Juglans regia Chandler and Howard) were evaluated. Free fatty acids, peroxides, and oxidative stabilities exhibited significant changes. After the storage period, γ-, δ-, and α-tocopherols in Howard oil significantly reduced by 42, 56, and 100% at 5 °C, while 23 °C showed 48, 42, and 100% losses, respectively. For Chandler oil, storage at 5 °C reduced γ-, δ-, and α-tocopherols by 19, 24, and 100%, while 23 °C caused 42, 45, and 100% losses, respectively. Storage of Howard kernels, up to month four, significantly reduced total phenolics by 9 and 18%, at 23 and 5 °C, respectively, whereas Chandler also reduced by 9 and 27%, at 23 and 5 °C, respectively. Additionally, 14 phenolic compounds were profiled in kernels, where flavonoids were dominant than phenolic acids. At the end of month four, the dominant phenolic compound was gallic acid at 23 °C . Additionally, positive correlations were observed between rancid sensory perceptions and oxidative volatiles. Storage conditions are crucial for maintaining the sensory and nutritional attributes of walnuts during postharvest management.The impact of storage temperature and time on quality of two walnut cultivars ( Juglans regia L.) has increased from 2.96 million to 3.32 million metric tonnes from 2017 to 2020, with China, United States, and Iran being the key producers [Global production of walnut  respond to storage conditions for quality deterioration is limited. Thus, this study investigated the impact of different storage factors  on the accumulation of chemical oxidative markers in different fractions (kernel and oil) of two popularly grown California walnut cultivars Juglans regia—‘Chandler’ and ‘Howard’) were harvested at full maturity stage in September; we obtained them from a grower and processor in California.Fresh kernels of two walnut cultivars  was stored in opened transparent containers inside a temperature-controlled chamber at 50% RH under florescent light. Samples were stored at 5 and 23 °C for a total of 4 months. At the end of each storage month, kernels (1 kg) from each temperature condition were collected for chemical and sensory analyses. Kernel oil extraction was conducted within 3 h after each sampling period.®; East Windsor, NJ, USA), transferred into a 600 mL beaker, and then dried in an oven at 105 °C, until a constant weight was reached. Afterwards, dried samples were kept in a desiccator to reach room temperature and then reweighed.Moisture content was evaluated as described by Grilo and Wang . ApproxiOil from dried kernels was extracted according to the method of Grilo and Wang . Walnut 232 and K268), oxidative stability, and free fatty acids were estimated as described by Grilo and Wang [232, and K268, respectively. Oxidative stabilities of kernel and oil were measured with a Rancimat apparatus 617 . In summary, 3 g of oil and 0.5 g of milled kernel samples were oxidized at 110 °C, with 20 l/h of airflow.Changes in peroxide value (PV), UV absorbances . Subsequently, the obtained oil mixture was stored at −20 °C for 1 h to allow for separation of oil from the organic phase of the mixture. After this, the organic extract was filtered with 0.45 µm (nylon) and analyzed using UPLC-DAD. Agilent 1290 Infinity II LC system with a diode-array detector and Agilent ZORBAX Eclipse Plus C18 column  were used to conduct the analysis, with a mobile phase of methanol:water (96:4), injection volume of 20 µL, and flow rate of 1.0 mL/min. The DAD signal was recorded at 292 nm, after each run time of 12 min. Identification of tocopherols were conducted by comparing their retention time to their respective pure standards .Tocopherols were extracted as outlined by Gimeno et al.  and modiv/v/v), vortexed (2 min), sonicated (5 min), and centrifuged  subsequently. Obtained supernatant was stored in the dark, and re-extraction from pellets was repeated twice. Afterwards, all three obtained supernatants were combined, and methanol was evaporated with a vacuum evaporator at 30 °C. Dried extracts were redissolved in 1 mL methanol prior to analysis. TPC was quantified by the Folin–Ciocalteu method, using a gallic acid calibration curve (ranges 10.1–504.6 µg/mL) [Total phenolic content (TPC) of investigated kernels was evaluated according to the method of Ojeda-Amador et al. . Briefly6 µg/mL) . Resultsv/v) formic acid. The column was an Eclipse Plus C18 column , with a security guard ultra C18 guard column (4.6 mm × 2 mm) of the same material. The gradient included: isocratic at 2% B for 10 min, then 2 to 37% B in 27 min, isocratic at 37% B for 5 min, then from 37 to 40% B in 18 min, from 40 to 60% B in 10 min, from 60 to 100% B in 20 min, isocratic at 100% B for 14 min, then from 100 to 2% B in 1 min, and isocratic at 2% B for 7 min. Column temperature was 35 °C, and the total run time was 112 min at a flow rate of 1 mL/min and injection volume of 3 µL. The UV/vis absorption spectra were recorded at 280 nm , 310 nm (anacardic acids), and 350 nm (flavonols). Results were expressed as mg/kg of kernel or oil.Redissolved phenolic extracts were membrane-filtered with 0.45 µm cellulose filter before HPLC-DAD analysis, as described by Ersan et al. . HPLC-DAVolatile compounds from kernel and oil were extracted by solid-phase microextraction (SPME) . BrieflyBoth walnut kernel and oil samples obtained from each storage condition were evaluated using the objective sensory method of descriptive analysis. The panelists selected for this project had many years of experience using this methodology for evaluating food and beverage products. The panelists  participated in a two 2.5 h sessions to develop sensory terminology to describe the walnut kernels and the walnut oils. After reaching a consensus, the panelists selected seven attributes for the walnut kernels and fifteen for the walnut oils. The descriptive terms for the kernels were: honey aroma, cardboard aroma, rancid aroma, crunchy, rancid flavor, bitterness and astringency/drying. The fifteen attributes for the walnut oils comprised seven aroma and eight flavor attributes: overall aroma intensity, buttery aroma, fresh nuts aroma, toasted/burnt aroma, grainy aroma, cardboard/woody aroma, rancid aroma, overall flavor intensity, sweetness, fresh nuts flavor, toasted flavor, grainy flavor, cardboard flavor, rancid flavor and greasy/thick texture. For the walnut kernels, eleven samples plus one control (month 0) were evaluated over two sessions: Chandler on one day and Howard on a separate day. A modified Latin square design was used each day to randomize the presentation of the twelve samples among the eight panelists.Kernels were served in clear, tulip-shaped wine glasses of 220-mL capacity which were coded with 3-digit random numbers. Two kernel halves were placed in each glass and then covered with a 5.7 cm diameter plastic Petri dish for at least 2 h prior to evaluation. The tests were conducted in a room illuminated with “daylight” fluorescent lighting. All samples were served at room temperature (20–23 °C). The panelists were separated by dividers and were not allowed to communicate during the session. Panelists expectorated the samples and rinsed with bottled water between tastings. Red seedless grapes were also available as a palate cleanser. Each judge rated the intensity of the attributes for the different samples using structured 10-point (0 to 10) scales anchored at the ends with terms “low” and “high” or “smooth” and “grippy.” Previously agreed upon reference standards were available in each booth. Similarly, the eleven walnut oils samples, in addition to a control, were evaluated over two sessions: Chandler on one day and Howard on a separate day. A modified Latin square design was used each day to randomize the presentation of the twelve samples among the eight panelists.The oils were served in clear, tulip-shaped plastic wine glasses of 133-mL capacity which were coded with 3-digit random number codes. Approximately 12 mL of oil were poured in each glass and then covered with a 4.5 cm diameter plastic cover for one hour prior to evaluation. The plastic cups with the oils were put on metal trays and the trays were placed on heating mats in each sensory booth. All samples were served at a temperature of 28 ± 2 °C. The tests were conducted in a room illuminated with “daylight” fluorescent lighting. The panelists were separated by dividers and were not allowed to communicate during the session. Panelists expectorated the samples and rinsed with bottled water between tastings. Each judge rated the intensity of the attributes for the different samples using structured 10-point (0 to 10) scales anchored at the ends with terms “weak” and “strong,” or “low” and “high.” Previously agreed upon reference standards were available in each booth. p ≤ 0.05. Correlation analysis between oxidative volatiles and sensory attributes was performed by Pearson’s correlation test. XLSTAT (version 2013) was used for partial least squares regression analysis to evaluate the relationship between chemical oxidative markers and sensory perception of rancidity.All chemical analyses described in this study were conducted in triplicates. Statistical analyses were performed using SPSS software . Biochemical markers and sensory attributes were analyzed using a two-factorial model (time and temperature) at five  and two (5 and 23 °C) levels, respectively. Significant differences were tested by Tukey’s multiple comparison test at p < 0.05) in quality parameters were observed during walnut storage. For moisture content, Howard kernels recorded the highest value (7.88 g/kg), with 5 °C/month 4, with this observation being significantly higher by 34, 52, and 47.59%, compared to their respective 23 °C  and raw samples. Similar to Howard, the moisture content for Chandler kernels showed an increasing trend, with 5 °C along the storage period. Overall, the Chandler moisture content at 5 °C/month 4 was 1.7 and 1.75 times higher than with 23 °C  and the raw kernel samples, respectively. The high moisture content observed with 5 °C can be attributed to the high humidity at this temperature, which resulted in the absorption of water by the kernels. This observation is crucial because it can lead to early deterioration and loss of quality.Postharvest storage conditions influence food quality by inducing undesirable changes in biochemical make-up . As depip < 0.05) the FFA by 33.33% at both 5 and 23 °C. Irrespective of cultivar type, the low FFA observed with raw samples may be linked with the low moisture contents of their kernels, compared to their respective stored kernels. This may have limited lipase activity for FFA formation [Production of free fatty acids (FFA) is due to the hydrolysis of triacylglycerols through hydrothermal conditions or the action of lipases . FFA resormation . Additioormation .p < 0.05) along storage. PV from Howard showed the highest value of 1.80 meq O2/kg at month 4 for both 5 and 23 °C, with this observation being 52.78 and 48.33% higher, compared to their respective controls. For Chandler, although PV at month 1 was significantly higher at 5 °C (1.06 meq O2/kg), compared to 23 °C (0.93 meq O2/kg), prolonging storage time up to month 4 increased PV to 1.80 meq O2/kg at 23 °C. According to Buransompob et al. [2/kg. Thus, it can be postulated that, storage conditions investigated in this study are appropriate to enhance walnut shelf-life stability during postharvest. Interestingly, a current work published by our research group observed higher PV ranges for Chandler (1.0–4.3 meq O2/kg) and Howard (1.8–6.4 meq O2/kg) between 1 to 28 weeks of storage at 25 °C [Peroxide value (PV) is an indicator of the formation of hydroperoxides as primary oxidative products . As expeb et al.  high quaat 25 °C . Therebyat 25 °C .232) is associated with the presence of hydroperoxides, conjugated dienes, carboxylic compounds, and conjugated dienes, whereas absorption at 268 (K268) is a representation of the presence of secondary products formed from oxidative compounds discovered at K232 [232 values for Howard oils ranged from 0.90–2.50, with the peak value observed at 23 °C/month 4. This observation was significantly (p < 0.05) higher than observed with 5 °C/month 4 (by 38%) and control by 64%. Following a similar trend, higher levels of storage conditions (temperature and time) reported higher K232 values in Chandler oil, with 23 °C/month 4 showing the highest value of 1.94 than oil samples from 5 °C/month 4 (1.39) and control (1.06). Additionally, although K268 for Chandler oil at 5 °C was highest at month 1 (0.14), it showed a decreasing trend up to month 4, whereas its observation at 23 °C/month 4 showed a significant increment. For Howard oils, the highest value of K268 (0.18) was recorded at 23 °C/month 3 and 4. It should be highlighted that, the simultaneous synergy between increasing values of K232 and K268 at 23 °C/month 4 indicate a continuous oxidation under high storage temperature and time [232 in walnut (cv. Pedro) oil stored at 5 °C  and 22 °C .UV absorption at 232  by 45.47%  and 42.40% (for γ-tocopherol). Interestingly, α-tocopherol was absent at month 4 for both investigated temperatures irrespective of cultivar. This observation could be due to the preferential utilization of α-tocopherol against oxidative stress over other investigated tocopherols during storage. According to the literature, α-tocopherol is the most reactive and less stable form of tocopherol, followed by γ- and δ-tocopherols due to its lower bond dissociation energy required for abstraction of hydrogen from its hydroxyl group than other tocopherols [p < 0.05) reductions of γ- (by 29.13%) and δ- (by 27.08%) tocopherols at 23 °C/month 4, compared to 5 °C, although 23 °C presented higher concentrations  at month 1. This trend was not the case for Howard, hence demonstrating the influence of genetics on tocopherol changes during postharvest handling. Additionally, higher tocopherol reductions at 23 °C indicate accelerated oxidation and the need to utilize available tocopherols as antioxidants.Furthermore, γ- and total tocopherols quantified for 5 °C Chandler oil were comparable to their control throughout the storage period, whereas, at 23 °C, they were comparable to the control, until month 4, where it reduced significantly , the main enzyme responsible for the oxidation of phenolic compounds into quinones for browning . PhenoliCu2+/Cu+ . HoweverCu2+/Cu+  reportedCu2+/Cu+ .p-coumaric, and chlorogenic acids), flavanols , and flavonols . Concentration of flavonoids  were more pronounced, compared to phenolic acids. The dominant phenolic compound for both Howard and Chandler was gallic acid, which was higher in Chandler (981.68 mg/kg) than Howard (703 mg/kg) at 23 °C/month 4. This observation was significantly higher (p < 0.05), compared to its concentration at 5 °C by 12.35% for Howard and 21.04% for Chandler, as well as its initial concentration prior to storage by 33.50% for Howard and 75.21% for Chandler. Besides gallic acid, other dominating phenolics included p-coumaric acid, caffeic acid, gallocatechin gallate, epicatechin gallate, quercetin, and rutin, with their concentrations dependent on storage temperature and time. At 23 °C/month 4, concentrations of gallocatechin gallate, rutin, p-coumaric acid, and caffeic acid were higher than at 5 °C by 10.76, 23.68, 33.89, and 51.69%, respectively. All other profiled phenolic compounds ranged between 17.34–20.74 mg/kg. The higher phenolic levels at 23 °C explained more oxidative stability at elevated temperatures. This result demonstrates the protective role of phenolics against oxidation by competing for free radicals and other endogenous pro-oxidants [The 14 phenolic compounds identified in kernels were grouped into phenolic acids  changes in unsaturated and saturated fatty acids. The previous literature has established the dense representation of unsaturated fatty acids in walnuts, among which, PUFAs are the most dominant [Investigated storage temperatures showed insignificant , and E-2-heptenal (13.60 mg/kg) were observed at 5 °C, and they were not significantly different from their respective results at 23 °C for Howard. Thus, it can be inferred that the evolution of the above-mentioned volatiles in Howard kernels was independent of the storage temperature, but dependent on storage time. Additionally, all six profiled alcohols in Howard kernel showed significant (p < 0.05) changes during storage. For instance, where 1-pentanol, 1-hexanol, and 1-octen-3-ol were at a maximum at 5 °C/month 4, the maximum evolution of 1-penten-3-ol, Z-2-penten-1-ol, and propanol were recorded at 23 °C/month 4 and 23 °C/month 3, respectively, and they were 3.32, 1.60, and 5.30 times higher, compared to observations at 5 °C. Kernel volatile data obtained from Chandler showed no significant (p > 0.05) storage temperature effects, with respect to heptanal, E-2-hexenal, and octanal, but reflected an influence of storage time.Results of this study confirm the comment of Frankel , that isFor all investigated walnut cultivars and storage conditions, this study showed hexanal and 1-hexanol as the highest elicited aldehyde and alcohol. This can be attributed to the significant distribution of oleic and linolenic acids among profiled fatty acids. These fatty acids are prone to oxidation, giving rise to aldehydes such as hexanal . It is ap > 0.05), compared to kernels. Of the 23 profiled volatiles, five  were peculiar to kernels, and two  were peculiar to oil. From p < 0.05) higher at 5 °C/month 4 than with 23 °C/month 4 by 40.51, 45.38, and 87.81%, respectively, in Howard oil.Twenty-three volatiles were detected in oils from both cultivars, although their concentrations were significantly lower (p > 0.05), compared to 23 °C/month 4. Overall, the results showed volatile concentrations to be dependent on cultivar, walnut fraction, storage temperature, and time. Since these volatiles are associated with distinct aroma descriptors, it can be postulated that, storage conditions impact different odor perceptions in walnut oil and kernel. For instance, the presence of hexanal and 1-hexanol in oils signify tallow and resin odors during storage [Volatiles from Chandler oil  showed s storage .p < 0.05) from months 2 to 4 (p < 0.05) perceptions for rancid taste, rancid aroma, astringency, and bitterness as early as month 1. Linking this trend to its pre-discussed volatile data, it can be postulated that the rancidity at 5 °C is detected when the propanoic acid, octanal, E-2-heptenal, pentanal, and hexanal reach 0.50, 1.17, 2.18, 35.95, and 766.62 mg/kg, respectively, whereas rancidity at 23 °C is detected when the aforementioned volatiles reach levels of 0.29, 0.57, 1.65, 14.11, and 153.59 mg/kg, respectively.Changes in sensory properties of walnut kernels and oils are presented in s 2 to 4 a–d. ContE-2-heptenal, butanal, pentanal, propanol, and hexanal reach 1.18, 2.94, 4.59, 25.22, 122.14, and 993.69 mg/kg, respectively, whereas, at 23 °C, oil rancidity is detected when the cited volatiles reach 0.68, 5.34, 3.73, 43.43, 90.48, and 675.32 mg/kg, respectively.This further indicates that hexanal is the dominating volatile for kernel rancidity detection, although it also exhibits synergistic actions with other rancid-inducing compounds. With respect to Howard oils, storage at 5 °C did not show rancidity until month 4, whereas oil at 23 °C was perceived rancid from month 3. Thus, the rancidity of Howard oil at 5 °C can be detected when oxidative volatiles, such as octanal, E-E-2,4-nonadienal, propanoic acid, octanal, pentanal, E-2-heptenal, butanal, propanol, and hexanal reach 0.09, 0.41, 0.51, 1.53, 2.22, 2.25, 22.30, and 279.43 mg/kg, respectively. In addition, the detection of oil rancid flavor in Chandler at 23 °C/month 3 received the highest score. Thus, rancidity in Chandler oil was perceived at 23 °C, when octanal, butanal, E-2-heptenal, pentanal, propanol, and hexanal reached 1.00, 4.58, 6.28, 55.68, 93.23, and 954.13 mg/kg, respectively. Considering the increasing rate of hexanal with rancidity detection, hexanal is postulated to be the most appropriate volatile marker for walnut quality prediction. Mexis et al. [For Chandler, kernels stored at 23 °C recorded the lowest scores for honey aroma, compared to kernels stored at 5 °C a–d. Addis et al.  also invr = 0.918, p < 0.01), nonanal , 1-hexanol , E-2-pentenal , and 1-octen-3-ol . Astringency at 5 °C showed high positive correlations, with 19 volatile compounds, of which, the four most highly correlated were 1-hexanol , nonanal , 1-pentanol , and 1-octen-3-ol . Additionally, rancid aroma was positively correlated with 19 volatiles, with the highest correlating compounds being nonanal , hexanal , 1-hexanol , 1-pentanol , and pentanal . This study confirmed a direct relationship between the increasing oxidative volatiles and reduction of walnut kernel oxidative stability.To further analyze the relationship between volatile compounds and sensory attributes, Pearson correlation coefficients were obtained with values of 22 volatiles . For bitr = 0.922, p < 0.01; r = 0.920, p < 0.01, respectively). However, at 23 °C, rancid flavor and aroma were positively correlated with 17 and 19 volatiles, respectively. Pentanal showed the highest positive correlation  with rancid oil flavor at 23 °C, whereas 5-hepten-2-one and 6-methyl were the most highly positively correlated  with rancid aroma, followed by hexanal  and pentanal . In summary, rancidity in walnut oils at 5 °C was highly linked with hexanal, whereas rancidity at 23 °C was not only linked with hexanal, but also the synergistic activities of other oxidative volatiles.Unlike that observed with kernels, the oxidative stability of walnut oils showed higher positive correlations with volatiles at 23 °C than at 5 °C . At 5 °Cx-variable) and sensory attributes (y-variable) of kernels and their loading plots , and oil rancid perception was strongly linked with volatiles produced at 23 °C. Additionally, the study revealed high positive correlations between bitterness, astringency, rancidity, and oxidative volatiles, such as hexanal, pentanal, and propanol. To maintain the positive sensory attributes and market value of walnuts, while extending their shelf-life, it is recommended to store oxidation-prone cultivars, such as Howard, at lower temperatures, compared to oxidation-resistant cultivars, such as Chandler.


Cohnella sp. LGH. Here, we identify and characterize the following depolymerization pathway in strain LGH through the genomic and enzymatic analysis. In the pathway, neoagarotetraose was depolymerized by a novel α-neoagarooligosaccharide (NAOS) hydrolase CL5012 into 3,6-anhydro-α-L-galactose (L-AHG) and agarotriose; Agarotriose was further depolymerized by a novel agarolytic β-galactosidase CL4994 into D-galactose and neoagarobiose; Neoagarobiose was finally depolymerized by CL5012 into L-AHG and D-galactose. Although α-agarase has not been identified in strain LGH, the combined action of CL5012 and CL4994 unexpectedly plays a critical role in the depolymerization of agarotetraose, one theoretical product of α-agarase hydrolysis of agarose. In this pathway, agarotetraose was depolymerized by CL4994 into D-galactose and neoagarotriose; Neoagarotriose was then depolymerized by CL5012 into L-AHG and agarobiose. Furthermore, another novel endo-type β-agarase CL5055 was identified as an isozyme of AgaW with different pH preference in the hydrolysis of agarose into α-NAOSs. Strain LGH seemed to lack a common exo-type β-agarase responsible for the direct depolymerization of agarose or neoagarooligosaccharide into neoagarobiose. These results highlight the diversity of agarolytic manner in bacteria and provide a novel insight on the diversity of agarolytic pathways.Previously, we have reported that an endo-type β-agarase AgaW was responsible for the hydrolysis of agarose into the major product neoagarotetraose in a terrestrial agar-degrading bacterium  O-linked β-D-galactose and 4-O-linked 3,6-anhydro-α-L-galactose (L-AHG)  . D-galachia coli . Agarosehia coli . Recentlhia coli . Furtherhia coli . Thus, thia coli . Due to hia coli , anti-dihia coli , and anthia coli , agarosehia coli . TherefoVibrio sp. EJY3, Agarivorans gilvus WH0801, Colwellia echini A3T, and Paraglaciecola hydrolytica S66T from marine environments (Streptomyces coelicolor A3(2) from terrestrial environments   VejABG cleaved β-1,4 linkage of AOSs into D-galactose and shorter-chain α-NAOSs  . The α-Nniege G7 , AgaWH11s WH0801 , and BpGplebeius . Recentl α-NAOSs . At pressp. EJY3 , AgWH2A s WH0801 , and GH2rmis NP1 . In 2021rotriose . The exorotriose . Besidesrotriose , and Patrotriose . Only anrotriose .Cohnella sp. LGH was previously isolated from soil and an endo-type β-agarase AgaW responsible for the hydrolysis of agarose into the major product neoagarotetraose was characterized  Co., Ltd., China. The Amicon ultra-2 and ultra-15 centrifugal filter unit (3-kDa cutoff size) was purchased from Millipore. Plasmid pET29a (+) and pBAD-HisA were stored in Soil Ecology Lab. Cohnella sp. LGH was isolated from farmland soil in NanJing, China. LB medium contained 1% NaCl, 1% Tryptone, and 0.5% Yeast extract. MM medium contained 0.1% NaCl, 0.2% (NH4)2SO4, 0.15% K2HPO4, 0.05% KH2PO4, 0.01% CaCl2, 0.01% MgSO4, and pH 7.2. MS medium contained MM medium and 0.5% glucose. NAOS medium contained MM medium and 0.5% hydrolysis product of AgaW acting on agarose.g for 30 min. The supernatant was desalted by a CommaX™ G-25 column  to obtain crude enzyme. The crude enzyme (10 μl) was incubated with 10 μl of 2 mg ml–1 NAOSs  at 30°C for 4 h, respectively. The reaction products were analyzed by TLC.The NAOSs were prepared by β-agarase AgaW as previously described . Strain De Novo Assembler (Newbler) v2.8, rRNA and tRNA genes were detected using Barrnap version 0.4.2 software and tRNAscan-SE version 1.3.1, respectively. The protein-coding sequences were predicted by Glimmer version 3.02 software using default parameters. The annotation of the predicted open reading frames was determined by using BLAST searches of non-redundant protein sequences from the NCBI, Swiss-Prot, COG, and KEGG databases.Strain LGH was grown in LB medium and its genomic DNA was extracted by Universal Genomic DNA Extraction Kit (TaKaRa). The genomic DNA of strain LGH was constructed as Illumina PE library (∼300 bp) and Roche454 MP library (∼1 Kb) using the TruSeq™ DNA Sample Prep Kit and TruSeq PE Cluster Kit v3-cBot-HS. After filtering ineffective and low-quality data, the Illumina PE library contained 3,831,007 total high quality read pairs with 204-fold depth of coverage and the Roche454 MP library harbored 79,364 total reads with fourfold depth of coverage. The two libraries were assembled by 454’s GS Premix Ex Taq™ (TaKaRa) was used in qPCR reaction and the 96-well plate with resulting mixture was performed by Step One Plus Real Time PCR system . The values of 2–ΔΔCt were calculated to estimate the differences of gene expression. ΔΔCt = (Cttarget gene – Cthousekeeper gene)experimental group − (Cttarget gene – Cthousekeeper gene)control group. The primers used for qPCR are listed in LGH was grown in MS and NAOS medium until the late exponential phase. Total RNA was extracted using a MiniBEST Universal RNA Extraction Kit (TaKaRa), and the concentration and purity of extracted RNA was measured by NonoDrop 2000 (Thermo Fisher Scientific). Then, approximately 0.5 μg RNA was added to synthesize cDNA using a PrimeScript™ RT reagent Kit (TaKaRa). TB Green cl4969, cl4994, cl4996, cl5009, cl5012, cl5015, cl5037, and cl5055) were amplified by PCR using genome of strain LGH as template. Two reported genes (agawh117 and agawh2a) were amplified by PCR using genome of A. gilvus WH0801 as template. The primers used for cloning are listed in E. coli BL21 (DE3) pLysS cells. Clones were screened by 50 μg ml–1 kanamycin and plasmid sequencing and cl5015 was also cloned into pBAD-HisA. The recombinant plasmid was transformed into E. coli TOP10 and then screened by 100 μg ml–1 ampicillin.Eight candidate genes  was added to induce the gene expression at 16°C for 16 h. The overexpression of pBAD-CL5015 was induced by 1 mM L-arabinose. Cells were harvested and resuspended in 10 mM Tris-HCl buffer (pH 7.0) and then lysed by sonication. Centrifugation at 15,000 × g for 30 min was performed to remove cell debris. Supernatant was purified by a 2-ml volume of NTA-Ni2+ column (Sangon) at 4°C. The binding buffer contained 300 mM NaCl, 50 mM sodium phosphate, 10 mM imidazole, 10 mM Tris base (pH 8.0), and the elution buffer was composed of 300 mM NaCl, 50 mM sodium phosphate, 250 mM imidazole, and 10 mM Tris base (pH 8.0). The purified enzymes were desalted, concentrated with a Millipore Amicon ultra-15 centrifugal filter unit, and stored in 10 mM potassium phosphate buffer (pH 7.0). The concentration of recombinant enzyme was quantified by a protein assay kit with bovine serum albumin as a standard.The confirmed recombinant strains were grown at 37°C in LB medium with 50 μg ml–1 α-agarase AgaWS5 was incubated with 1.5 ml of 1.5% agarose at 40°C in 1 mM potassium phosphate buffer (pH 7.0) for 12 h. The reaction mixture was cooled at 4°C for 10 min, was centrifuged at 15,000 × g for 30 min, and then the supernatant was filtered with a Millipore Amicon ultra-2 centrifugal filter unit for protein removal. The filtered liquid containing agarotetraose was collected and stored in −20°C. The agarotetraose was then identified by TLC and mass analysis.Approximately 0.5 ml of 5 mg ml–1 CL5055 was mixed with 350 μl of 0.5% agarose at 40°C ranging from 0 to 1,440 min. The products were determined by TLC and mass spectrometry. To further determine the depolymerization of NAOSs by CL5055, neoagarobiose, neoagarotetraose, and neoagarohexaose (10 μl of 2.5 mg ml–1) were mixed with 10 μl of 2 mg ml–1 CL5055 at 40°C for 2 h, respectively. The products were then determined by TLC and mass spectrometry. The hydrolysis activity of CL5055 was also quantified by the 3,5-dinitrosalicylic acid (DNS) method, which had been described previously  were incubated with 10 μl of 2.5 mg ml–1 agarotriose at 30°C for 4 h, respectively. In the assays, all the enzymatic reactions were incubated in 10 mM potassium phosphate buffer (pH 7.5). The products were then measured by high performance liquid chromatography (HPLC).In the ABG-mediated depolymerization assays, agarotriose was used as substrate. CL4969, CL4994, CL5037, AgWH2A (10 μl of 2 mg ml–1) were mixed with 10 μl of 2.5 mg ml–1 neoagarobiose or neoagarotetraose at 30°C for 4 h, respectively. The products were measured by high performance liquid chromatography-mass spectrometry (HPLC-MS).In the α-NAOS hydrolase-mediated depolymerization assays, neoagarobiose and neoagarotetraose were used as substrates, respectively. CL5012, CL5015, AgaWH117 (10 μl of 2 mg ml–1) were incubated with 5 μl of 2.5 mg ml–1 agarotetraose solution at 30°C for 2 h, respectively. CL5012 was further incubated with the supernatant of the reaction product of CL4994 at 30°C for 2 h. All products were measured by HPLC-MS.To determine the depolymerization ability of CL5012 and CL4994, both proteins . Spots were visualized by using 10% (by volume) H2SO4 in ethanol and heating at 105°C for 10 min. In addition, the developing solvent for agarotetraose was n-butanol-ethanol-water solution .For TLC analysis, reaction products were applied to silica gel 60 TLC plate , and then developed by –1; nebulizer gas pressure (nitrogen) was at 30 psi; and capillary voltage was at 4,000 V. Negative ions were acquired in full scan mode in the range of m/z 100–1,000 molecular mass units for identification within a 1–s scan time interval.For mass spectrometry analysis, the spots of TLC plate corresponding to the hydrolyzed products were scraped out and dissolved in 80% acetonitrile for drying. The molecular mass distribution of the products was determined using an Agilent 6460 triple-quadrupole mass spectrometer equipped with electrospray ionization (ESI) under negative-ion ionization conditions. The ESI-MS conditions were as follows: all injections were 5 μl; drying gas temperature was at 350°C; drying gas flow (nitrogen) was at 10 L min–1; column temperature was 30°C; detector temperature was 30°C; and gas flow was 1.6 L min–For HPLC analysis, the reaction products were determined by Agilent Infinity 1260 (Agilent Technologies) equipped with an evaporative light scattering detector G4260B . The chromatographic column was Alltech Prevail Carbohydrate ES column (250 × 4.6 mm). The HPLC conditions were as follows: all injections were 10 μl mobile phase, acetonitrile-water (8:2 by volume) had a flow rate of chromatography of 1 ml min–1; column temperature was 30°C; drying gas temperature was 350°C; and capillary voltage of mass was 4 kV. Negative ions were acquired and mass spectra were in the range of 100–1,000 m/z.For HPLC-MS analysis, the reaction products were determined by Agilent high-performance liquid chromatography triple-quadrupole mass spectrometer  equipped with ESI under negative-ion ionization conditions. The chromatographic column was Alltech Prevail Carbohydrate ES column (250 × 4.6 mm). The HPLC-MS conditions were as follows: all injections were 10 μl mobile phase, acetonitrile-water (8:2 by volume) had a flow rate of chromatography of 0.75 ml min–1 methoxyamine hydrochloride dissolved in pyridine at 75°C for 30 min, then 60 μl of N-methyl-N-(trimethylsilyl) trifluoroacetamide was added to each sample and incubated at 37°C for 30 min. After centrifuging at 15,000 × g for 30 min, supernatant was collected for analysis. Bruker 450-GC 320-MS system , equipped with a DB5-MS column , was used to analyze reaction products. The temperature program was as follows: all injections were 1 μl and the initial temperature was 100°C for 2 min, then was increased to 230°C for 15°C min–1, and maintained at 230°C for 20 min. The temperature was then increased to 300°C at 20°C min–1, and this temperature was maintained for 10 min. Electron ionization was performed at 70 eV, and the temperature of the ion source and transfer line was 230°C. Mass spectra was in the range of 50–500 m/z.For GC-MS analysis, the reaction mixtures were centrifuged at 15,000 × g for 30 min, then the supernatant was collected and lyophilized. The dried samples were derivatized by adding 50 μl of 20 mg ml2HPO4-citrate buffer of pH 3.0–5.0, 10 mM potassium phosphate buffer of pH 6.0–7.0, 10 mM Tris-HCl buffer of pH 8.0–9.0, and a 10 mM glycine-NaOH buffer of pH 10.0–12.0.To determine the optimal pH for each enzyme, the enzymatic reactions were performed in different conditions. CL5055 was determined at 45°C in a pH range from 3.0 to 12.0 at an interval of 1; CL4994 was determined at 30°C in a pH range from 4.0 to 10.0 at an interval of 1; CL5012 was determined at 35°C in a pH range from 4.0 to 10.0 at an interval of 1. The pH stability was determined after preincubation of enzymes in different buffers for 1 h. Meanwhile, the preincubation temperatures depended on enzymes, CL5055 at 45°C, CL4994 at 30°C, and CL5012 at 35°C. Buffers used in this section were: 10 mM NaThe optimal temperatures of each enzyme were determined in different conditions. CL5055 was determined in 10 mM Tris-HCl buffer (pH 9.0) at a temperature range from 30 to 65°C at an interval of 5; CL4994 was determined in 10 mM potassium phosphate buffer (pH 7.0) at a temperature range from 20 to 45°C at an interval of 5; CL5012 was determined in 10 mM Tris-HCl buffer (pH 8.0) at a temperature range from 20 to 50°C at an interval of 5. The temperature stability was determined after preincubation of enzymes at different temperatures for 1 h. The preincubation pH depended on enzymes, CL5055 at pH 9.0, CL4994 at pH 7.0, and CL5012 at pH 8.0.The effects of potential inhibitors or activators on the enzyme activity were determined by adding various metal ions and chemical reagents at a final concentration of 10 mM. The relative activity was defined as a percentage of the activity obtained in the absence of an additive. Three independent determinations were performed for all above measurements in this section.–1 CL5055 was incubated with agarose at a final concentration of 0.5–10 mg ml–1 in 10 mM Tris-HCl buffer (pH 9.0) at 45°C for 10 min. One activity unit (U) of CL5055 was defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute at 45°C. Approximately 25 μg ml–1 CL4994 was incubated with agarotriose at a final concentration of 0.5–7.5 mg ml–1 in the 10 mM potassium phosphate buffer (pH 7.0) at 30°C for 10 min. Owing to the incomplete separation of D-galactose and neoagarobiose  at 35°C for 10 min. One U of CL5012 was defined as the amount of enzyme required to produce 1 μmol of L-AHG per minute at 35°C. Kinetic parameters were calculated from non-linear regression data analysis against various substrate concentrations using origin 8.0 and three independent determinations were performed.To determine the kinetic parameters of each enzyme, approximately 50 μg mlarobiose , one U o–1 CL5055 was mixed in 15 ml of 10 mM potassium phosphate buffer (pH 8.0) with 0.5% agarose as substrate. The reaction was incubated at 45°C at various reaction times ranging from 0 to 120 min and the efflux time was measured at each time. The experiment was repeated three times.To determine the kinematic viscosity of the CL5055, the Ubbelohde viscometer was used. 20 μl of 2 mg mlp-nitrophenyl-α-D-galactopyranoside and p-nitrophenyl-β-D-galactopyranoside, respectively. Twenty microliter of 2 mg ml–1 CL5055 was incubated in 700 μl of 10 mM potassium phosphate buffer (pH 8.0) with each substrate at a final concentration of 3 mg ml–1 at 45°C for 2 h. The reaction was stopped by addition of 500 μl of 1 M Na2CO3. The activity of the enzyme was measured spectrophotometrically at 420 nm by evaluating the release of p-nitrophenol from the hydrolysis of the artificial chromogenic substrate. Three independent determinations were performed.The substrate specificity of the CL5055 was measured by using two artificial chromogenic substrates, CL4969, QTH40778.1; CL4994, QTH40796.1; CL4996, QTH40798.1; CL5009, QTH40807.1; CL5012, QTH40809.1; CL5015, QTH40812.1; CL5037, QTH40833.1; CL5055, QTH40851.1.Accession numbers of enzymes involved in this study, NCBI accession numbers: Cohnella sp. LGH was capable of utilizing agarose as a sole carbon source for growth and showed strong agarolytic activity. One endo-type β-agarase AgaW was responsible for the hydrolysis of agarose into the major product of neoagarotetraose and has been identified and characterized from strain LGH  analysis further showed that the transcriptional levels of five candidate genes, containing cl4994, cl4996, cl5009, cl5012, and cl5055, increased when strain LGH utilized α-NAOSs as a sole carbon source for growth  from S. Coelicolor A3(2) (ABD81917) from S. degradans 2-40T (CD08992)   and SdNAns 2-40T , but alsCD08992)  which weCD08992) . We thens WH0801 . One spoto L-AHG . Anotheralactose . CL5012 alactose . One proto L-AHG . Anotherrotriose . The comrotriose .2+, Ca2+, and Mn2+. On the contrary, Zn2+ and Cu2+ could obviously increase its enzymatic activity, suggesting that Zn2+ or Cu2+ might be involved in the catalytic reaction of CL5012 , 70.66 U mg–1, 43.5 s–1, and 4.8 × 103 M––1 s––1, respectively. According to the classification analysis of dbCAN2 server,1 CL5012 was further classified into GH117 family.We then detected the enzymatic properties of CL5012 by utilizing neoagarobiose as the substrate. CL5012 showed the maximum activity at pH 8.0, and its relative activity dropped quickly at pH beyond 6.0 and 9.0. It was stable at a pH range from 6.0 to 8.0 and almost lost activity at pH 4.0 after being pre-incubated for 60 min . The prof CL5012 . AccordiWP_014232195) from Vibrio sp. EJY3 (MG456856) from A. gilvus WH0801 (COG3250) of the β-galactosidase like protein family . The results suggested there should be other protein responsible for the agarotriose degradation in LGH. We then utilized two reported agarotriose hydrolase sequences belonging to the family of β-galactosidase like protein, the ABG VejABG , 79.41 U mg–1, 52.9 s–1, and 4.4 × 103 M–1s–1, respectively. The classification analysis of dbCAN2 server showed that CL4994 was belong to GH2 family.Agarotriose was further utilized as the substrate to determine the enzymatic properties of CL4994. CL4994 exhibited maximum activity at pH 7 and retained more than 80% of maximum activity at a pH range from 5.0 to 8.0. CL4994 was stable at a pH range from 6.0 to 8.0, retaining at least 80% of initial activity for 60 min pre-incubation . The optand EDTA . AccordiCatenovulum sediminis WS1-A and AgaD from Thalassomonas sp. LD5  were present in its sequence (KR296705) in strain LGH , 186.43 U mg–1, 8.6 × 103s–1, and 1.9 × 107 M–1s–1, respectively. Combined to its protein sequence phylogenetic tree analysis  and the 3,6-anhydrogalactonate cycloisomerase VejACI (accession NO. H2IFX0). In strain LGH, CL4996 shared 44.5% sequence identity to VejAHGD and was present the same aldehyde dehydrogenase family domain (accession NO. pfam00171) as VejAHGD (CD03316) as VejACI  and strain LGH only harbored one agarolytic pathway. S. coelicolor A3(2) harbored the common agarolytic pathway but lacked the auxiliary agarolytic pathways  can be found in the article/GL and JW designed the studies. GL, RG, SW, and JW analyzed the genome sequence. GL, RG, SC, and JL cloned and purified enzymes. GL and SW performed the qPCR analyses. GL, WX, ZZL, XS, JX, QZ, and ZHL characterized the enzymatic properties. GL wrote the manuscript. JW, ZW, and FH revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lactiplantibacillus plantarum BCH-1 has been evaluated. Bioactive compounds were extracted by ethyl acetate and were fractionated by gradient column chromatography from crude extract. Based on FT-IR analysis followed by GC-MS and ESI-MS/MS, the active compound was identified to be Bis-(2-ethylhexyl) phthalate. Antibacterial potential was evaluated by disk diffusion against E. coli (12.33 ± 0.56 mm inhibition zone) and S. aureus (5.66 ± 1.00 mm inhibition zone). Larvicidal potency was performed against Culex quinquefasciatus Say larvae, where Bis-(2-ethylhexyl) phthalate showed 100% mortality at 250 ppm after 72 h with LC50 of 67.03 ppm. Furthermore, after 72 h the acetylcholinesterase inhibition was observed as 29.00, 40.33, 53.00, 64.00, and 75.33 (%) at 50, 100, 150, 200, and 250 ppm, respectively. In comet assay, mean comet tail length (14.18 ± 0.28 μm), tail DNA percent damage (18.23 ± 0.06%), tail movement (14.68 ± 0.56 µm), comet length (20.62 ± 0.64 µm), head length (23.75 ± 0.27 µm), and head DNA percentage (39.19 ± 0.92%) were observed at 250 ppm as compared to the control. The current study for the first time describes the promising antibacterial and larvicidal potential of Bis-(2-ethylhexyl) phthalate from Lactiplantibacillus plantarum that would have potential pharmaceutical applications.Lactic acid bacteria produce a variety of antibacterial and larvicidal metabolites, which could be used to cure diseases caused by pathogenic bacteria and to efficiently overcome issues regarding insecticide resistance. In the current study, the antibacterial and larvicidal potential of Bis-(2-ethylhexyl) phthalate isolated from  Staphylococcus aureus (VRSA) and methicillin-resistant S. aureus (MRSA), have established a great interest in the discovery of natural products for the control of drug-resistant pathogens and new diseases + along with their sodiated ionic peak at m/z 413.3 [M + Na]+ and potassium adducts at m/z 429.3 [M + K]+. Additionally, several other daughter ionic peaks, e.g., m/z 279.1, 181.2, 167.1, and 149.0 were also obtained  suggested that BEHP showed the highest toxicity with low LC50, which decreases with time  after 72 h of exposure, while 61% and 78% mortality were noted after 24 and 48 h, respectively . The morith time . The LC5C. quinquefasciatus larvae was determined at different concentrations after 72 h  with control were used to observe DNA fragments migration by agarose gel-electrophoreses  and 4′,6-diamidino-2-phenylindole (DAPI) were mainly purchased from Sigma-Aldrich, St. Louis, MO, USA. Silica gel (70–230 mesh) and Thin-layer chromatography (TLC) silica gel 60 F₂₅₄, aluminum sheet 20 × 20 cm2 were purchased from Merck, Darmstadt, Germany. The de Man, Rogosa & Sharpe (MRS) broth/agar, Glycerol , Nutrient agar/broth, HPLC grade organic solvents , Ethanol), Sodium phosphate, Acetylcholine chloride, Fast blue B salt, Ethylenediaminetetraacetic acid (EDTA), Sodium chloride (NaCl), Tris, Triton X-100, NaLactiplantibacillusplantarum BCH-1 (KX388380) strain previously isolated from fermented rice rinsed water with lactose as a carbon source was cultured on MRS agar/broth at 37 °C  in distilled water, while the dried collected fractions  were redissolved  in DMSO (a polar aprotic solvent that dissolve both polar and nonpolar compounds)  mixture and run, using direct injection mode at 9 μL/min. The capillary temperature was set at 288 °C. The mass range was selected at m/z 100 to 1000 in positive ionization mode for data acquisition. The collision induced dissociation energy (CID) was manually selected in the range of 5 to 30 eV for obtaining favorable fragmentation. The sheath and auxiliary N2 gases were also adjusted manually. For the data analysis and structural elucidation, Xcalibur™  and ChemDraw  software were used.For further confirming the metabolite of F2 fraction and elucidating its molecular structure, the ESI-MS/MS  analysis was carried out by following the protocol described earlier  with a fC. quinquefasciatus were introduced to each concentration of the test compound in beakers containing 100 mL of water. The beakers were labeled properly and covered with muslin cloth. Each test was performed in triplicate with a control group and their mortality was recorded after 24, 48, and 72 h. The immovable dead larvae that showed no response with sharp pin probing were removed from beakers and stored in microcentrifuge tubes containing ethanol [Stock solution  was prepared by dissolving 100 mg of F2 fraction in 1 mL DMSO and the volume was raised to 100 mL with distilled water. From the stock solution, different dilutions of 50 ppm, 100 ppm, 150 ppm, 200 ppm and 250 ppm were prepared in 100 mL deionized water. The abovementioned concentrations of bioactive fraction (F2) having resultant compound were prepared to perform bioassay following the WHO protocol , while a ethanol  to deterC. quinquefasciatus larvae were washed with distilled water and dried on blotting paper. After washing thoroughly, these larvae were homogenized by using cold sodium phosphate buffer  with Teflon hand homogenizer and subsequently centrifuged at 6000 rpm (4430× g)  for 20 min at 4 °C. The supernatant was used for the estimation of AChE. All glassware and solutions used for homogenization purpose, were placed at 4 °C before use and the prepared homogenate was also placed on ice until used for AChE estimation [For Acetylcholinesterase enzyme (AChE) estimation, 6000 rpm 30× g (Z3w/v) was added to stop the reaction. The sample and control (without enzyme solution) were placed in a spectrophotometer  at 405 nm to observe optical density (OD) [For this purpose, 50 μL of enzyme solution was added to 50 μL of 2.6 mM acetylcholine chloride (as substrate) with 1 mL of sodium phosphate buffer  and incubated at 25 °C for 5 min. Finally, 400 μL of Fast blue B salt (0.3% ity (OD) .Enzyme inhibition percentage was calculated by the following formula:w/v) normal melting agarose (NMA). After drying, the stored sample of mosquito larvae suspended in 140 μL of 0.75% (w/v) low melting agarose (LMA) was layered over prepared glass slides (frosted ends) and left for drying at room temperature. Slides were covered with a coverslip (24 × 60 mm) and placed at 4 °C for 10 min to harden the agarose layer. After that, the coverslip was removed and slides were immersed in cold lysis solution  Triton X-100, pH 10 and 5% (v/v) DMSO) at 4 °C for 2 h in dark. For DNA unwinding, slides were placed in an electrophoretic tank  filled with cold electrophoretic buffer  for 25 min and electrophoresis was performed in the same buffer for 20 min at 25 V and 300 mA (0.73 V/cm). After electrophoresis, the slides were stained with 20 μL of DAPI (1 μg/mL) per gel after gel neutralization by washing twice with 0.4 mM Tris (pH 7.5). The slides were scored under a fluorescence microscope  using Komet 5.5 Image Analysis System . A total of 100 randomly selected (50 cells per 2 replicate slides) per treatment were scored [Comet assay was performed for the determination of DNA damage  with slie scored ,53.n = 3). For antilarval activity, Abbot’s formula [50. Corrected mortality data were subjected to ANOVA using SPSS software package for Windows  and means were separated through Tukey’s HSD (Honest Significant Difference) test. The value of p ≤ 0.05 was considered statistically significant [Zones of antibacterial activity inhibition were measured as mean ± standard deviation of three replicates  phthalate (BEHP) was isolated from LAB species


The photophysical characterization showsthe presence of two emission bands in solution assigned to the 3π–π* transition from the N^C^N ligandsmixed with 3MLCT/3ILCT transitions (higher energyband) in deaerated samples. The formation of excimers has also beenidentified as a broad band at longer wavelengths [near-infrared (NIR)emission] that becomes the main emission band for compounds containingphenanthrene as the chromophore. NIR emission behavior has also beenexplored using acetonitrile/water mixtures, and the presence of aggregatesthat emit at ca. 650 nm has also been detected.Two series of Pt(II)-cyclometallatedcompounds containing N^C^Ntridentate and alkynyl-chromophore ligands have been synthesized andstructurally characterized. The N^C^N ligands differ on the presenceof R Two series of Pt(II) complexes have beensynthesized andtheir photophysical data carefully analyzed in solution and powder.Aggregation studies were performed in acetonitrile/water mixtures.Infrared emitters were obtained through excimers’ formationor aggregation. They present square-planar geometriesthat can undergo face-to-face intermolecular interactions throughligand–ligand and Pt(II)···Pt(II) interactions,which can give rise to new electronic excited states that producered-shifted emission from triplet metal–metal-to-ligand charge-transfer(3MMLCT) or excimeric 3IL excited states inaddition to the one arising from the mononuclear Pt(II) moiety.12 These assemblies are of great relevance to modulate the resultingphotophysical properties, both regarding their energy and emissionintensity by subtle changes to their environment. It should be takeninto consideration that aggregation can happen in the ground state(dimers) or the excited state (excimers). Additionally, Pt(II), asa heavy atom, induces strong spin–orbit coupling, favoringthe population of the T1 triplet excited state by enhanced intersystemcrossing from S1 → T1 and producing phosphorescence at roomtemperature.13The d3The use of cyclometallatingligands is a convenient strategy tofavor luminescence since the strong field of these ligands tends tofavor emission efficiencies as they raise the energies of deactivatingmetal-centered states, reducing non-radiative deactivation pathways.Tridentate cyclometallated ligands are particularly relevant sincethey have been observed to induce higher rigidity on the complex withrespect to bidentate ligands, inhibiting distortion and reducing non-radiativedeactivation processes.15 In particular, C^N^N and N^C^N coordination modes are more commonlyfound in tridentate cyclometallating ligands, and they have been observedto exhibit intense luminescence and versatile emissive excited states,including not only intraligand (IL) (π–π*) excitedstates but also the excimeric excited states.18 Among them,N^C^N coordination seems to favor higher emission intensities andquantum yields.9 Fewer efforts have beenmade on the analysis of the co-ligand that occupies the fourth coordinationposition at the Pt(II) center, even though they can also have an influenceon their resulting assemblies and luminescent properties.19The natureof the cyclometallatedligands and co-ligands and theionic or neutral character of the molecules are extremely relevantto modulate the absorption and emission properties. Thus, the studyof Pt(II)-cyclometallated compounds is currently a highly relevantresearch field in order to achieve high luminescence  quantum yields,color tunability, and stability.20 IR and NIR emission is of vital importance inseveral relevant applications such as full-color displays, remotesensing of environmental conditions, night-vision displays, bio-chemosensors,in vivo imaging, light-fidelity (Li-Fi) communication, or securityauthentication devices, and it is mostly explored with pure organicmolecules.25 Different strategies can be followed to modulate the final emissionof the complexes to red, with excimers’ formation and aggregation-inducedemission (AIE) being popular design methodologies.30An interesting tangent in the field isthe modulation of the chemicalstructures and assemblies to shift the emission to the red since,for example, OLEDs and materials that emit in the infrared (IR) ornear-infrared (NIR) region represent a challenging target due to thefavored deactivation processes in low-energy emissive populated states.In this work, we have designedand synthesized two series of Pt(II)-cyclometallatedcomplexes containing tridentate N^C^N ligands with an alkynyl chromophoreas the co-ligand occupying the fourth coordination position. The differentchromophores have been chosen in order to evaluate how the electron-withdrawingcharacter (fluorine), soft atom (sulfur in thiophene), or extendedaromaticity  can affect theresulting packing through intermolecular contacts affecting theirluminescence. Additionally, the two series of compounds differ onthe presence of a H or F atom at the central benzyl ring, which couldalso confer different intermolecular forces in the packing. The differencesin the resulting photophysical properties depending on the N^C^N ligandand co-ligands have been analyzed in detail together with the resultingAIE processes, with those being observed to shift the emissions upto ca. 700 nm, thanks to excimer and aggregate formation.1 and 2 were reacted with differentalkynyl aromatic ligands with sodium hydroxide as a base.31 Final compounds 1x and 2x were obtained as orange solids with moderate yields (54–80%)after precipitation and washing with water, methanol, and hexane.All compounds were synthesizedby following the route summarized in 1HNMR spectroscopy showed the disappearance of the terminal alkynylproton (R2-C≡C-H)from the corresponding alkynyl aromatic ligand. This observation togetherwith the appearance of the aromatic protons of the new ancillary ligandconfirms the correct substitution of the chlorido and formation ofthe desired platinum complexes. A significant downfield shift in thepyridine proton (Hf) of ca. 0.10–0.25 ppm is observedwhen compared with the precursors (9.36–9.38 ppm). However,its coupling constant with the platinum atom is not affected sincethe ligand trans to the nitrogen of the pyridinebond is not exchanged . For family 2x, all compounds were also characterizedby 19F NMR spectroscopy, observing only one signal as atriplet due to the coupling with the two adjacent aromatic protonsof the central ring. A second signal is present in the NMR spectrumof compound 2b due to the aromatic p-fluorobenzene moiety .Characterization of the synthesized compoundsby –1) vibrationaswell as the disappearance of the band assigned to the stretching ofthe terminal proton of the free alkynyl moiety (around 3300 cm–1) was determined by IR spectroscopy. Further confirmationof the successful formation of the products was checked by electrosprayionization [ESI(+)] mass spectrometry finding the protonated molecularpeak for all cases .The C≡C (around 2070 cm1e and 2c (Table S1) by slow diffusion of methanol or hexane, respectively,into a concentrated dichloromethane solution of the compound. Compound 1e presents one single molecule in the asymmetric unit, whilefor compound 2c, two crystallographically independentmolecules are observed -cyclometallatedcompounds.33 The aromatic ring attached tothe alkynyl moiety is almost perpendicular to the cyclometallatedunit for compound 1e (85.3°), while for the twomolecules in the asymmetric unit of compound 2c, theyare observed in two different conformations with angles of 52.1°and 83.3°.Single crystals suitable forX-ray diffraction analysis were grownfor compounds observed 1 and 2. Figures S25 and S26). These dimeric structuresare arranged in a head-to-tail fashion, which does not permit thepresence of metallophilic interactions in 1e (with aPt···Pt distance of 5.0521(5) Å). A similar distancewas observed for Pt2···Pt2 of 4.9888(4) Å in 2c. However, in 2c, a much shorter Pt···Ptdistance of 3.4654(4) Å was observed for Pt1···Pt1,which is just below the combined van der Waals radii (3.5 Å)for this interaction.31 As depicted in 1e, these dimer-like structures are due to the presence ofπ–π stacking (3.36 and 3.35 Å) and C···H(2.85 Å) intermolecular short contacts, which results in an interplanardistance of 3.0 Å. Additional C–H···πintermolecular contacts of 3.03 Å between the phenanthrene groupsof two adjacent molecules can be identified, which could be responsiblefor different emissive properties (see below). However, for compound 2c, we can only observe F···H: 2.45 Åand C···H: 2.87 Å interactions, leading to a higherinterplanar distance of 3.2 Å  were recorded in 5 × 10–5 M dichloromethane solutions at 298 K, and the resultsare shown in Figures S27–S29 and 1π–π* IL transitionsof the cyclometallated 1,3-di(2-pyridyl)benzene, which are also recordedin the absorption of the free ligands L1 and L2 , overlapped, in some cases,with 1π–π* IL transitions of the alkynylaromatic moiety. In the lower energy region (350–450 nm), lessintense bands are observed, which can correspond to mixed charge-transferand ligand-centered character transitions, according to the literature.36The absorption spectraof all compounds .36 The large Stokes shift and the quenching of the emissionin the presence of oxygen support their phosphorescent emission assignment, provingthat the phosphorescent emission of these compounds is due to theperturbation from the platinum atom. Another band centered at 700nm is observed for all final platinum complexes 1a–e and 2a–e, which can be assigned to excimer formation.37 The excimer assignment can be done, thanks to the excitation spectrathat resemble the same pattern (that fits the absorption spectra)when the emission is collected at the two maxima . The lack of excimer formationrecorded for 1 and 2 together with the differentexcimer intensities recorded in the different cases indicates themain role of the alkynyl-R2 chromophore in the excimerformation. This excimer is observed to be more favored for series 2 complexes and mainly for the more extended chromophore,phenanthrene, in compound 2e. In fact, excimer emissionhas been previously reported in the literature for other LPt-Cl analoguesto 1 and 2 only at higher concentrations.38 The total phosphorescence quantum yields (QY)increase in deaerated solutions, reaching values up to 60%, similarto the values reported for analogous compounds.39 Global QY values have been split into their monomer and excimercontribution . Thus, although no significant differenceshave been observed in the global QY values of the platinum complexeswhen the two series of compounds are compared, some significant differencescan be observed when the emission efficiency is split between thetwo contributions (monomer and excimer). In this case, the principal parameters that seem to have the main effect are the extended aromaticity(a larger contribution of the excimer for 1e and 2e) and the H or F atom at the central N^C^N tridentate ligand,with a larger effect in the presence of a fluorine in the excimerformation. This may be due to the resulting less electronic densityin the phenanthrene aromatic ring, making them more suitable for establishingintermolecular contacts.The samples were excited attheir lower energy absorption band,and the recorded emission spectra display a vibronically structuredband centered around 500 nm 5 that casignment1. The frribution 1, and itecursors S31.36 Th41 The recordedvalues for the final platinum complexes are, in general, in the sameorder as those recorded for the platinum precursors 1 and 2. Phenanthrene derivatives 1e and 2e are again a particular case that displays longer decaytime values than the rest of the compounds as previously observedfor other platinum cyclometallated compounds containing this chromophore.4 Time-resolved phosphorescence spectra were studiedfor these two compounds (1e and 2e), andthe resulting kinetics show the clear predominance of the monomeremission with respect to the excimer at increasing time-gating inagreement with the longer decay times recorded .Phosphorescence emission lifetimes were recordedfor both the monomerand the excimer emission transitions and are of hundreds of nanoseconds.These values increase to a few microseconds in deaerated solutions,and they are in the range of those reported previously in the literaturefor similar compounds 2 and supTable S2). According to these values,the increase in the recorded quantum yields from the monomer emissionband in deaerated samples can be ascribed mainly to a significantdecrease of the non-radiative deactivation pathway rate. We can observethat knr values are one order of magnitudesmaller than those in air-equilibrated samples, while kr values stay similar. Additionally, looking at the excimeremission band, we can observe that the knr values for phenanthrene derivatives 1e and 2e are smaller than those calculated for the other platinum complexes,in agreement with the significant increase in the emission intensityof this band.Radiative and non-radiative rate constants have beencalculatedin all cases from the experimental quantum yields and lifetime values. This emission band can be assigned to the emissionof the π–π stacked aggregated forms, which is inaccordance with the observation of the dimer formation in the obtainedcrystalline structures.31 The phosphorescencequantum yields have moderate values up to 12%, and the correspondinglifetimes are of hundreds of nanoseconds  or similar to  those previously obtained inair-equilibrated solution samples.In the solid state (powder), all compounds presenta broad emissionband centered between 579 and 626 nm .Density functional theory (DFT)calculations in solution, using the B3LYP functional, 6-31G*/LANL2DZbasis set, and CPCM solvation model  results in a shortening of the distance between the platinumand the ligand situated in a trans position withrespect to the metallated carbon, while the bond length between themetallated carbon and the platinum remains constant, as the one betweenthe platinum and the nitrogen. Finally, changes in the nature of thearomatic ring bonded to the alkyne moiety only result in small variationsin the distance between the alkyne carbon and the aromatic carbonbonded to it.We have analyzed the modificationsin the platinum environmentupon modifying the nature of the ligands. Thus, the substitution ofthe chlorido ligand by an aryl alkynyl moiety results in an incrementof the distance between the platinum and the cyclometallated carbonand a very slight increment of the platinum–nitrogen bond length.The change of a hydrogen atom for a fluorine in the cyclometallatedligand  absorption spectra using dichloromethane as asolvent. The most intense transitions are shown in Table S4. Figure S40a,b shows theenergy and the nature of the orbitals involved in these transitionsfor complexes 1 and 1a-1e,as well as the energy of the corresponding transitions. All of thesetransitions are of π–π* type.TD-DFT calculationswere performed on the systems 1 and 2. These orbitals, for complex 1, are shown in Highest occupiedmolecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO)transitions are only observable in complexes 1a. Thus, this transition is ILCT/LLCT in character.In the remaining systems, the lowest energy absorptioncorrespondsmainly to a HOMO–1 to LUMO+1 transition. The former is basedmainly on the alkyne moiety and the central ring of the cyclometallatedligand, with a very small contribution of Pt, while the latter isbased mainly on the three rings of the same ligand, as shown in 1a-1e and 2a-2e show an absorption band in the 380–390 nm interval.We have assigned it to a HOMO–1 - > LUMO transition. Aspreviouslystated, HOMO–1 has a contribution of the alkyne moiety andthe central ring of the cyclometallated ligand, while LUMO is centeredon the N-substituted rings of the pincer ligand; thus, this transitionis also ILCT/LLCT in character. 1c complex.Complexes 1d and 2d) andphenanthrenyl (1e and 2e) substituents alsofeature a band in the 309–319 nm interval. There are severaltransitions that can contribute to this band, but the one that isalways present is HOMO–3→ LUMO+1 (HOMO–3→LUMO in 2e). Both orbitals are centered in the pincerligand, as shown in 1d complex, so this transition canbe regarded as ILCT.Systems with naphthyl  show a change in the absorption band that presentsa shoulder shifted at higher wavelengths. This suggests that uponthe addition of water, new aggregated species is formed, which ispresent in the ground state.To tryto induce the red-shifted emission in air-equilibrated samples, waterwas tested as a bad solvent to induce aggregation by preparing severalacetonitrile/water mixtures. The absorption spectra of the compoundsin acetonitrile and 90%:10% water/acetonitrile . Excitation spectraalso support this assignment since they display a different profilefrom the recorded one for the monomer .The resulting behavior is observedto be strongly dependent on the alkynyl-RS45).At 90% water content, its emission reaches the maximum intensity,and, in some cases, it is predominant to the monomer, even becomingthe sole emission in compound 2e affording a completelyred-shifted emission. In fact, AIE seems to be more favored in series 2 complexes .The total phosphorescence quantum yield increaseswhen increasingthe water percentage, as expected for an AIE behavior. This is alsosupported by the increasing contribution of the aggregates’quantum yield when it is present by splitting the global QY valueinto the corresponding contributions of the monomer and the aggregatedforms. The phosphorescence lifetime values of both monomer and aggregatesbecome larger with increasing water content in all compounds, and,in all cases, τkr and knr values (Tables S7 and S8). Additionally, the more efficient AIE effect is directly relatedto the kr rate constant. In the case ofthe aggregates, AIE behavior is directly related to an increase of kr rate constants. A significant increase of kr is detected (up to 5-fold in series1 and more than one order of magnitude in series 2) in the emission of the aggregates, while the contribution of knr is much less important. That is, the aggregationprocesses induced by the addition of water in the acetonitrile solutionsfavor the intermolecular contacts and the close distance between themolecules, making the environment more rigid and favoring the efficiencyof the radiative emission process as it is evidenced in the increaseof the kr constants.It can be observedthat the non-radiative deactivation processesare more favored in both the monomer and the aggregates in agreementwith the calculated N^C^N Pt(II)-cyclometallatedcompounds are emissive in both thesolution and solid state with different emission origins. Room temperaturephosphorescence emission is extremely favored in agreement with therecorded large Stokes shifts and long lifetimes. The presence of IRemission bands can be modulated by various methodologies includingthe formation of excimers and AIE to produce red-shifted emissions,where AIE can be favored by using a mixture of good and bad solvents,and excimer formation can be directly obtained in a homogeneous solutionand the solid state (powder).2O–CH3CN 1:1 to introduce the sample. IR spectra were recorded in KBr dispersionon an FT-IR 520 Nicolet spectrophotometer. NMR spectra were recordedin CDCl3 at the Unitat d’RMN of the Universitatde Barcelona with a Mercury 400 spectrometer . Chemical shifts are given in δ values(ppm) relative to tetramethylsilane (1H) or CFCl3 (19F), and coupling constants J aregiven in Hz. Multiplicity is expressed as s (singlet), d (doublet),t (triplet), q (quadruplet), qi (quintuplet), and m (multiplet). Numberingschemes for the characterized compounds are displayed in Electrospray mass spectra were obtained atthe Unitat d’Espectrometria de Masses (Universitat de Barcelona)in an LC/MSD-TOF spectrometer using H2Cl2 with a Cary100 scan 388 Varian UV spectrometer. Emission and excitation spectrawere recorded in a Horiba Jobin-Yvon SPEX Nanolog-TM spectrofluorometerat 298 K using 5 ×10–5 M solutions and in thesolid state.UV–visspectra were recorded in CHEmission quantum yields were determined with aHamamatsu QuantaurusQY absolute photoluminescence quantum yield spectrometer C11347.Luminescence lifetimes were measured on a JYF-DELTAPRO-NL equipmentupon excitation of the samples with a 390 nm NanoLED and collectingthe decays through a cut-off filter of 450 nm.L1) and 2,2′-dipyridine(L2) and platinum compounds 1, 1a, and 2 were prepared as reported elsewhere.42All reagents were obtainedfrom commercial sources and used as received. Ligands 1,3-di(2-pyridyl)benzene was obtained as an orange solid from0.025 g (0.052 mmol) of compound 2, 0.011 g (0.104 mmol)of phenylacetylene, and 0.004 g (0.104 mmol) of sodium hydroxide.Yield: 0.017 g (61%).Compound : δ 9.52 ; 7.96 ;7.64 ; 7.58 ; 7.27 ; 7.23–7.26; 7.19 . 19F NMR : δ −118.62 . MS-ESI+: m/z 989.13 [2M-C8H5]+, 546.09[M + H]+, 444.05 [M-C8H5]+. IR: ν 2085.34 (C≡C).1b) was obtained as an orange solid from0.020 g (0.043 mmol) of compound 1, 0.010 g (0.086 mmol)of 1-ethynyl-4-fluorobenzene, and 0.004 g (0.086 mmol) of sodium hydroxide.Yield: 0.017 g (71%).Compound : δ 9.49 ; 7.93 ; 7.69 ; 7.51–7.56 ; 7.24–7.26 ; 6.97 . 19F NMR : δ −116.03 . MS-ESI+: m/z 971.14 [2M-C8H4F]+, 546.09 [M + H]+, 426.06 [M-C8H4F]+. IR: ν 2081.07 (C≡C).2b) was obtained as an orange solid from0.020 g (0.042 mmol) of compound 2, 0.010 g (0.084 mmol)of 1-ethynyl-4-fluorobenzene, and 0.003 g (0.084 mmol) of sodium hydroxide.Yield: 0.013 g (54%).Compound : δ 9.50 ; 7.97 ; 7.65 ; 7.53 ; 7.28 ; 7.24–7.26 ; 6.97 . 19F NMR : δ −115.88 , −118.14 . MS-ESI+: m/z 1007.12 [2M-C8H4F]+, 564.08 [M + H]+, 444.05[M-C8H4F]+. IR: ν 2085.34 (C≡C).1c) was obtained as an orange solid from0.020 g (0.043 mmol) of compound 1, 0.009 g (0.086 mmol)of 3-ethynylthiophene, and 0.004 g (0.086 mmol) of sodium hydroxide.Yield: 0.014 g (61%).Compound : δ 9.50 ; 7.93 ;7.68 ; 7.52 ; 7.36 ; 7.18–7.26. MS-ESI+: m/z 959.11 [2M-C6H3S]+, 534.06 [M + H]+, 426.06 [M-C6H3S]+. IR: ν 2076.81 (C≡C).2c) was obtained as an orange solid from0.020 g (0.042 mmol) of compound 2, 0.009 g (0.084 mmol)of 3-ethynylthiophene, and 0.003 g (0.084 mmol) of sodium hydroxide.Yield: 0.015 g (65%).Compound : δ 9.51 ; 7.96 ;7.64 ; 7.36 ; 7.28 ; 7.21–7.25. 19F NMR : δ −118.47 . MS-ESI+: m/z 995.09 [2M-C6H3S]+, 552.05 [M + H]+, 444.05 [M-C6H3S]+ IR: ν 2081.07 (C≡C).1d) was obtained as an orange solid from0.020 g (0.043 mmol) of compound 1, 0.013 g (0.086 mmol)of 2-ethynylnaphthalene, and 0.004 g (0.086 mmol) of sodium hydroxide.Yield: 0.020 g (80%).Compound : δ 9.56 ; 8.05 ; 7.94 , 7.73–7.81 ; 7.67–7.72 , 7.54 ; 7.34–7.46; 7.21–7.26 . +MS-ESI: m/z 1003.16 [2M-C12H7]+, 578.12 [M+ H]+, 426.06 [M-C12H7]+. IR: ν 2085.34 (C≡C).2d) was obtained as an orange solid from0.015 g (0.033 mmol) of compound 2, 0.010 g (0.066 mmol)of 2-ethynylnaphthalene, and 0.003 g (0.066 mmol) of sodium hydroxide.Yield: 0.012 g (61%).Compound : δ 9.57 ; 8.05 ; 7.98 , 7.77–7.79 ; 7.66–7.68 ; 7.42 ; 7.29 ; 7.26–7.29 . 19F NMR : δ −118.47 . MS-ESI+: m/z 10039.15 [2M-C12H7]+, 596.11 [M + H]+, 444.05[M-C12H7]+. IR: ν 2085.34 (C≡C).1e) was obtained as an orange solid from0.020 g (0.043 mmol) of compound 1, 0.017 g (0.086 mmol)of 9-ethynylphenanthrene, and 0.004 g (0.086 mmol) of sodium hydroxide.Yield: 0.017 g (63%).Compound : δ 9.63 ; 8.98 ; 8.71 ; 8.65 ; 8.08 ; 7.95 , 7.84 , 7.72 ; 7.66 ; 7.53–7.61 ; 7.19–7.25 . MS-ESI+: m/z 1053.18[2M-C16H9]+, 628.14 [M + H]+, 426.06 [M-C16H9]+. IR: ν2064.01 (C≡C).2e) was obtainedas an orange solid from0.020 g (0.042 mmol) of compound 2, 0.017 g (0.084 mmol)of 9-ethynylphenanthrene, and 0.004 g (0.084 mmol) of sodium hydroxide.Yield: 0.020 g (74%).Compound : δ 9.64 ; 8.97 ; 8.71 ; 8.65 ; 8.08 ; 7.98 , 7.84 , 7.67 ; 7.57 ; 7.31 ; 7.24–7.28 . 19FNMR : δ −118.35 . MS-ESI+: m/z 1089.16 [2M-C16H9]+, 646.12 [M + H]+, 444.05 [M-C16H9]+. IR: ν 2072.54 (C≡C).1e and 2c by slow diffusion of methanol or hexane, respectively, in a dichloromethanesolution of the compounds.Single crystals suitable for X-raydiffraction analysis were grown for 1e and 2c were obtained using a Bruker-NoniusKappa CCD diffractometer with an APEX-II detector with graphite-monochromatizedMo Kα (λ = 0.71073 Å) radiation. Data collectionand reduction were performed using the program COLLECT46 and refinedby full-matrix least squares on F2 usingthe OLEX2 software,47 whichutilizes the SHELXL module.48Single-crystal X-ray data for m COLLECT and HKL 49 included in Spartan 20.50 The functional chosen was B3LYP,51 and the basis set was chosen as follows: 6-31G*for C, H, N, and Cl, including polarization functions for non-hydrogenatoms,52 and LANL2DZ53 for Pt. Solvent effects were considered using the CPCMmodel.54 No symmetry restrictions wereimposed. Optimized geometries are given in Table S9.Theoretical calculations wereperformed at the DFT level usingQ-chem 5.1,


BCL-2 inhibitor venetoclax (VEN) in combination with hypo-methylating agents (HMA) led to a significant improvement of response rates and survival. Further, application in the relapsed or refractory (r/r) AML setting or in context of allogeneic stem cell transplantation  seems feasible.Diagnosis of acute myeloid leukemia (AML) is associated with poor outcome in elderly and unfit patients. Recently, approval of the Fifty-six consecutive adult AML patients on VEN from January 2019 to June 2021 were analyzed retrospectively. Patients received VEN either as first-line treatment, as subsequent therapy , or at relapse after alloHSCT. VEN was administered orally in 28-day cycles either combined with HMA or low-dose cytarabine (LDAC).FLT3-ITD mutated AML with 3.4 (1.9–4.9) months versus 10.4 (0.8–24.3) months for non-mutated cases, . Patients harboring NPM1 or IDH1/2 mutations lacking co-occurrence of FLT3-ITD showed a survival advantage over patients without those mutations (11.2 (5–24.3) months versus 5.0 (0.8–22.1) months, respectively, . Multivariate analysis revealed mutated NPM1 as a significant prognostic variable for achieving complete remission (CR) . The most common adverse events were hematological, with grade 3 and 4 neutropenia and thrombocytopenia reported in 44.6% and 14.5% of patients, respectively.After a median follow-up of 11.5 (range 6.1–22.3) months, median overall survival (OS) from start of VEN treatment was 13.3 (2.2–20.5) months, 5.0 (0.8–24.3) months and 4.0 (1.5–22.1) months for first-line, subsequent line treatment and at relapse post-alloHSCT, respectively. Median OS was 11.5 (10–22.3) months from start of VEN when subsequent alloHSCT was carried out. Relapse-free survival (RFS) for the total cohort was 10.2 (2.2 – 24.3) months. Overall response rate  was 51.8% for the total cohort . Subgroup analysis revealed a significantly reduced median OS in Detailed analyses on efficacy for common clinical scenarios, such as first-line treatment, subsequent therapy (r/r AML), and application prior to and post-alloHSCT, are presented. The findings suggest VEN treatment combinations efficacious not only in first-line setting but also in r/r AML. Furthermore, VEN might play a role in a subgroup of patients with failure to conventional chemotherapy as a salvage regimen aiming for potential curative alloHSCT.The online version contains supplementary material available at 10.1007/s00432-022-03930-5. Acute myeloid leukemia (AML) in elderly unfit patients remains challenging and overall long-term prognosis is poor  benefit for the combination of HMA or low-dose cytarabine (LDAC) with VEN compared to single agent therapy  included 431 AML patients (286 azacitidine (AZA) plus VEN and 154 with AZA alone) with a median age of 76 years  approval in October 2020 and in May 2021 by the European Medicines Agency (EMA) as first-line therapy for patients not eligible for intensive treatment. LDAC-VEN combination has exclusively been approved by the FDA yet.Due to improved response rates and excellent tolerability, there are ongoing debates as to whether VEN combination strategies should be implemented into first-line treatment for subsets of newly diagnosed AML patients treating in a curative intent  setting. Encouraging results have been reported in small retrospective studies evaluating outcomes of r/r AML patients treated with VEN as single agent or in combination with other conventional agents .n = 18), (ii) subsequent line  (n = 23), and (iii) at relapse post-alloHSCT (n = 15). 27 patients (48.2%) had de novo, 28 (51.8%) secondary AML (sAML) derived from myeloproliferative diseases (MPN) or myelodysplastic syndromes (MDS) and one patient (1.8%) treatment-associated AML. Treatment response was adjudicated according to European LeukemiaNet (ELN) 2017 recommendations  first-line therapy  registry. The analysis was approved by local ethics committee of the University Hospital Jena, Germany (no. 3967–2/13 for SAL registry).2 intravenously daily on days 1–5, 5-azacitidine 75 mg/m2 subcutaneously for 7 consecutive days, repeated after 28 days each) or low-dose cytarabine     based on treosulfan or busulfan in combination with fludarabine with or without ATG prior to transplantation . For classification of hematological toxicity changes for neutrophils, platelets and hemoglobin compared to baseline values at start of VEN treatment have been assessed.Cytogenetic evaluation was performed using standard banding techniques, and karyotypes were described according to the currently valid International System for Human Cytogenetic Nomenclature .Response assessment was carried out separately for three disease settings: first-line, subsequent line  and relapse post-alloHSCT. Subgroup analyses were accomplished for patients achieving composite complete remission (CRc) and for distinct molecular aberrations  and survival from start VEN treatment to last follow-up or death from any cause. OS is defined as date of first diagnosis of AML to date of last follow-up and death from any cause. PFS is defined as date of initiation of VEN until progression/relapse or last follow-up/death from any cause. Relapse-free survival (RFS) was calculated for patients attaining CRc measured from date of remission to relapse or last follow-up.9/l, platelets ≥ 100 × 109/l). Partial remission was defined as 5—25% blasts in the bone marrow and a total reduction of blasts of at least 50% of AML blasts. Progressive disease (PD) was defined as increase of bone marrow and/or peripheral blast count or new extramedullary manifestations. Stable disease (SD) was defined when CRc, PR or PD are not met and last for at least 3 months.Overall response rate (ORR) was defined as CRc comprising complete remission, complete remission with incomplete hematological recovery (CRi) and complete remission with incomplete platelet recovery (CRp) and partial remission (PR). Additional response criteria were applied according to ELN 2017 guidelines . Transfusion dependence was determined by Gale criteria with greater than or equal to 2 units per month over the prior 3 months .Time-to-event analyses  were estimated using Kaplan–Meier method and compared using log-rank test. A total of 56 adult AML patients with a median age of 66.5 (range 34–83) years were included in this analysis. Table n = 27) presented with de novo AML, 51.8% (n = 29) with secondary AML (sAML) including one patient with treatment association (Table 48.2% (Eastern Cooperative Oncology Group (ECOG) performance status rated 31 patients (55.3%) as 0–1 and 25 patients (44.6%) as 2–3, respectively.n = 22) are summarized in Table S1.Cytogenetic risk profiling was performed for 55 of 56 patients (98.2%) and resulted in favorable, intermediate and adverse risk in 0 (0%), 34 (60.7%) and 21 (37.5%), respectively. Data for molecular aberrations were available in 54 of 56 (96.4%) AML patients. Risk stratification according to the current ELN criteria 2017 showed a distribution in favorable, intermediate and adverse risk in 5 (8.9%), 17 (30.3%) and 32 (57.1%) of patients, respectively . A third subgroup was treated with VEN due to disease relapse following alloHSCT (n = 15). Of note, 7 patients were successfully transitioned to alloHSCT after VEN treatment . Notably, one patient received VEN/HMA as first-line treatment before subsequent alloHSCT. Since all other VEN/HMA first-line patients (n = 17) were treated within a palliative setting, the only patient underwent alloHSCT was not included in survival analysis for first-line VEN treatment group.The majority of patients received VEN in combination with an HMA agent backbone, including 22 (39.3%) decitabine and 28 (50%) AZA (Table Median time of VEN treatment was 3.4 (0.6 – 22.1) months with a median application of 3 (1–18) treatment cycles (28 days). The overall mean drug dosage was 149 (20.9–362.5) mg. In 85.7% of all patients, concurrent azole medication was applied . Interruption of treatment was necessary in 16 patients (28.6%) being the majority due to grade 4 neutropenia (93.7%) and one patient due to severe nausea and emesis (6.2%).22 of 38 patients (57.9%) were refractory to any treatment line prior to VEN. In median, one CR (range 0–4) with a median of 1.5 (range 0–8) therapy lines could be achieved prior to initiation of VEN treatment. Information on prior or subsequent treatment regimens to VEN is indicated in Table Subgroup analyses with respect to treatment intention showed a median OS starting at VEN treatment initiation of 13.3 2.2–20.5) months, 5.0 (0.8–24.3) months, 4.0 (1.5–22.1) months for first-line treatment, subsequent line treatment and post-alloHSCT, respectively  months and PFS of 20.5 (2.6 – 24.3) months, respectively  months and not reached in patients who received CRc (2-year OS: 63%) . RFS for the whole cohort was 10.2 (2.2 – 24.3) months .FLT3-ITD was associated with an ORR of 12.5%, in contrast 71.4% in NPM1 and / or IDH1,2 mutated AML, respectively .An ORR of 51.8% was achieved for the whole population (data not shown). In detail, ORR of 61.1% was seen when VEN was applied as first-line treatment, 52.2% for subsequent line treatment and 42.8% at relapse post-alloHSCT Fig. . Median p = 0.598) and 6.4 (1.1–22.2) months vs. 5.0 (2.2–24.3) months for ELN high-risk vs. favorable + intermediate, respectively (p = 0.565).With respect to genetic subgroups, no difference in survival since VEN initiation was seen for cytogenetic (intermediate vs. high risk) and ELN risk groups (favorable + intermediate vs. high-risk) Fig. A, B: 6.4FLT3-ITD mutation  had a significantly reduced median OS of 3.4 (1.9 – 4.9) months compared to 10.4 (0.8 – 24.3) months for those without an activating FLT3-ITD mutation .Analysis of distinct molecular subgroups revealed that patients harboring NPM1, IDH1, or IDH2 mutations without co-occurring FLT3-ITD mutations with the remaining cohort, an increased sensitivity to VEN-based therapy has been demonstrated: 11.2 (5 – 24.3) months versus 5.0 (0.8–22.1), respectively   and 42.5 × 109/l (12–280) on days 0 and 100 of treatment, respectively. Transfusion dependence for platelets was decreasing from 62.9% to 47.2% and for red blood cells (RBC) from 75.9% to 55.5% during the first 100 days of treatment. Median neutrophil count was 0.39 × 109/l (range 0.1–12.5) and 0.2 × 109/l (range 0.1–3.8) at days 0 and 100 of treatment, respectively. On day 60, 17 of 49 patients (36%) showed an increase of the neutrophil count compared to baseline value.Furthermore, assessment of blood count and transfusion (in-)dependence was performed. As demonstrated in Table The median of the mean VEN dosage of the whole cohort was 149 mg/d (20.9 – 362.5) with a median treatment duration of 105 (18 – 674) days (Table n = 22) were compared to those with > 100 mg (n = 34). Kaplan–Meier analysis demonstrates a median survival of 6.4 (1.5–17.7) months and 8.1 (1.1–24.3) months for patients having received a mean daily dose of ≤ 100 mg and > 100 mg, respectively (p = 0.357)   and VEN at relapse post-alloHSCT.p < 0.001) , median OS was 5.0 (0.8 – 24.3) months and in case of relapse post-alloHSCT 4.0 (1.5 – 22.1) months, respectively.Clinical scenarios, such as failure of conventional induction chemotherapy or relapse post-alloHSCT, remain a challenge. Especially in genetically high-risk patients, re-induction by conventional chemotherapy is associated with poor response rates  months in FLT3-ITD-mutated versus 10.4 (0.8 - 24.3) months in FLT3-wildtype patients, respectively.Across genetic risk groups, no difference in survival was observed when stratified by cytogenetics and ELN criteria. Detailed molecular genetic analyses revealed a significantly worse outcome in n = 10)  had favorable responses to VEN which have also been documented by others  compared to doses higher than 100 mg (34 patients), no significant difference was seen with a median survival since VEN treatment of 6.4 (1.5–17.7) months or 8.1 (1.1–24.3) months, respectively. Although not statistically significant, patients treated with higher doses of VEN had a greater likelihood of achieving a CRc (44% versus 27%). Measuring individual VEN plasma concentrations and considering concurrent azole medication would yield a much more reliable result for exposure–efficacy relationship. Furthermore, genetic heterogeneity of AML, such as upregulation of In conclusion, VEN treatment shows impressive response rates when applied as first-line treatment suggesting VEN-based combination approaches also for curative intended AML patients either characterized by relevant comorbidities or harboring unfavorable cytogenetic risk factors associated with a high rate of induction failure. Furthermore, in r/r AML including relapse after alloHSCT, it represents a reasonable therapeutic approach. Those who fail on VEN treatment and are not eligible for intensive therapy have a poor prognosis and alternative treatment strategies are required.Supplementary file1 (DOCX 91 KB)Below is the link to the electronic supplementary material.