additive exPlanation (SHAP) approach,22.116 The geographic location ofGVB and the dominant winds ensure minimal anthropogenic contaminationfrom local emissions while also capturing long-range transported pollutionair masses.16 Aerosol size distributions weremeasured by a scanning mobility particle sizer  and an aerodynamic particle sizer. Accordingto the specification sheets, typical single-channel uncertainty inaerosol number concentration measured by the SMPS and APS are ±20%and ±10%, respectively. The SMPS and APS are attached to thesame inlet, which follows EUSAAR-ACTRIS protocol and is positionedabout 4 m above the ground.17 The two instrumentswork with a time resolution of 10 min. Data are averaged over 1 hperiod and then reported at an hourly time resolution. In the presentstudy, hourly data are averaged into daily data. The percentages ofvalid data for daily SMPS and APS from March to October 2015 are 81.2and 93.9% (Table S1), respectively.Aerosolsampling and measurements were performed from March to September 2015at the Gruvebadet (GVB) Observatory , an Italian station located at about 800 m south-westfrom the town of Ny-Ålesund in the Svalbard archipelago. Marchto September is the operating period of the station. Lack of datain winter months is due to less maintenance of the instruments duringthe polar-night period. In the north-eastern direction toward theNy-Ålesund research village, a clean area was established andmotorized activity and other potentially contaminant activities wereforbidden.10 filter samples were collected, using a TecoraSkyPost sequential sampler equipped with a PM10 samplinghead, operating following the EN 12341 European protocol. The percentagesof valid data for ions and metals from daily filter samples are 90.2and 89.8% (Table S1), respectively. Inorganicanions  andcations  and selected organic anions[methanesulfonate (MSA) and oxalate] were analyzed using a three-Dionexion chromatography system equipped with electrochemical-suppressedconductivity detectors.18 Analytic uncertaintyis typically below 5%. The concentrations of metals  were determined byan ELEMENT2  inductivelycoupled plasma mass spectrometry (ICP–MS) instrument, a double-focusingmagnetic sector field (SF) ICP-SF-MS coupled with a desolvation systemprovided with a microflow nebulizer.19Daily PM20 Surfacenet solar radiation, total cloud cover, total precipitation, snowfall,and boundary layer height were obtained from the Copernicus ClimateChange Service (C3S), available at: https://cds.climate.copernicus.eu/cdsapp#!/dataset/reanalysis-era5-single-levels (last accesses: September 2021).Meteorological parameters, including wind speed, wind direction,relative humidity, and ambient temperature, were recorded hourly ata height of 10 m (a.g.l.) on the Amundsen-Nobile Climate Change Towerin the neighborhood of the site.2.2+ and Ca2+ were each apportioned to a sea salt (ss)fractionand a non-sea-salt (nss) fraction.5 Inaddition, SO42– was apportioned to seasalt (ss-SO42–), mineral dust , biogenic (bio-SO42–), and anthropogenic fractions (anthr-SO42–) following previous studies.16 The particle volumeconcentrations (dV) at each size bin from SMPS andAPS and mass concentrations of chemical species  were combined in a concentrationmatrix for the PMF model. To obtain an optimal solution with physicalmeaning, we applied relatively low uncertainties to chemical componentsbut high uncertainties to SMPS and APS to make sure that factors resolvedby the model were well separated mainly based on chemical composition. Details about settings for the PMF modelingand its limitation can be found in the Supporting Information, Text S1.Source apportionmentof the daily chemical composition data from filter samples complementedby SMPS and APS measurements was performed using the U.S. EnvironmentalProtection Agency (US-EPA) positive matrix factorization (PMF) 5.0.Size distributions from SMPS and APS were not merged due to the absenceof overlapping size bins from the two instruments, so the data withdiameters <500 nm were mobility diameters and those with diameters>500 nm were aerodynamic diameters. Before PMF modeling, Na2.321 wasused to calculate 7 day22 hourly backward trajectoriesarriving at an altitude of 100 m (a.g.l.) at the GVB from March toOctober 2015. Meteorological data (.gbl) from the National Centersfor Environmental Prediction (NCEP) and the National Center for AtmosphericResearch (NCAR) reanalysis data set were used to run the HYSPLIT model.Trajectories were clustered into six clusters based on an angle-baseddistance matrix, which is provide by “trajCluster” functionin the “Openair” package in R.23 The back trajectories are divided into above or below the mixinglayer height . Following a previousstudy,5 daily surface types in the NorthernHemisphere are temporally allocated with the back trajectories. Eachdata point from the hourly back trajectories is labeled as (i) sea,(ii) sea ice, (iii) snow, (iv) land, or (v) above ML. In the presentstudy, relative fractions of the surface types  were calculated disregarding periods that the airmass spent above ML.The Hybrid Single Particle Lagrangian IntegratedTrajectory (HYSPLIT 5.0.0) modelN is the total number of trajectories, ck is the concentration measuredupon arrival of trajectory k, and τijk is the residence time of trajectory k in grid cell . A high valueof i,j) could, on average, cause highconcentrations at the sampling site. The CWT analysis was performedusing the “trajLevel” function in the “Openair”package in R.23To investigate potential source regionsleading to total particle concentration from each resolved source,the back trajectories were gridded to 1 ° × 1 ° gridcells and linked to particle concentrations by the following equation2.424 Here, webuilt RF models using “RandomForestRegressor” functionprovided by “scikit-learn”25 in a python environment. PNs of the PMF-resolved factors are independentvariables for the RF models. Explanatory variables include ambienttemperature, boundary layer height, relative humidity, surface pressure,total cloud cover, snowfall, surface net solar radiation, total precipitation,wind speed, wind direction, air mass clusters, and relative fractionof the accumulated time for surface types (height below the mixinglayer), that is, the time a back-trajectory air parcel spends oversea, sea ice, snow, and land. The hyper-parameters for the RF modelwere tuned using a function of “GridSearchCV” from the“scikit-learn” library. More details about the RF modeland selection of the hyper-parameters are shown in the SupportingInformation, Text S2. To investigate howspecific processes drive the different aerosol factors, the SHAP approachwas applied to interpret the RF models.15 SHAP is a game theoretic approach that is able to fairly distributethe anomaly of the total concentration among different parameters14xi,j is the value of feature j for sample i. K is the total numberof different parameters. f is the SHAP value offeature xi,j, indicating the contribution of xi,j to yi. The average value of the model predictions on allthe samples is ybase,baseline of the model. If f > 0  < 0), it means that the value of parameter j on sample i can increase (decrease) the aerosolconcentration on sample i relative to the base value.The higher value of |f| represents a higher impact/importanceof xi,j on the corresponding measured aerosol concentration. The SHAP approachwas implemented by the “shap” python package.14Random forest (RF) modeling has been applied widely to reproduceair pollutant concentrations.33.1Table S1) ofparticles from 10 nm to 20 μm, though missing diameter rangeof 470–542 nm due to measurement limitation (Text S3), allows us to understand their monthly variations(The good data coverage (riations1. Enhanc–3) and at ∼170nm (321 ± 198 cm–3), respectively. However,particle volume (dV/dlogDp) exhibited a bimodal distribution in boththe accumulation mode at ∼250 nm (1.4 ± 0.9 μm3 cm–3 in March and 1.5 ± 0.8 μm3 cm–3 in April) and the coarse mode at ∼3μm (6.0 ± 4.6 μm3 cm–3 in March and 3.9 ± 2.4 μm3 cm–3 in April). In May, a bimodal pattern was observed for both PNSDand PVSD. For PNSD, in addition to a peak in the accumulation modeat ∼160 nm with 218 ± 56 cm–3, anothermuch larger peak was found in the nucleation mode at ∼20 nmwith 753 ± 618 cm–3, attributable to localNPF events6 with concurrent enhanced biogenicaerosols  in May.27PNSD in March and April showed accumulation-modepeaks (dN/dlogDp)at ∼200 nm (262 ± 194 cm–3) in June to ∼40 nm(420 ± 374 cm–3) in August, with July (367± 326 cm–3 at ∼25 nm to 388 ± 309cm–3 at ∼40 nm) in the middle showing a transitionpattern with double peaks at ∼25 nm and ∼40 nm. PNsin September and October are lower but PVs—peaking in the coarsemode at ∼3 μm (5.5 ± 6.1 μm3 cm–3 in September and 4.9 ± 4.7 μm3 cm–3 in October)—were higher than the othermonths. On average, the coarse-model aerosols contributed ∼0.3%to particle number but ∼81.7% to particle volume concentrations.PVSD during June–August exhibited similar features;however,higher concentrations in the coarse mode of 10–20 μmwere observed in June, likely due to dust-relevant spikes during June11–13, 2015. For PNSD, there is a shift in the peak from ∼25nm  as the optimal fit toboth the chemical and PNSD/PVSD data . The PMF model successfully reproduced chemicalspecies (Table S3) and total particle volume/numberconcentrations from SMPS and APS measurements, with high R2, and the scaled residuals are within the range of ±1for all size bins . We identifiedthe well-separated  factorsas nucleation, biogenic, secondary, trace metals, anthropogenic, mineraldust , sea salt, andblowing snow aerosols  than at GVB .Data S1). Monthly variation of PN10–25 nm and PN25–100nm was dominatedby the nucleation factor  with monthly average contributions of 91.3 ± 6.4%(78.8% in March—97.6% in July) and 46.3 ± 20.0% (16.0%in March—70.1% in May), respectively. At least some particlessmaller than 30 nm in diameter can be activated as CCN in the Arctic;29 therefore, nucleation has a high potential toinfluence cloud formation in the summertime Arctic. Secondary aerosolsmade a more significant contribution (28.8 ± 9.4%) to PV25–100 nm than the nucleation factor (13.9 ±9.1%), as secondary aerosols dominated the larger ultrafine particlebins (62.6–100 nm)  and PV100nm–1μm . In May, biogenic aerosol was themain source with average contributions of 66.0% to PN100nm–1μm and 67.1% to PV100nm–1μm, primarily dueto high biogenic emissions, such as high dimethylsulfide (DMS) concentrationsobserved in Svalbard in May 2015.27 However,secondary aerosols generally dominated PN100nm–1μm (52.2 ± 14.2%) and PV100nm–1μm (32.4± 13.9%) during June–October. These results demonstratethe complexity of the sources and processes influencing the accumulation-modeaerosol.Monthly variation of PNfactors2, includ1–20μm (41.8 ± 9.3%) and PN1–20μm (49.9 ± 9.6%) in almost all seasons,which is consistent withthe occurrence frequency-based results.5 Higher sea-salt concentrations were observed in spring/autumn thanin summer . The peakof sea salt concentration in September is likely due to the higherfraction of air mass traveling over open ocean compared to the othermonths . However, the secondpeak in March is more difficult to explain. A possible source is frompack ice30 and snow,31 considering the high wind speeds associated with air massestraveling over sea ice and snow .For coarse-mode particles, sea salt aerosol dominatedboth PV10nm–20μm, respectively.Although sea salt and blowing snow contributed notably to super-micrometerparticles in both number and volume , theycontributed very little to sub-micrometer and total particle number and 32.0 ± 9.8% (18.6% in May—39.5%in July) to the overall PVe number2. These 10nm–20μm, which is comparable to 6.9 ±5.5% based on an occurrence frequency-based study.5 Nucleation makes a major contribution  to overall PN10nm–20μm. Compared to nucleation, the contributions from the other factorsto PN10nm–20μm are much smaller: 21.9 ±10.1% from secondary, 7.5 ± 5.1% from biogenic, 6.1 ± 4.1%from mineral dust , 6.1 ± 10.6% from anthropogenic, 3.2± 3.3% from blowing snow, 2.2 ± 1.5% from trace metal factor,and 1.0 ± 0.9% from sea salt. The sources of the trace metalfactor are not clear , but it contributed only 1.8 ± 0.8% to PV10nm–20μm and 2.2 ± 1.5% to PN10nm–20μm.Mineral dust  contributed7.8 ± 4.2% (2.8% in August—15.0% in June) to the overallPV3.3Figure S9. The anthropogenicfactor appears to be associated with air masses arriving from northernEurasia, particularly during the Arctic haze period . Snow-covered regions  were potential source regions for both sea salt and blowingsnow in spring and autumn, which is consistent with the enhanced seasalt and blowing snow aerosols in these months . Dust particles in Svalbard are associated withair masses  from (i) northernEurasia in spring, (ii) northern Alaska in summer, (iii) the ArcticArchipelago and coastal regions of Greenland in autumn, and (iv) localsources , which have been identified as potential highlatitude dust source regions.33Potential source regions of the aerosol factors in spring (includingMarch–May), summer (including June–August), and autumn seasonsare shown by CWT in Figure S9), with the former linked toopen ocean  and the latter to Arctic continental regions. Unlike the above aerosol factors,high CWT values for nucleation and biogenic aerosols were found inthe High Arctic and marginal ice zone in summer and spring (primarily in May), respectively, implying enhanced emissionor formation from sea ice.44 This is consistentwith sea ice being an important source of alkylamines and the precursorsof sulfur and iodine oxoacids, all of which can efficiently participatein NPF processes.35 However, the High Arctic and marginal ice zone did not contributemuch to nucleation aerosol in spring and autumn , implying that sea ice exposure alone is not sufficientto promote the nucleation processes.Thepotential source-region map for secondary aerosol shows that the peakin summer and that in spring are related to different source regions; therefore,the responses of each of the meteorological parameters on every predictionfor the nine aerosol factors were quantitatively investigated (S12–S14) by the SHAP approach. None ofthe meteorological parameters have the same impacts on all the aerosolfactors, further highlighting the importance of breakdown of totalaerosol into different aerosol factors.Understanding potential drivers of the aerosol factors rather thantotal aerosol is crucial as each aerosol factor responds to changesin environmental processes differently. The relationships betweeneach aerosol factor identified in this work and meteorological/environmentalvariable are shown in Figure S12), consistentwith their key roles in photochemistry.37 Temperature-dependentgas precursor emissions and high-temperature days coincident withhigh solar radiation may potentially explain the positive responseof nucleation aerosol to temperature. A sea ice trajectory fractionof 0 was observed as an activation threshold for nucleation aerosol(38 Both enhanced sea ice exposureand high solar radiation appear to be key drivers of nucleation aerosolnumber (S12 and S14). Boundary layer height and windspeed also appear to be significant drivers for nucleation aerosol,though with a negative response (S13 and S14). Enhancement of nucleation aerosol in July may be associated withlow boundary layer height and low wind speed , indicating the importance of local NPF.This is consistent with results from the nearby Zeppelin, Svalbard.44 Overall, solar radiation, boundary layer height,and wind speed are the main drivers governing the monthly variationin nucleation aerosol .For nucleation aerosol, the most important parameterwith positiveresponses is solar radiation, followed by temperature and sea iceexposure  S12, cons27 and aerosol precursor emissions), surface pressure, and temperatureare the most important parameters  responsible for the monthly variation in biogenic aerosol . It is known that MSA concentrationsare related to temperature both directly through DMS oxidation40 and indirectly through emissions from melting sea ice.26 Our results show that temperature influencesthe monthly variation in biogenic aerosol with a nonlinear response. The first and second peaksin the monthly variation  ofsecondary aerosol were mainly attributed to low surface pressure  in April and high temperature  during July–August, respectively .The monthly variation in secondary aerosol was mainly governedby temperature, boundary layer height, and surface pressure (S12–S14). More air masses traveling overopen ocean and continental regions during August–October comparedto the other months  may haveled to enhanced contributions from both natural and anthropogenic sources.The drivers of the trace metal factorare more difficult to determine. Low humidity and high wind speed facilitateddust emissions (S12 and S13). The differences betweenthe two types of mineral dust are: (i) high temperature-driven enhancementof mineral dust_1 in June and July  and (ii) more contributions from mineral dust_1 to particles largerthan 10 μm relative to mineral dust_2 . This isbecause meteorological conditions during cold seasons are conduciveto long-range transport of anthropogenic pollution from northern Eurasia, leading to Arctic haze.4 An inflection point at ∼0 °C wasobserved  of sea salt and blowingsnow factors. However, wind speed and air masses traveling over openocean were more important for the sea salt factor than for the blowingsnow factor . In addition,large snowfall could facilitate blowing snow aerosol .Boundary layer height andair masses traveling over sea ice andsnow were the common parameters controlling the monthly variations are similar to those for nucleationaerosol and sea salt aerosol, respectively.As expected, the drivers fortotal aerosol in number and volume(3.5Data S2) for the nine aerosol sources will beuseful for future source apportionment studies at the high latitude.Sea salt and blowing snow aerosols contributed substantially to totalparticles in mass/volume; however, their contributions to sub-micrometerand total particles in number are small. Since particle numbers withdiameter greater than 40 nm are highly correlated to number of CCNin the Arctic,41 we presented results for PN40nm–20μm in 40nm–20μm as a proxy) withmonthly average contributions of 40.4 ± 13.1 and 20.2 ±12.8%, respectively. Considering recent evidence that NPF-derivedparticles can be activated as CCN smaller than 30 nm,29 the potential contribution from nucleation aerosol is probablyeven greater.This study has quantifiedsources of both particle number and volume from 10 nm to 20 μmin Svalbard, the first source apportionment study of this kind atthe high latitude. A range of aerosol factors were identified, includingnucleation, secondary, trace metal, biogenic, anthropogenic, sea salt,and blowing snow aerosols, though they can be overlapped. 1Nucleation: with theshift from a polar to a marine environment and change in Arctic cloudproperties, the response of nucleation aerosol to Arctic warming ishighly uncertain. An increase in frequency of nucleation events wasfound to be correlated with decrease in sea ice extent, which mightbe related to enhanced organic nitrogen/amine emissions from meltingsea ice regions.35 However, the much greater impactof solar radiation on nucleation than sea ice exposure (S14) should be considered for better understanding the direct and indirectresponses of nucleation to sea ice melting.exposure 3 and S142Biogenic and secondary aerosol: Our results suggest that biogenic aerosol is likely to increaseas a result of sea ice melting in a warming Arctic but decreases whenthe average temperature is above ∼0 °C. Such a nonlinearresponse to temperature may explain the inter-annual variation inbiogenic aerosol42 and affect nucleationprocesses43 because the biogenic factoris a potential source of nucleation aerosols . Boundary layer height, temperature,and air mass cluster are the key parameters , and sea ice is an important source region for potentialCCN (rameters S14. BounBy an explainable machine learning technique,we showed a highly nonlinear and variable response of aerosols ofdifferent origins to environmental parameters and thus ongoing Arcticwarming. In particular:12 Our findings pointto the key aerosol processes to focus on when quantifying the sensitivityof aerosol sources and responses to environmental drivers.In summary, this study presents a framework of a receptormodelcoupled with machine learning to understand sources and drivers ofwide-range particles at a high Arctic site, and the results show thateach aerosol factor will be affected by environmental change differently,highlighting the complexity of Arctic aerosol. Further measurementsin winter seasons and at other Arctic sites will be needed to developan Arctic-wide understanding.


Pull-down assays showed Myo10 binding to α5 but not the β1 subunit, αvβ3, and αvβ5 integrins. Several of these integrins colocalized in nascent adhesions in the filopodial tip and shaft. Using conformation-specific antibodies, we found that β1 integrin, but not α5 or αvβ3 integrins, were activated following 1-h mechanical stretch. Cadherin -11 (CDH11), a cell adhesion molecule, did not bind to Myo10, but was associated with filopodia. Interestingly, CDH11 was downregulated on the TM cell surface following 1-h mechanical stretch. In glaucoma cells, CDH11 protein levels were increased. Finally, mechanical stretch caused a small, yet significant increase in Myo10 protein levels in glaucoma cells, but did not affect cellular communication of fluorescent vesicles via filopodia-like tunneling nanotubes. Together, these data suggest that TM cell adhesion proteins, β1 integrin and CDH11, have relatively rapid responses to mechanical stretch, which suggests a central role in sensing changes in IOP elevation in situ.The trabecular meshwork (TM) is the tissue responsible for regulating aqueous humor fluid egress from the anterior eye. If drainage is impaired, intraocular pressure (IOP) becomes elevated, which is a primary risk factor for primary open angle glaucoma. TM cells sense elevated IOP via changes in their biomechanical environment. Filopodia cellular protrusions and integrin transmembrane proteins may play roles in detecting IOP elevation, yet this has not been studied in detail in the TM. Here, we investigate integrins and filopodial proteins, such as myosin-X (Myo10), in response to mechanical stretch, an  Since tormation . Myo10 pormation . This imFilopodia and integrins are mechanosensors likely involved in sensing changes to the TM biomechanical environment caused by elevated IOP. Therefore, we investigated the effects of mechanical stretch on filopodia-associated proteins, Myo10 protein interactions, and cellular communication via TNTs in normal and glaucomatous TM cells.Normal (NTM) and glaucoma (GTM) cells were cultured from TM tissue dissected from human cadaver eyes  using established techniques . Donor i2 overnight at 4°C. The tubes were then placed in a magnetic holder so Myo10 antibody-coated magnetic beads remained in the tube while they were washed thoroughly with PBST. After the final wash, 1x SDS-PAGE sample buffer was added to the tube, samples were boiled, and immunoprecipitated proteins were separated by SDS-PAGE. Following transfer, nitrocellulose membranes were probed with either integrin β1, α5, αvβ3, αvβ5, or CDH11 primary antibodies . Following washing with phosphate buffered saline with 0.05% Tween (PBST) and blocking with bovine serum albumin, the Myo10-antibody beads were incubated with TM RIPA lysates with 10 mM MgCltibodies . Secondain situ  and 10 µM verapamil, which increases fluorescent signal of SiR-actin, was added to the cultures . SiliconRIPA lysates were harvested on ice from confluent NTM and GTM cells, or from TM cells that were mechanically stretched for 24 h. A 24 h time point was used so that changes in protein levels could accumulate. After harvesting, protease cocktail inhibitor was added to the RIPA lysates, proteins were separated by SDS-PAGE and Western immunoblots were performed with the indicated primary antibodies . Total E5 cells/ml on FlexCell membranes. Cells were allowed to adhere for 2 h and then the membranes with attached cells were mechanically stretched for 24 h. Cells were fixed and immunostained with rat monoclonal anti-CD44, clone IM-7  and Alexa-fluor 594-conjugated donkey anti-rat secondary antibody (ThermoFisher) to visualize the cell membrane. Confocal z-stacked images were acquired and the total number of DiO (green) and DiD (red)-labeled cells in each image were counted. The number of TM cells containing at least five vesicles of the opposite color (transferred vesicles) was then made a percentage of total cell number, as described previously  and technical (>n = 3) replicates were performed for all the experiments. The n’s are reported in the figure legends. Data are reported as mean ± standard error. One-way ANOVA with a post-hoc Tukey test, or a paired Students t-test, were performed. Significance was set at p < 0.05.Biological (>Previous studies indicated that β1 integrins and α5 integrins are transported along filopodia by Myo10 . Here, wTo determine if Myo10 and these integrins interacted, we performed pull-down assays using a Myo10 antibody. As shown in in vitro method to mimic elevated IOP (Mechanical stretch is an ated IOP . Integriated IOP , but thiated IOP . To deteated IOP . There wated IOP . This inIn addition to integrins, we investigated the distribution of filopodial proteins Eps8 and mDia2 in non-stretched vs. stretched TM cells. In non-stretched TM cells, mDia2 was localized to the base, shaft, and tip of filopodia , while ECDH11 is a cell adhesion protein, which is highly expressed in TM cells . The filCDH11 is up-regulated in diseases associated with excess ECM deposition such as invasive breast cancer, scleroderma, and liver fibrosis . GlaucomWe also investigated whether mechanical stretch affected Myo10 protein levels. Consistent with our previous observations , Myo10 lIn this study, we show that mechanical stretch, a proxy for elevated IOP, affects the composition and distribution of proteins in TM cell filopodia and vesicle transfer in TNTs. Specifically, we show that TM filopodia contain Myo10, integrins α5β1 and αvβ3, and to some extent αvβ5 integrin, and CDH11, Eps8, and mDia2. Mechanical stretch specifically activated β1 integrin within 1 h, as shown with conformation-specific monoclonal antibodies, suggesting that certain integrins become activated as a rapid response to the stretch and distortion caused by elevated IOP. In addition, mechanical stretch increased the number of CDH11-negative TM cells suggesting that other cell surface adhesion molecules are also affected. In contrast, mechanical stretch had no effect on vesicle transfer via TNTs in either NTM or GTM. Together, these data suggest that filopodial-associated and cell-surface proteins are dynamically altered in response to their biomechanical environment, but these mechanical cues do not significantly impact cellular communication via TNTs. In addition, it provides additional evidence that the function of TNTs and filopodia are different, as previously suggested .Since only heterodimer integrins, not individual subunits, are expressed on the cell surface, and the α5 subunit only pairs with the β1 subunit, localization of the α5 subunit must indicate the presence of the α5β1 integrin heterodimer in filopodia . LocalizUsing conformation-specific antibodies , we showIn this study, we show that Myo10 may play a role in transporting α5β1 integrins to the filopodial tip, as suggested in several cell types . The disThe pull-down assays also showed positive interactions of Myo10 with αvβ3 and αvβ5 integrins. These data expand the repertoire of integrins that bind to Myo10. αvβ3 integrin is involved in phagocytosis, TGFβ signaling, and is activated by dexamethasone in TM cells . In addiCDH11 was found associated with filopodia/TNTs connecting TM cells. However, CDH11 immunostaining was not specifically localized to nascent adhesions of the filopodia shaft or tips, but instead, CDH11 was detected along the entire length of the filopodia. Recently, CDH11 was found to localize to focal adhesions, where it binds fibronectin via an interaction with syndecan-4 . CDH11 iMyo10 protein levels had small, but significant increases in mechanically stretched GTM cells. During normal filopodia dynamics, Myo10 travels towards the tip of the filopodia during actin assembly and growth, and displays rearward movement as the filopodia is retracted to the cell . BecauseIn summary, we have shown that Myo10 binds to a wider range of integrins than previously reported. In addition, mechanical stretch specifically and rapidly activates β1 integrin, while reducing CDH11 on TM cell surface. These data provide novel information regarding the cellular structures and transmembrane adhesive proteins that TM cells use to sense changes to the biomechanical environment such as those changes caused by elevated IOP.