The experimental results showed that ZZO could inhibit inflammation, remodel fibrous tissue, and promote angiogenesis in the DU area, facilitating wound healing in DU mice. Moreover, the PI3K/AKT signaling pathway was indeed activated by ZZO treatment, promoting macrophage M2 polarization. In addition, we used molecular docking technology to evaluate the binding sites between ZZO and the PI3K/AKT pathway. The results showed that ISO has a good binding interaction with AKT. Moreover, ISO promoted M2 polarization in macrophages in a dose-dependent manner in vitro. Our study found that ZZO could promote DU wound healing by inhibiting inflammation, which was achieved by macrophage M2 polarization through activating the PI3K/AKT pathway. Further studies have demonstrated that ISO plays major role in the above process. These findings provide a theoretical basis for further preclinical evaluation and lay a foundation for nano-gel compound treatment with ZZO.Diabetic ulcer (DU) has been recognized as one of the most prevalent and serious complications of diabetes. However, the clinical efficacy of standard treatments for DU remains poor. Traditional Chinese medicine (TCM) shows a positive therapeutic effect on DU. Specifically, Zizhu ointment (ZZO) has been widely used to treat DU in long-term clinical practice, but the exact mechanism by which it promotes DU wound healing remains unknown. In this study, network analysis and high-performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS) were conducted to identify the active compounds of ZZO. We detected isovalerylshikonin (ISO), mandenol, daidzein, kaempferol, and formononetin in both network analysis and UPLC-HRMS. Moreover, ZZO could ameliorate DU by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and inflammation signaling pathways, according to the results of KEGG analysis. We established a DU mouse model with a high-fat diet and streptozotocin injection  Diabetic ulcer (DU) is regarded as one of the most prevalent complications of diabetes, with a global prevalence of 6.3% , leadingAstragalus mongholicus Bunge , Arnebia guttata Bunge , Asini corii colla , Borneolum , and Calamus draco Willd. —is helpful in clinical practice (ZL201010186284.X).Traditional Chinese medicine (TCM) has become popular in the treatment of DU at multiple levels and in multiple pathways . In a sypractice . We recrpractice . Recentlvia inhibiting Notch4 signaling and promoting the M2 polarization of macrophages  of ZS, ZC, XJ, HQ, EJ, and BP were 7: 3: 3: 6: 5: 1, and the ratio of the components of ZZO to the base materials was 1: 8 . The ZZOUPLC-MS analysis was accomplished by Shanghai Applied Protein Technology. Purified ZZO samples of 0.02 g were weighed and placed in 1.5 ml centrifuge tubes. The samples were dissolved in 1 ml of 70% methanol, then vortexed for 30 min and centrifuged . The supernatant was collected and vacuum freeze-dried. The residue after lyophilization was again dissolved in 2 ml of 40% methanol. The samples were vortex mixed, rotated, and centrifuged , and the supernatant was collected. The ZZO extracts were analyzed using a UPLC-HRMS system . UPLC separation was conducted on an ACQUITY UPLC HSS T3 column  at a flow rate of 0.3 ml/min. HPLC-grade solvents and additives were from ThermoFisher Scientific (United States). The gradient program using 0.1% formic acid in water (phase A) and 0.1% formic acid acetonitrile (phase B) was adopted as follows: 95% A at 0 min to 2% A at 17 min, 95% A at 17.2 min, lasting for 2.8 min, then back to original condition. The column temperature was 35°C and the injection volume of ZZO solution was 2 μL. MS analysis was conducted using the positive ion mode. The instrument was calibrated using external standards before analysis to ensure a mass accuracy of better than 3 ppm throughout the experiment. The source parameters were as follows: spray voltage of 3.8 kV, capillary temperature of 320°C, sheath gas flow rate of 45 arb. units, Aux gas flow rate of 20 arb. units, spare gas flow rate of 0 arb. units, and probe heater temperature of 370°C. A full MS scan (m/z 90–1300) with a resolution of dd-MS2 was used.http://tcmsp-e.com.tcmsp.php), the largest database to include data for TCM pharmacology  , and the GeneCards database (https://www.genecards.org/) using the keywords “diabetic foot ulcer” and “diabetic ulcer” , and only items with p-values <0.05 were selected , including the non-diabetic group  and the DU group without or with ZZO treatment. DU groups were fed a high-fat diet comprised of 60% calories  for three weeks and subjected to STZ  for one week (2 gauze with excipients or ZZO (50 mg/cm2). The dressing was changed daily. All animal experiments were approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine  and correspond to the National Institutes of Health guide for the care and use of Laboratory animals .Adult male C57BL/6 mice  were purchased from Shanghai Model Organisms Center, Inc. The mice were housed in the Experimental Animal Center of Shanghai University of TCM in a specific pathogen-free environment under a 12 h light/dark cycle with freely available water and food. Mice were randomly assigned to each group .To assess the condition of wound healing in mouse backs, the wound was photographed by a digital camera  on days 0, 3, 7, and 14 . The wouThe wound tissues were fixed with 4% paraformaldehyde  for 48 h. Following standard paraffin embedding and sectioning, the wound tissues were subjected to hematoxylin and eosin  and Masson staining . Stained tissues were analyzed using a digital slide scanning system (Precipoint M8).To detect the co-localization of macrophages, a double IF was performed. After routine deparaffinization, rehydration, and antigen repair, the sections were incubated with 3% bovine serum albumin . Then a rabbit anti-F4/80 antibody  mixed with a mouse anti-arginase 1 or a mouse anti-CD68 , and a mouse anti-ARG1 mixed with a rabbit anti-p-AKT  were incubated overnight at 4°C. Tissue sections were washed with PBS three times and cultured in the dark with donkey anti-mouse Alexa Fluor 488  or donkey anti-rabbit Alexa Fluor 488  and donkey anti-rabbit Alexa Fluor 555  or goat anti-mouse Alexa Fluor 555  for 2 h at room temperature at the next day and then washed three times with PBS. Nuclei were counterstained with DAPI  for 10 min and then washed with PBS; antifade mountant  was added to each coverslip, and the coverslips were placed on the slides. Positive signals were captured using a confocal microscope  and further analyzed with the Adobe Photoshop CS software program. Analysis of fibrosis and angiogenesis was performed by dual IF with mouse anti-α-SMA  and rabbit anti-CD31 antibodies . The methods were as described previously.GAPDH as an internal control, quantitative PCR analysis was performed to quantify the relative mRNA expression of targeted genes. The result of qPCR from the threshold cycle (Ct) and use the 2−△△Ct method was defined to calculate the relative expression level. The primer pairs specific for various genes used in our experiments are listed in Trizol  was used to extract RNA according to the manufacturer’s instructions. NanoDrop ND-1000 was used to determine the concentration of RNA, and the extracted RNA was stored in a refrigerator at −80°C. Using 500 ng total RNA as a template, cDNA was synthesized with the cDNA Synthesis Kit . Samples were stored at −20°C and subjected to qPCR using a StepOnePlus Real-Time PCR System (Applied Biosystems). Each qPCR sample was performed in a 10 μL reaction containing 2xSYBR Green qPCR Master Mix , 10 nM forward and reverse primers, and 2 μL cDNA in triplicate. The qPCR protocol was executed for 45 cycles, with each cycle consisting of denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Using Wound tissues and cells were lysed in a RIPA lysis buffer  containing proteinase and phosphatase inhibitor cocktails . An equal amount of protein sample (20 μg) from each group was loaded on a 7.5% or 10% SDS-PAGE gel  along with standard molecular weight markers , followed by transfer onto a polyvinylidene difluoride (PVDF) membrane , which was then blocked with 5% BSA for 2 h. Membranes were incubated overnight at 4°C with a rabbit anti-p-AKT antibody , a rabbit anti-p-PI3K antibody , a mouse anti-PI3K antibody , a rabbit anti-AKT antibody , a rabbit anti-Arg1 antibody , a rabbit anti-CD206 antibody , a rabbit anti-CD163 antibody , and a rabbit anti-β-actin antibody  in 5% BSA. After rigorously washing with TBS containing 0.1% Tween-20 , membranes were incubated at room temperature for 1 h with a goat anti-rabbit HRP-conjugated IgG antibody  or a goat anti-mouse HRP-conjugated IgG antibody . Subsequently, membranes were washed three times with TBST. Target proteins were visualized using a super-sensitive electrochemiluminescence (ECL) reagent  with a Molecular Imager ChemiDoc XRS System (Tanon).2 conditions. M1 macrophages were induced by lipopolysaccharide  cells were purchased from American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin under 37°C, 5% CORAW264.7 cells treated with ISO-L  or ISO-H (100 μM) were grown on coverslips in 24-well plates to about 30% confluency. After 24 h of incubation, cells were fixed with 4% PFA for 30 min. After washing with PBS three times, cells were stained with a mouse anti-CD68 antibody  and a mouse anti-ARG1 antibody  overnight at 4°C. Next, cells were gently rinsed three times with PBS and incubated in the dark with donkey anti-mouse Alexa Fluor 488  for 2 h at room temperature. After rigorous rinsing, cells were stained with Alexa Fluor 555-phalloidin  for 30 min at 37°C. Subsequently, nuclei were counterstained with 4′,6-diamidino-2-phenylindole  and mounted with an antifade mounting medium . Photographs were taken using a confocal microscope .RAW264.7 cells were seeded into a 6-well plate and then treated with ISO-L or ISO-H. After the cells grew to a 100% confluent layer, 2 ml of serum-free DMEM medium was added. A 10μL pipette tip was used to make a straight scratch on the upper surface. Then, cell debris was removed with PBS, and images were taken at 0 and 48 h using an inverted microscope.t-test. p-values <0.05 were considered to indicate statistical significance.Statistical analyses were conducted using GraphPad Prism . One-way ANOVA and two-way ANOVA were used for comparisons between multiple groups, and pairwise comparisons within groups were analyzed by the Student’s Based on the TCMSP and HIT, 111 compounds from ZS, HQ, ZC, EJ, BP, and XJ were obtained. A total of four duplicate compounds were included, including oleanolic acid, rhamnocitrin, daidzein, and formomonetin. Moreover, there were found to be 38 compounds in ZC, 61 compounds in HQ, 5 compounds in BP, 5 compounds in ZS, 4 compounds in EJ, and 2 compounds in XJ. The overall compounds in ZZO are summarized in Among the six active herb medicines, 1067 targets were retrieved from target prediction databases. A total of 1132 candidate targets for DU were obtained from the Genecard, Drugbank, and OMIM databases. Eventually, 199 common targets  were conAKT1(degree = 72), tumor necrosis factor , SRC proto-oncogene , epidermal growth factor receptor , caspase 3 , prostaglandin-endoperoxide synthase 2 , estrogen receptor , peroxisome proliferator-activated receptor gamma , matrix metallopeptidase 9 , mechanistic target of rapamycin kinase , mitogen-activated protein kinase 1 , kinase insert domain receptor , peroxisome proliferator-activated receptor alpha , interleukin-2 , and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha .The top 100 common target genes were imported into the STRING database, and medium confidence of PPI was input to Cytoscape 3.9.1 for analyzing and constructing the PPI network . In the p < 0.05). The overall results of the GO analysis are summarized in The 100-core putative ZZO targets were subjected to GO and KEGG analysis. The results revealed that 3380 biological process (BP), 251 cellular component (CC), and 3288 molecular function (MF) terms, and 136 pathways, were enriched among the targets  with the AKT target. The results from the molecular docking software are shown in α-SMA and CD31 revealed that the amount of newly formed blood vessels and collagen fibers were remarkably higher in the ZZO group compared with the DU group , as well as anti-inflammatory factors  by RT-PCR, and the results fully correspond with the in vivo findings  mice induce NF-κB activation and IL-1β release, which is characteristic of the M1 phenotype (The multicomponent–multitarget methods of TCM offer various therapeutic activities for DU. As discussed in the introduction, our previous studies have verified that ZZO can promote wound healing in DU though the specific compounds and potential mechanisms have remained unclear. Thus, we adopted network analysis to determine the active compounds and anti-DU mechanisms of ZZO. The results show that ZC and HQ play key roles in anti-DU ZZO. After excavating related targets, the PI3K/AKT pathway was identified for further study. PI3K is activated under physiological stimuli and binds to receptor tyrosine kinase provided with a ligand binding domain. A central component in the PI3K pathway is the serine/threonine-specific protein kinase AKT, with multiple downstream target proteins . The PI3henotype . Our resin vitro. Similarly, ISO promotes the transition of RAW264.7 cells into the M2 phenotype in a dose-dependent manner.Moreover, M2 macrophages can recruit fibroblasts directly or by the secretion of chemokines, such as TGF-β, which promote collagen deposition and damage repair . MeanwhiIn conclusion, we conducted an integrative network analysis and experimental evaluation of ZZO against DU. We evaluated the activation of the PI3K/AKT pathway under ZZO treatment. We found that ZZO can promote M2 polarization by activating the PI3K/AKT pathway and then secreting IL-4, IL10, and VEGF to promote collagen deposition and angiogenesis . However


Biodiesel plants are struggling to find value added applications for fatty acid methyl esters (FAME). One option for FAME valorization would be dibasic esters, which can be transesterified with 2-ethylhexyl (2EH) or other alcohols to produce lubricant basestocks and achieve the most widespread viscosity grades VG46 and VG32. Biocatalytic, metathesis and other synthetic pathways are available to produce dibasic esters. Using a ruthenium-based catalyst, methyl oleate was converted into monounsaturated dibasic ester by metathesis and reached VG22 after transesterification with 2EH in this investigation. Synthesized 2EH esters of other dibasic acids showed distinct viscometric trends. Their correlation implied that FAME from gondoic and erucic acids should result in VG32 and VG46 respectively, if converted into 2EH dibasic esters. Pour points demonstrated excellent low temperature fluidity and resistance to heat thinning when monounsaturation was retained. Oxidative stability properties remained acceptable, volatility was lower than that of VG46 mineral oils. Mixed alcohols, acids and esters can also be used for meeting VG specifications or achieving higher biobased contents. Currently petrochemical ester basestocks dominate in high performance hydraulic fluids (HF). However, fractionation of FAME into high-erucic/gondoic esters in biodiesel plants can produce a valuable biobased feedstock for large volume manufacture of HF and other lubricants. Monounsaturated dibasic esters were obtained by FAME metathesis and tested for viscosity, extreme temperature and other lubricant properties. Their 2EH derivatives can produce 100% bio-derived basestocks for widespread heavy duty hydraulic fluids. However, the demand for fractionated FAME is still limited, partly due to slow markets, such as the lubricant industry.With petroleum prices fluctuating around relatively low levels, biodiesel plants often have operational difficulties. Attempts are made to reduce raw material costs, streamline the manufacture and valorize the products, among which fatty acid methyl esters (FAME) are by far the most critical. As the feedstock, oils with unusual fatty acid (FA) profiles are sometimes used. While saturated FAME are often removed to improve low temperature fluidity, other FAME of various chain lengths or double bond positions typically remain intermixed with the dominating methyl oleate and linoleate. Only a few biodiesel plants consider the opportunity to fractionate FAME based on their chain lengths. Traditionally oleochemical industry focuses on free FA as a raw material for various niche applications. Segregation of individual free FA is quite complicated, because they are not easily fractionated and distillation often results in decarboxylation.2etc. No dominant ester has emerged as a basestock yet, since these esters are still manufactured in batch processes. Many of them are difunctional, e.g. neopentyl glycol esters, dimer acid esters,3 phthalates, main constituents of estolides,4 glycol oleates, adipates, azelates, sebacates,5etc. The latter three belong to so called “dibasic esters”, which are colloquially defined as the esters of linear α–ω dicarboxylic acids. It might be noted that dibasic esters should be synthesized using C5 or preferably longer alcohol chains, such as 2-ethylhexyl (2EH). While in usage, shorter moieties tend to produce volatile carboxylic acids or ketones due to hydrolysis and oxidation of the esters. This is problematic in HF because of objectionable odors, corrosion issues and other side effects.Among many lubricant raw materials, basestocks for hydraulic fluids (HF) represent an opportunity of high volume and substantial value for bio-derived chemicals. A broad selection of different esters is already utilized there, such as polyol esters, mellitates, estolides, soaps3 worldwide.6 Formal VG specifications define HF viscosity at 40 °C, so VG46 should fall between 41.4 and 50.6 mm2 s−1, while VG32 between 28.8 and 35.2 mm2 s−1. Their heat thinning trends are also very important for heavy duty applications, because less heat-dependent viscosity assures better efficiency. In lubricant technology viscosity reduction with heating is defined by Viscosity Index (VI). Typically, mineral HF attain VI ∼100, ester-based HF thin down slower with VI ∼130 and vegetable oils stand out with VI ∼200 due to excellent resistance to heat thinning. Approximate variation of viscosity with temperature for VG22, VG32 and VG46 is shown in Despite broad availability of biobased esters, HF usually utilize petrochemical esters for better costs and performance. Viscosity grades VG46 and VG32 are by far the most dominating volume-wise, totaling over a million m7 and are employed to predict viscometric properties of dibasic esters theoretically.In essence, lower VG might be more favorable if equipment operates at higher temperatures, but uses HF of high VI. Consequently, despite lower volumes, VG32 is also very important, as manifested by wide usage of rapeseed oil in HF. Squalane and di-2EH-sebacate (26S10 in this report) are often used as control fluids in lubricant researchetc.). Additives for dibasic esters and vegetable oils are well established among HF formulators, so the latter properties rarely become extremely challenging.The basestocks also need to address low temperature solidification and oxidative degradation, mostly related to acidification, vapor losses and residue formation. Cold properties can be improved by pour point depressants and oxidative stability by antioxidants, but they still mostly depend on the intrinsic basestock properties. A number of other technical aspects are also important for formulated HF. However, most of them are either dictated by basestock manufacture, such as clarity, color or contaminants, or can be resolved through the formulation with additives and system adaptation  are compared to innovative dibasic esters with or without monounsaturation as products of FAME metathesis and transesterification with 2EH or other alcohols. The focus is devoted to FAME of oleic, gondoic and erucic acids, the former being a predominant component of biodiesel plant products. The latter FAME are present in various quantities depending on the origins of transesterified oils. Erucic acid may even be the most abundant FA in some rapeseed varieties, crambe and other non-food oils. Synthesis and viscometric testing provides sufficient data to identify which FAME would be the most suitable for conversion into VG32 and VG46 basestocks. Many properties have to be attained by fully formulated HF, such as lubricity or corrosion inhibition, but they can mostly be controlled by additives and manufacture practices. Therefore, in this investigation the experiments are limited to low temperature fluidity, volatility and oxidative stability testing, i.e. the properties which are the most relevant to the basestock itself.In this investigation a series of conventional 2EH dibasic esters  or mono-unsaturated (“U”). The tail of the code denotes the number of C atoms in the dibasic acid. For example, thapsic acid (α–ω C16:0) and α–ω C18:1 dicarboxylic acid are assigned “16” and “18” respectively. Sometimes several dibasic esters are bundled into one group, such as “26Sxx” (2EH esters of saturated dibasic acids), “xxS18” (octadecanedioates) or “xxU18” (octadecenedioates).In addition to dibasic esters, several oils are studied for comparison, representing reference compounds and commercial basestocks, N,N-dimethylaminopyridine , dichloromethane  and dicyclohexyl carbodiimide  were purchased from Sigma-Aldrich. Perstorp Inc. (Sweden) kindly provided a free sample of 2-propylheptanol.Oils 26S9, 26S10 and 26S12, representing fully saturated α–ω dibasic esters , squalane, thapsic  and 1,20-eicosanedioic acids were purchased from TCI Chemicals (Belgium). Low Erucic Acid Rapeseed oil (LEAR), distributed by the grocery chain Maxima (Lithuania) was purchased in a local store. Polyalphaolefin PAO8 was received from Ineos Oligomers (USA) as Durasyn 168. Mineral oil RL208 was manufactured by Shell and purchased from Weber Reference Oils (Germany). Reagent grade 2-octanol (97%), 2-ethylhexanol , 2-butyloctanol (95%), 4 −1) was selected to represent mol. wt of LEAR, since LEAR is mostly composed of oleate glycerides (over 60 mol%). Several researchers investigated PAO8 composition chromatographically, concluding that C40H82 (563 g mol−1) should be the most abundant hydrocarbon.17 In mineral oils it would not be reasonable to identify any particular molecule to represent the whole mixture. So a typical practice to calculate the average mol. wt of mineral basestocks using ASTM D2502 was employed, relying on their viscosities at 100 °F (i.e. 37.8 °C) and 210 °F (i.e. 98.9 °C). The improved approach which also uses density values18 was employed:d is density (g mL−1) at 15.6 °C; v100F and v210F are kinematic viscosities (mm2 s−1) at 37.78 °C and 98.89 °C respectively.Some reference oils contained various compounds of different mol. wt Synthetic and vegetable oils usually have an identifiable component, which is much more abundant than others. Glycerol trioleate  in dichloromethane with dicyclohexyl carbodiimide.The dibasic esters were mostly synthesized from methyl oleate by following the scheme in 1H NMR : δ 3.98–3.84 , 2.23  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>OCH2–), 1.60–1.45 , 1.34–1.08 , 0.82 , 0.81 .13C NMR : δ 174.09 (CO), 66.61 (–O–CH2), 38.77 (CH), 34.45 (COC̲H2–), 30.43, 29.63 29.60, 29.48, 29.28, 29.17, 28.92, 25.06, 23.81, 22.97 (–CH2–), 14.03 (–CH3), 10.99 (–CH3).Syntheses of 2EH esters of thapsic acid (26S16) and eicosandioic acid (26S20) have not yet been reported. Thapsate was synthesized in a solution of thapsic acid  and DCM (500 mL) by adding 2EH alcohol , DMAP  and DCC . Reaction mixture was stirred for 72 hours at 35 °C. Precipitated solid was filtered off, DCM was removed under reduced pressure. Residue was dissolved in hexane (200 mL) and solution was eluted  through a pad of silica gel (5 cm height). Solvent was evaporated on rotary evaporator, and residual volatiles were removed under reduced pressure (0–1 mm Hg). Obtained di(2-ethylhexyl) hexadecanedioate (code 26S16) appeared as light amber liquid . 1H NMR : δ 4.05–3.94 , 2.31 , 1.69–1.50 , 1.41–1.23 , 0.91 , 0.90 .13C NMR : δ 174.09 (CO), 66.61 (–O–CH2–), 38.77 (CH), 34.46 (COC̲H2–), 30.44, 29.69, 29.68, 29.65, 29.61, 29.48, 29.28, 29.17, 28.93, 25.06, 23.81 and 22.97 (–CH2–), 14.03 (–CH3), 10.99 (–CH3).Synthesis of 2EH ester of eicosandioic acid (26S20) was carried out in a solution of 1,20-eicosandioic acid  and DCM (500 mL) by adding 2EH alcohol , DMAP  and DCC . Reaction mixture was stirred for 72 hours at 35 °C. Precipitated solid was filtered off, DCM was removed under reduced pressure. Residue was dissolved in hexane (200 mL) and solution was eluted (ethyl acetate and hexane mixture 1 : 19 vol.) through a pad of silica gel (5 cm height). Solvent was evaporated on rotary evaporator, and residual volatiles were removed under reduced pressure (0–1 mmHg). Obtained di(2-ethylhexyl) eicosanedioate (code 26S20) appeared as light amber liquid . cis- and trans-isomers, so the obtained xxU18 esters cannot be considered as individual organic compounds. Distribution between the cis- and trans-isomers in 1U18, which was used for synthesizing xxU18 was analyzed by NMR and GC as described,16 see trans-/cis-isomers was 73.3/26.7 and 73.7/26.3 respectively, showing an excellent agreement. Roughly, trans-isomers are nearly three times more abundant than cis-isomers.It should be noted that due to metathesis-based synthesis monounsaturated dibasic esters represent a mixture of i.e. pour point. Kinematic viscosities were measured by adapted ASTM D445 procedure using Cannon-Fenske capillary viscometers, maintaining 40 °C or 100 °C temperatures with ±0.1 °C accuracy. In this report the term “viscosity” refers exclusively to kinematic viscosity, unless specified otherwise. Viscosity values from literature sources were also used for discussion. When dynamic viscosities were extracted, they were divided by density and converted into kinematic viscosities. When viscosities at 100 °F and 210 °F were extracted, they were converted into kinematic viscosities at 40 °C and 100 °C respectively using ASTM D341 protocol. Viscosity Index (VI) was calculated using ASTM D2270 methodology. Pour points were measured using an adapted ASTM D97 procedure for low-volume samples.19 As required by the standard, sample pouring was inspected at 3 °C increments using +21 °C as a starting temperature. Each sample was tested at least twice, but no attempt was made to make runs at other starting temperatures. As implied by the ASTM D97 standard, the measurement procedure of pour points below −57 °C was not extended to lower temperatures.The procedures include measurements of viscosities and low temperature fluidity, −1 was applied and initially the thermal ramping of 50 °C min−1 was used for the temperature range of 50–235 °C. Afterwards, the ramping was reduced to 5 °C min−1 until reaching 249 °C and then the sample was held for 4 minutes under isothermal conditions. Oxidative degradation was screened using a thin film oxidation procedure as described in detail previously.20,21 Vapor losses were measured gravimetrically by placing the oil sample in a film of 500 μm thickness on a steel coupon and heating it at 120 °C under ambient humidity. The coupons were weighed periodically to monitor the vaporization rate. Polymerization also took place during the tests and some samples formed insoluble residues. The duration until more than half of the residual film volume was no longer soluble in acetone was recorded. Two or more runs were carried out for each oil both in NOACK volatility and thin film oxidation tests.The procedures include measurements of volatility using thermogravimetric analysis (TGA) and oxidative stability using thin film oxidation tests. NOACK volatility protocol of ASTM D6375 was employed by using Pyris TGA1 instrument (PerkinElmer). Initially, 9–10 mg oil samples were placed in a platinum crucible of 5.5 mm ID and a 2.8 mm height. Reference oil RL208 was used for calibration to establish the air flow and heat ramping parameters. The flowrate of 20 mL min4.Out of many lubricant properties, viscosity is often the most important, in particular for HF. Therefore, this report devotes most attention to the relationships between molecular architecture of dibasic esters and their viscosities. One of the major goals is to establish which particular FAME is suitable for conversion into VG32 or VG46 basestocks. These viscosity grades are by far the most popular in HF.6,13 for the most promising dibasic esters. Their conclusions are summarized in the below subchapters along with the results of the oxidative stability experiments.Many other properties are also important for the basestocks, such as low-temperature fluidity, volatility, oxidative stability, heat capacity, ability to dissolve additives and perform well in lubricity, corrosion and other bench tests. Good performance in many of these tests have already been demonstrated in previous reports13 or acquired from suppliers, show a rather broad range of properties. Viscosities of some these dibasic esters were also reported by other researchers.22 Their data23 along with the results of this study, including volatility and pour point measurements, are itemized in Synthesized dibasic esters, including those described previously−1 for PAO8 and squalane respectively. Compared to 563 and 422.81 g mol−1 in 7 to represent basestocks. Their viscometric, extreme temperature and degradation trends are compared to those of dibasic esters in the sections below.It should be noted that it was necessary to convert some viscosities, extracted from literature, or assume average mol. wt for some oils using ASTM D341 or D2502 as described above. For verification, the conversion yields 550 and 395 g molAdipate, azelate and sebacate dibasic esters have been used in lubricants for many decades. However, their viscosity is too low to be utilized as basestocks for VG32 and VG46 hydraulic fluids. Since 2EH alcohol is the most popular and usually the most cost-effective choice in dibasic esters, 2EH esters with saturated dibasic acids are given higher priority industrially. Its biobased version is also available on the market. Viscosities of 2EH esters of various dibasic acids clearly depend on their mol. wt, 25 or hydrocarbons.26v = 1.2232·exp(0.0054·mol. wt) with R2 = 0.9704 at 40 °C and v = 0.6093 exp(0.004 mol. wt) with R2 = 0.916 at 100 °C. If viscosities at 40 °C per VG specifications are considered, the trendlines imply that 32 mm2 s−1 would be reached at 604.5 g mol−1 and 46 mm2 s−1 at 671.7 g mol−1. These values approach those of possibly industrially viable self-metathesis products, α,ω-C22:1 from FAME with Δ11 (e.g. methyl gondoate C20:1-Δ11) and as α,ω-26:1 from FAME with Δ13 (e.g. methyl erucate). Considering the former, a similar value of 32.85 mm2 s−1 at 40 °C for 2EH-terminated mixed diesters of monounsaturated α,ω-dicarboxylic acids HOOC–C20H38–COOH has already been reported.27 Despite the same mol. wt those acids contain methyl branching, which might affect viscosity somewhat, but the difference is not likely to be large. This confirms that the esters of 2EH and dicarboxylic α,ω-C22 or α,ω-C26 acids with or without methyl branches should have excellent viscosities for VG32 or VG46. It should be noted that if biobased 2EH is used, its esters with α,ω-C22 or α,ω-C26 would constitute 100% bio-derived VG32 or VG46 respectively.The relationship between viscosity and mol. wt appears to approach exponential both at 40 °C and 100 °C. This agrees well with the expectations, as reported by other researchers for fatty esters28 and pour point depressants29 should lead to a viscosity increase by 10–20%. The thickening effect would comfortably accommodate the formulation into the needed VG. Therefore, gondoate- and erucate-based FAME might provide a valuable feedstock for the manufacture of high volumes of HF. Among industrial crops, gondoic acid is present at significant levels in Simmondsia chinensis (jojoba), Camelina sativa (camelina) and several other oilseeds. Availability of erucic acid is much broader, including high-erucic rapeseed, wallflower, mustard, Crambe abyssinica (crambe) and other sources. If these oils are processed in biodiesel plants, distillation of FAME can be a viable option for obtaining methyl erucate feedstocks of sufficient purity for metathesis as well. It should be noted that biocatalytic pathways to selectively hydrolyze and transesterify erucic a.30 or convert erucic a. in to α,ω-C22:1 have already been reported,31 which provides one more incentive to fractionate FAME and produce dibasic esters for HF.As a matter of fact, formulated HF contain additives, such as antioxidants, anti-wear agents, rust inhibitors and many others, including several wt% of relatively high mol. wt polymers. Addition of 1–2% of viscosity index improversvs. 17U18 or 26S18 vs. 26U18. This agrees with reports on viscosities of stearate, oleate and linoleate esters,25 which find lower viscosity with increasing unsaturation. If 17S18 vs. 26S18 or 17U18 vs. 26U18 are compared, no significant dependence of viscosity on either methyl or ethyl branches could be observed. It seems that alcohols heavier than 2-butyl octanol would be beneficial to approach VG46. However, it is very problematic to use heavy alcohols for industrial (trans-)esterification. Even under high alcohol excess, the reaction typically does not go to completion, with significant levels of partial esters and free alcohols still present. These need to be distilled off and it becomes necessary to employ deep vacuum and high temperatures. The high temperatures needed for distilling alcohols heavier than ∼C13H27OH lead to the side reactions of progressively larger scale. In addition, high costs of such alcohols and the lack of biobased options present even further limitations. Therefore, the approach of using heavier alcohols and high-oleic FAME appears less viable than the one above, based on 2EH and high-erucic FAME, see Another option to achieve VG32 or VG46 viscosities might be esterification with other alcohols, heavier than 2EH. Esters of several Guerbet alcohols with saturated and monounsaturated C18 dibasic acids are compared, It should also be noted that viscosities of dibasic esters appear significantly lower than those of hydrocarbons of similar mol. wt, cis-unsaturation , are the main factors that contribute to VI. Monounsaturation seems to benefit VI in dibasic esters as well, when comparing 17S18 vs. 17U18 or 26S18 vs. 26U18. Slow heat thinning is very important for HF, leading to better energy savings, less wear and improved longevity of hydraulic systems. This tendency of dibasic esters, fortified by lower-than-expected volatility and viscometric suitability for VG32 or VG46, are very helpful in utilizing those basestocks for heavy duty HF.Oil refiners continuously strive to achieve higher VI for mineral basestocks. Conventional crudes commonly have VI below 100, therefore, hydroisomerized and other deeply refined basestocks have been developed. Nevertheless, they rarely exceed VI of 140, even PAO8 has VI = 135. Dibasic esters demonstrate clearly higher VI than those of mineral oils or synthetic hydrocarbons. In most cases heat thinning of dibasic esters is slower than that of polyol esters or other niche basestocks. Vegetable oils, such as LEAR with VI = 229, represent the only basestock type that can offer higher VI, but that comes at the expense of oxidative stability and low temperature fluidity. Molecular linearity and abundance of unsaturation, in particular vice versa, it has to operate for long hours in sunny hot conditions. Under those circumstances volatility and oxidation may become important, as discussed below. With respect to another thermal extreme, HF should resist crystallization and solidification at low temperatures.Viscometric parameters are usually most important for the lubricant basestocks, but a series of other properties must also be considered. In case of HF, the range of operating temperatures must also be assessed. Hydraulic equipment might need to start up in cold winter and i.e. it defines the temperature at which the liquid stops pouring when cooled down gradually. Pour points usually go up with increasing mol. wt, but branching, double bonds and other structural features also have strong influence. Some trends become evident when comparing pour points of dibasic esters with those of conventional basestocks, In order to assess low temperature fluidity, pour points are typically measured. A pour point can be perceived as an opposite of the melting point, Pour point dependence on mol. wt is evident when comparing the 26Sxx homologues. Exact values are also listed in the 32 and recorded 1.8%. Evaporation of xxS18 and xxU18 dibasic esters appears comparable, 33 Nevertheless, the acquired test results confirm that emissions from FAME-derived dibasic esters are much lower than those from mineral basestocks. The dibasic esters should be considered similar to the recognized low volatility basestocks, such as PAO8 or LEAR. Formulations with these basestocks demonstrate increased flash points, absence of objectionable odors and improved resistance to fire hazards, all of which are very beneficial for HF.At another thermal extreme of high temperatures, volatile losses are not desirable in HF. So the presence of low mol. wt components should be avoided, because volatility cannot be significantly reduced by using some additives. A standard TGA test for the basestock volatility is performed using a NOACK protocol at 250 °C. Mineral oil RL208 shows by far the fastest evaporation at 11.7% despite its viscosity being above VG32 or most of the tested esters. Since mineral oils contain broad diversity of hydrocarbons and heterocycles with varying mol. wt, it can be expected that some constituents are more volatile and evaporate more rapidly. Synthetic basestock PAO was specified at 2.8% NOACK volatility34 Antioxidant-fortified LEAR is a frequent choice for heavy duty HF, because operating temperatures of hydraulic systems are lower as a result of better heat capacity in esters than hydrocarbons.35 Therefore, this report mostly focuses on the qualitative ranking of oxidation trends of dibasic esters by comparing them to the conventional basestocks. One problematic outcome of HF degradation is oxidative decomposition, which is measured using a thin film procedure in this study. In order to minimize the effects of thermodynamic evaporation, saturated and monounsaturated dibasic esters of very similar mol. wt are compared side-by-side, Oxidative stability of HF is important, but not as critical as in engine oils, gear oils or many other lubricants. Frequently, heavy duty equipment can use vegetable oil-based HF for much longer durations than mineral oil HF despite dramatically poorer results of the former in standard bench oxidation tests.Samples are tested in duplicates and all recorded values are displayed without averaging. Contribution of monounsaturation to the decomposition rates is still quite evident, but it does not appear that this could be extremely detrimental. Some difference between 2EH and 1-methylheptyl moieties can be identified. Dibasic esters with the latter seem to yield lower vapor losses, which agrees with the expectation that secondary ester linkages in 17S18 and 17U18 resist decomposition better than the primary ones in 26S18 and 26U18 respectively. However, more testing would be needed to prove this statistically.36 the decomposition rates can still be compared to other basestocks. However, vapor losses of mineral basestocks should not be considered as oxidative decomposition under this protocol, because NOACK tests suggest that RL208 and 350SN might have relatively high amounts of lower mol. wt fractions. Therefore, only other dibasic esters as well as synthetic and vegetable basestocks are outlined. PAO and LEAR appears to produce volatiles somewhat slower, Despite expectations that monounsaturation should accelerate oxidative scission with respect to aliphatic chains,27 which found their stability fully sufficient for HF. Advantages of monounsaturated dibasic esters against LEAR would be even more evident in thinner films, where not only oxidative, but electrochemical aspects become important.37 Bearing in mind that LEAR is often used as a basestock for heavy duty HF, residue formation trends of monounsaturated dibasic esters are not worrisome. In general, their resistance to oxidative degradation should be fully sufficient to assure excellent stability.Another outcome of HF degradation is the formation of insoluble residues. Thin film experiments show that all saturated dibasic esters remain liquid until the end of testing, even despite major vapor losses. For example, the vapor losses of 26S9 amount to more than 85% after 450 h, but the remaining film is still fully liquid. Mineral oils, PAO8 and squalane also show similar trends, etc.) or contaminants .A number of other technical aspects have to be considered when evaluating a specific dibasic ester as a candidate for HF basestock. Lubricant blenders expect to see some data on lubricity, corrosion resistance, water rejection, elastomer compatibility, biobased contents, biodegradation and maybe other aspects. The latter three strongly depend on the alcohol moiety, but if biobased 2EH alcohol is utilized, they should not be overly problematic. Lubricity, corrosion inhibition, oxidative degradation and water rejection are very dependent on basestock uniformity and functional additives. Unfortunately, these properties might be affected strongly negatively by various foreign components in the formulation. Therefore, optimization of additive types and concentrations should only be carried out when the selection of basestock is finalized. Therefore, manufacture-related parameters should be well established, such as amounts of byproducts 45 the formulation should meet VG46 specifications as well.Many pathways have been reported to produce 2-butyl octanol from bio-derived resources. It was obtained at 77% yield from 1-hexanolAnother approach to obtain 100% bio-derived HF basestocks could utilize a commercial 2EH from renewable resources and larger dibasic acid moieties. As discussed above, 38 of VG32 formulations with of α,ω-C18:1, which was esterified with 1 : 1 blend of 2EH and 2-butyloctanol show good additive compatibility and imply great opportunities for high performance HF, utilizing monounsaturated dibasic esters. Furthermore, the benefits of monounsaturation are evident not only in major improvements of VI and low temperature fluidity. Tribological benefits of monounsaturated compounds are already recognized for their contribution to tribofilm formation.46 Therefore, monounsaturated basestocks become very appealing to HF blenders.Mixed alcohols can also be used for esterification. Previous results47 and provide high amounts of erucic and gondoic acids, respectively. Valorization of fractionated FAME would add more value for biodiesel plant products, open up more markets for biobased materials and produce further socio-economic benefits due to excellent performance of monounsaturated dibasic esters in heavy duty lubricants.The feedstock for manufacturing such basestocks can be FAME, produced at existing biodiesel plants from a variety of oils, including industrial crops, and fractionated using conventional distillation. Among non-food oilseeds, crambe and camelina are especially promising, because they can be cultivated on low-value land5.i.e. FAME with Δ9 double bond) led to VG22 (after transesterification with 2EH), while the data correlation suggested that such conversion of FAME with Δ11 unsaturation (e.g. methyl gondoate C20:1-Δ11) should lead to VG32 and FAME with Δ13 unsaturation (e.g. methyl erucate) to VG46 basestocks. Metathesis and transesterification of methyl oleate into VG32 was carried out by employing heavier alcohols than 2EH, just industrially that might require higher processing temperatures and deeper vacuum.A novel category of dibasic esters, based on monounsaturated linear α–ω dicarboxylic acids, was synthesized from FAME using metathesis during the initial stage. Viscosity grades for mainstream hydraulic fluids were targeted, mostly employing 2EH alcohol for subsequent transesterification. Viscometric measurements showed that the metathesis of methyl oleate (A series of basestock-related properties was tested and compared to widely used mineral oils, rapeseed oil and synthetic hydrocarbons. Resistance to heat thinning and VI was much higher, especially in monounsaturated esters, compared to conventional basestocks. Methyl branching improved low temperature fluidity somewhat, while ethyl and longer branches were dramatically more effective, as were double bonds. Monounsaturation was not excessively detrimental to oxidative decomposition and the dibasic esters were many times more resistant to oxidative solidification than LEAR. Their volatility was much lower than that of mineral oils and appeared comparable to synthetic basestocks. Consequently, their formulations can exceed high performance requirements, featured by petrochemical HF, while being able to approach the bio-derived contents of vegetable oil HF, which are not considered heavy duty due to rapid oxidation. High-erucic FAME might be particularly suitable as a feedstock for such monounsaturated dibasic esters and industrial considerations should be made for their commoditization.There are no conflicts to declare.


Plasmodium falciparum parasites is important to track emerging and new mutations and trends in established mutations and should serve as an early warning system for antimalarial resistance. Dried blood spots were obtained from a Plasmodium falciparum malaria survey in school children conducted across eight counties in western Kenya in 2019. Real-time PCR identified 500 P. falciparum-positive samples that were amplified at five drug resistance loci for targeted amplicon deep sequencing (TADS). The absence of important kelch 13 mutations was similar to previous findings in Kenya pre-2019, and low-frequency mutations were observed in codons 569 and 578. The chloroquine resistance transporter gene codons 76 and 145 were wild type, indicating that the parasites were chloroquine and piperaquine sensitive, respectively. The multidrug resistance gene 1 haplotypes based on codons 86, 184, and 199 were predominantly present in mixed infections with haplotypes NYT and NFT, driven by the absence of chloroquine pressure and the use of lumefantrine, respectively. The sulfadoxine-pyrimethamine resistance profile was a “superresistant” combination of triple mutations in both Pfdhfr (51I 59R 108N) and Pfdhps (436H 437G 540E), rendering sulfadoxine-pyrimethamine ineffective. TADS highlighted the low-frequency variants, allowing the early identification of new mutations, Pfmdr1 codon 199S and Pfdhfr codon 85I and emerging 164L mutations. The added value of TADS is its accuracy in identifying mixed-genotype infections and for high-throughput monitoring of antimalarial resistance markers.Molecular surveillance of  Plasmodium falciparum kelch 13 (Pfk13), though at low frequencies (Chloroquine and sulfadoxine-pyrimethamine (SP) resistance in Africa was historically imported from Southeast Asia , 2. Addiquencies , 5. Bothquencies  conductequencies . Thus, mPlasmodium falciparum chloroquine resistance transporter (Pfcrt) mutation K76T (Pfmdr1) , but the effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine and artemether-lumefantrine (AL) have not been clearly defined  treatment failure in the Greater Mekong subregion, in Cambodia, Thailand, and Vietnam  and selects for partner drug resistance , 8. Mutaion K76T , 11 and (Pfmdr1)  mutation(Pfmdr1) . Pfcrt a defined . Furtheremisinin –17. Addi Vietnam . In addi Vietnam .Pfdhfr) and dihydropteroate synthase (Pfdhps), the two key enzymes in the folate biosynthesis pathway, which mediate resistance to the antifolate drugs sulfadoxine and pyrimethamine, respectively, have been reported in numerous studies , termed a fully resistant combination, that is characteristic of East African parasites  were malaria positive by RDT. The dried blood spots (DBSs) from RDT-positive children were screened by 18S rRNA reverse transcription-PCR (RT-PCR) to identify 1,263 (56%) samples with detectable levels of DNA to take forward for TADS. The median (range) RT-PCR cycle threshold (TC) for the samples was 31 (19 to 39). An initial random set of 192 samples were screened, and over 50% of the samples did not yield a PCR product for the genes. Therefore, to generate a feasible number of amplicons in duplicate for TADS, samples with low parasitemia above the median TC of 31 and those from schools with a low representation of positive samples (<10) were excluded. In total, 500 samples were used to generate amplicons in duplicate for Pfdhfr, Pfdhps, Pfk13, and Pfmdr1 genes. Sequence data were obtained from 322, 225, 196, and 98 samples, respectively  genotyped, and mixed infections could not be accurately determined using the capillary sequencing method and thus were not analyzed (Three SNP loci were genotyped in pulation . The majpulation  33), le, lePfmdranalyzed .Pfdhfr, five SNPs were genotyped. The mutation T85I is unique, since it has, to our knowledge, not been described before (Pfdhfr triple mutant (51I 59R 108N) was also prevalent in the study population at 78%. All other haplotypes, including the wild type, were rare, though remarkably, 22% of the infections were classified as quadruple mutant based on the number of mixed-genotype infections containing the quadruple haplotype  were 100% wild type. The mutations A569S, previously described in Uganda at <1% frequency  should be surveyed temporally to monitor a rise in frequency and evidence of directional selection. TADS improved the detection of Pfdhfr codon 164, to identify quadruple mutants (and the “superresistance” dhfr-dhps) combination , resistant (mutant), or mixed-genotype infections. Newly defined rare variants  was thought to be at fixation due to the absence of sensitive (NCS) parasites between 2006 and 2013 in symptomatic children in the coastal region of Kenya, where malaria is endemic  and Pfdhps triple mutant (437G 540E 581G), suggest that these superresistant combination parasites will escape SP treatment; however, SP in intermittent preventive treatment for malaria in pregnancy (IPTp) remains efficacious (dhfr 59R 108N and dhps 437G 540E)  dominated (Pfdhfr (51I 59R 108N) and Pfdhps (436H 437G 540E) triple mutants, which still renders SP ineffective. Similar to mutations found in Uganda and Tanzania, these mutations do not appear to have a clear impact on the efficacy of SP in IPTp, since IPTp-SP is thought to be effective in clearing parasites in Kenya  . This saand 2008 . In contin Kenya . The gredhfr and dhps quintuple-, sextuple-, and septuple-mutant parasites confer resistance to SP, making the drug ineffective for treatment. However, SP in IPTp continues to be effective, despite the widespread and high prevalence of mutant parasites. Thus, the mechanism through which SP is effective as a chemoprophylactic drug is not well understood. It may be due to the long half-life of the drug combination of 4 to 7 days  have maintained school-based malaria infection surveys since 2009 to track changes in transmission intensity –61. Betw– in 2015  and a pr in 2015 . At eachTrained interviewers asked each participating child about details related to their age, bed net use, and any illness on the day of the survey. Each child was asked to provide a finger-prick blood sample for a rapid diagnostic test (RDT) (CareStart) and an ∼50-μL dried blood spot (DBS) on Whatman 3-mm filter paper (Sigma). The DBS samples were allowed to air dry for at least 1 h and individually packed in zip-lock bags, with a desiccant, to prevent cross-contamination and delivered in a cooler box to the nearest health care facility for short-term storage, before shipment to the KEMRI-Wellcome Trust Research Programme laboratories.The study protocol received ethical approval from the KEMRI and National Ethics Review Committee (number KEMRI/SERU/ESACIPAC/11/3822). Additional approval was provided by the appropriate county-level health and education authorities, who were briefed about the survey. At the school level, parental consent was based on passive, opt-out consent rather than written opt-in consent owing to the low risk and routine nature of the study procedures . Individg for 5 min, and 120 μL of the DNA-containing solution was stored at −20°C for further analyses.Parasite DNA was extracted from two areas of the ∼50-μL DBS using the Chelex-saponin method . Each DBM19173) . A 25-µL final quantitative PCR (qPCR) volume was prepared as follows: 2.5 µL each of P. falciparum 18S rRNA forward and reverse primers (10 pmol/µL), 0.625 µL of 18S minor groove binder (MGB) probe (10 pmol/µL), 12.5 µL 2× TaqMan universal PCR master mix, 6.75 µL of sample or 3D7 control samples, with the remaining volume PCR clean water. The reaction was done under the following qPCR cycling conditions: 50°C for 2 min (AmpErase step), 95°C for 10 min (hot-start activation), and then 45 cycles of 95°C for 15 s (denaturing) and 95°C for 1 min . Eight sequencing controls were also prepared from P. falciparum laboratory reference isolates  (BEI Resources) and DNA representing different apical membrane antigen 1 (ama1) variants. The reference DNA isolates were mixed in proportions of 1:1:1, 1:0:0, 0:0.5:0.5, 0:0:1, 0.5:0.25:0.25, 0.44:0.33:0.22, 0.5:0.45:0.05, and 0.5:0.49:0.01 to determine the lowest limit of variant detection in the assay.The parasite DNA was amplified using the TaqMan probe on the ABI Prism 7500 HT real-time system (Applied Biosystems) following a previously described method . The TaqPfcrt (PF3D7_0709000), Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800), Pfk13 (PF3D7_1347700), and Pfmdr1 (PF3D7_0523000) and ama1 (PF3D7_1133400) from the DNA reference isolates were amplified using nested PCR. The first PCR assay was set up as a 10-µL final reaction volume as follows: 1 µL of template DNA (<50 ng), 0.14 µL Expand high-fidelity DNA polymerase (3.5 U/µL) , 0.3 µL forward and reverse 10 mM external primers (Table S1), 0.2 µL of 10 µM deoxynucleoside triphosphates (dNTPs) (Bioline), 1 µL each of buffers 2 and 4, and 6.56 µL of nuclease-free water. The nested PCR was prepared as described above, except that 1 µL of the first PCR assay product with multiplex identifier (MID) -tagged (Table S2) internal forward primers and untagged reverse internal primers (Table S1) were used. Each sample was amplified in duplicate with nonoverlapping MID tags. The following PCR cycling conditions were used: 94°C for 2 min, 10 cycles of 94°C for 15 s, 52°C for 30 s, and 72°C for 45 s, followed by an additional 20 cycles of 94°C for 15 s, 52°C for 30 s, and 72°C for 45 s and a final elongation step of 72°C for 5 min. Successful PCR amplification was confirmed using 1% (wt/vol) agarose gels stained with RedSafe nucleic acid staining solution (iNtRON Biotechnology DR).The drug resistance marker genes Pfmdr1 samples in duplicate and 22 samples without duplicates (14 pools), 314 Pfk13 duplicate samples and 98 samples without duplicates (79 pools), and the 8 ama1 control mixtures. These amplified products were purified using the Zymo ZR-96 DNA Clean & Concentrator-5 kit (Zymo Research) according to the manufacturer’s instructions, and products were then eluted in 30 µL of DNA elution buffer. In contrast, for the second run, the following amplicons were included: 83 samples in duplicate (18 pools) for Pfmdr1,  386 samples in duplicate (50 pools) for Pfdhfr, 422 samples in duplicate (59 pools) for Pfdhps, and the 8 ama1 control mixtures. These amplicons were purified using AMPure XP SPRI beads  as per the manufacturer’s instructions and eluted in 20 μL of PCR-grade water. Purified DNA from the two sequencing runs was quantified using a Qubit double-strand DNA (dsDNA) high-sensitivity (HS) assay kit (Invitrogen) according to the manufacturer’s instructions. Thereafter, the PCR amplicons were normalized to equal amounts of 1 ng each using EB buffer (Qiagen) followed by library construction. Library preparation was done using the Kapa Hyper prep kit and the Kapa dual-indexed adapter (KK8722) (Roche) as per the manufacturer’s instructions. Thereafter, a size selection cleanup was done using 0.8× AMPure XP beads  to select for amplicons with sequencing adapters and eliminate free adapters, primers, nucleotides, salts and DNA fragments not ligated to adapters. The adapter-ligated libraries were then amplified using Illumina primers and cleaned with 0.8× AMPure beads to select for fragments >450 bp in size. The libraries were quantified using a Qubit dsDNA HS kit on a Qubit fluorometer V3 (Invitrogen), and sizes were verified by the DNA 1000 assay kit using the 2100 Bioanalyzer (Agilent). The libraries were mixed in equimolar concentrations, denatured, spiked with 8% PhiX DNA, and finally sequenced using a MiSeq reagent kit v3 (Illumina) with an output of 2 × 300-bp paired reads.PCR amplicons were mixed to create amplicon pools of nonoverlapping MIDs. Since 16 unique MIDs were used, this allowed up to 8 samples per gene to be sequenced in duplicate in each sequencing library. The first sequencing run included 59 mdr1 (samples from sequencing run 1), samples with and without a replicate were included in the analysis, since the inclusion of the samples without duplicates did not impact the number of unique haplotypes identified. A conservative cutoff of 1% was set based on the lowest  lab isolate mixture, unless the haplotype was independently detected in other samples at >1%. Chimeric reads were considered PCR artifacts and discarded. The SNP and haplotype frequencies in the population were calculated as the number of samples that contained the SNP or haplotype over the total number of samples genotyped. Statistical analysis to generate SNP and haplotype frequency tables was carried out in R v4.0.3  primers failed to generate nested PCR amplicons; therefore, Pfcrt was amplified and sequenced using previously described primers and PCR conditions , and SNPs were identified and called based on the 3D7 reference sequences. Nucleotide positions that displayed a peak within a peak in the sequence chromatograms were noted as “mixed.” Consensus sequences were extracted from the sequence assemblies using CLC Genomics Workbench v9.5.3 and used to construct multiple-sequence alignments in Clustal Omega v1.2.1 (The MID tagged nditions , 32 on aa v1.2.1 , 68 to iPfdhfr, OM370904 to OM370913; for Pfdhps, OM370914 to OM370917; for Pfk13, OM370918 to OM370923; and for Pfmdr, OM370924 to OM370928. The Pfcrt sequence data have been deposited in GenBank under the following accession numbers: for codon 76, OM417816 to OM418014, and for codon 145, OM418015 to OM418379.The amplicon sequence data have been deposited in GenBank under the following accession numbers: for


BRCA1/2 mutation carriers aims to improve breast cancer (BC) prognosis. Our aim is to clarify the prognostic impact of tumor size in BRCA mutation carriers with a pT1 BC, which is currently unclear. We are especially interested in differences between pT1a, pT1b, and pT1c regarding the prognosis of node-negative breast cancer, the effect of chemotherapy, and the prevalence of lymph node involvement.Intensive screening in BRCA1/2-associated BC patients were selected from a nationwide cohort. Primary outcomes were 10-year overall survival (OS) per pT1a-b-c group and the effect of chemotherapy on prognosis of node-negative BC, using Kaplan–Meier and Cox models. Finally, we evaluated lymph node involvement per pT1a-b-c group.For this study, BRCA1/2-associated BC diagnosed between 1990 and 2017 were included, of which 679 had pN0 BC. After a median follow-up of 10.5 years, 10-year OS in patients without chemotherapy was 77.1% in pT1cN0 and lower than for pT1aN0  and pT1bN0 . OS was better with than without chemotherapy for pT1cN0  0.56, 95% confidence interval (CI): 0.21–1.48). Lymph node involvement was 24.9% in pT1c, 18.8% in pT1b, and 8.6% in pT1a.963 women with pT1 BRCA1/2-associated BC patients. The results suggest that early detection in BRCA1/2 mutation carriers of pT1a/b BC may reduce mortality and the need for systemic therapy.Smaller tumor size is associated with better OS and less lymph node involvement in pT1 The online version of this article contains supplementary material available (10.1007/s10549-022-06608-1. BRCA1 or BRCA2 mutation have life-time breast cancer (BC) risks up to 75%, and are often diagnosed with BC at a relatively young age [BRCA1/2 mutation carriers may opt for bilateral prophylactic mastectomy, reducing BC risk to almost zero, or participate in a tailored BC screening program [Women carrying a pathogenic germline BRCA1/2 mutation carriers, however, no trials investigating screening exist, and screening efficacy is only presumed to be similar to that of the general population [BRCA1/2 mutation carriers—breast density is higher than for women in regular screening programs, possibly affecting mammography efficacy. Therefore, and to avoid radiation exposure, MRI is already the modality of choice in young women. Additionally, debate is still ongoing whether the association between tumor size and outcome is as strongly present in BRCA1/2-associated BC [BRCA1 mutation carriers has been reported to be weaker than for sporadic BC or BRCA2-associated BC [BRCA1/2 mutation carriers.In the general population, randomized controlled trials (RCT) of mammography screening demonstrated a reduction in BC mortality . Consequpulation . At the iated BC –11. Moreiated BC . TogetheBRCA1/2 mutation carriers have previously been investigated, but so far no studies could directly demonstrate survival benefit from BC screening [BRCA1/2 mutation carriers are currently lacking, the purpose of the current study is to determine the prognostic value of this tumor characteristic within a population of BRCA1/2 mutation carriers. First, we evaluate whether tumor size, categorized as pT1a (0.1–0.5 cm), pT1b (> 0.5–1.0 cm), and pT1c (> 1.0–2.0 cm) according to the TNM classification of malignant tumors, UICC (TNM), is a good prognostic factor of survival in pT1N0 BRCA1/2-associated BC patients who did not receive chemotherapy. We are especially interested in the natural course of disease without chemotherapy and the possibilities to omit this treatment modality for patients with small node-negative tumors, for chemotherapy can severely impact quality of life. Second, we evaluate the effect of chemotherapy in BRCA1/2-associated BC. Third, as lymph node involvement has consequences for both treatment (regardless of tumor size), and prognosis in the general BC population, we evaluate the association between tumor size and lymph node status in BRCA1/2-associated BC patients.Screening programs tailored to creening , 14. GivBRCA1/2 mutations were identified through the departments of Clinical Genetics of the eight Dutch academic medical centers and the Netherlands Cancer Institute. All participating centers’ Medical Ethics Committees approved the study. Written informed consent was acquired from each participating woman, or a close relative or proxy for deceased individuals. Relevant data on patient characteristics, cancer diagnosis, tumor characteristics, and preventive strategies were retrieved and updated through linkage with the Dutch National Cancer Registry and the national pathology database, and from medical files and questionnaires. Therefore, this registry is a mixture of both retrospective data collection and prospective follow-up [Eligible participants were retrieved from the ongoing national HEBON (Hereditary Breast and Ovarian Cancer Research Netherlands) study cohort. In the HEBON study, members of families with pathogenic germline ollow-up . The latBRCA1 or BRCA2 mutation via testing, and diagnosed with pT1a, pT1b, or pT1c breast cancer between 1 January 1990 and 1 January 2017 (regardless of N-status). BC was diagnosed either before or after confirmation of BRCA1/2 mutation carrier status. Patients were excluded if they had distant metastases at BC diagnosis, had received neoadjuvant chemotherapy , or had any other primary tumor prior to BC diagnosis , skin basal cell carcinoma (BCC) or skin squamous cell carcinoma (SCC)). Further reasons for exclusion were insufficient data regarding tumor size, vital status, or date of death.We included only women carrying a proven pathogenic germline BRCA mutation status, and vital status. All reported tumor stadia were histologically determined. ER-status and PR-status were defined positive if immunohistochemical staining showed 10% or more of the tumor cells positive for the receptor. The HER2-status was positive if the immunohistochemical staining was 3 + , or 2 + with a positive in-situ hybridization (ISH) test.We retrieved dates of birth, BC diagnosis, DNA test, and death. We retrieved information on TNM classification current at the time of diagnosis, mode of detection, estrogen receptor (ER) status, progesterone receptor (PR) status, Human Epidermal Growth Factor Receptor 2 (HER2)-status, type of surgery, chemotherapy, HER2-targeted treatment, endocrine treatment, radiotherapy, risk-reducing salpingo-oophorectomy (RRSO), risk-reducing mastectomy (RRM), The primary outcome was 10-year overall survival (OS) from date of BC diagnosis. To allow for prevalent cases at date of genetic testing, we applied left truncation in the survival analyses . Time atWe used Kaplan–Meier survival analysis and the Log-Rank test to compare unadjusted OS curves of the pT1aN0, pT1bN0, and pT1cN0 subgroups not receiving chemotherapy, and of patients receiving chemotherapy vs no chemotherapy within the pT1N0 subgroups. Cox proportional hazards regression models were used to calculate hazard ratios (HR) and 95% confidence intervals (95% CI) for OS. We applied a subject matter knowledge-based model-building strategy, as we were interested in the etiological impact of tumor size on prognosis, and not in prediction. The following relevant clinical or pathological variables were included in the multivariable model: mutational status, ER-status, HER2-status, chemotherapy, endocrine therapy, RRSO, year of diagnosis, and age at diagnosis.Finally, we also investigated the percentage of lymph node involvement at time of BC diagnosis, stratified by tumor size. As early detection is a major determinant of lymph node involvement, we further stratified by screening status. Missing data for screening were imputed by using the timing of DNA test result respective of BC diagnosis, as women with a DNA test result before BC diagnosis are very likely to participate in screening .2 test to compare categorical variables and the Kruskal–Wallis test for continuous variables. All p values are two-sided.Descriptive statistics are shown as proportions or median and range. We used Pearson’s χStatistical analyses were performed using STATA 15.1.Because of missing data for several important covariables (such as the HER2-status), we implemented a multiple imputation model to allow for all variables to be included in the multivariable Cox models without losing observations. The imputation model was built in STATA 15.1, see supplemental materials (Online Resource 1).p < 0.05 for the time-varying term.The models were checked for interactions between the main variable of interest with all other included variables. We tested the proportional hazards assumption through the addition of time-varying effects to the model, using a cut-off of BRCA1/2-associated pT1 BC  and more often had an ER-negative tumor .We included 963 women with primary p = 0.08) and pT1b  patients (Table BRCA1 and BRCA2 combined), 10-year OS of pT1a patients (69.4%) was worse than for pT1b  and pT1c patients . In pT1aN0 patients, OS was worse for those receiving chemotherapy than for those without Table .For node-negative patients not receiving chemotherapy, adjusted HRs for overall mortality of 0.71 (95% CI 0.14–3.51) for pT1a and of 0.36 (95% CI 0.14–0.94) for pT1b were found when compared to pT1c patients (Table n = 963), 28.5% of patients had lymph node involvement at diagnosis. The proportion with positive lymph nodes increased with larger tumor size at diagnosis: 16.4% for pT1a, 20.9% for pT1b, and 32.1% for pT1c (data not shown). In the screened population , we found 20.9% lymph node involvement . The highest proportion of lymph node involvement was found in BRCA2 mutation carriers with a pT1c tumor (Online Resource 1).In the total pT1 population n = 96, 28.5% oBRCA1/2-associated BC patients. We found a strong indication that adjuvant chemotherapy improves survival for patients with a pT1cN0 tumor. Twenty-nine percent of the total population had lymph node involvement, its prevalence increasing with larger tumor size at diagnosis.The results of our study showed that, as is the case for non-hereditary BC, smaller tumor size was associated with improved OS in pT1N0M0 BRCA1 mutation carriers with node-negative tumors of 0.1–1 cm and of 1–2 cm  [BRCA1 mutation carriers between tumor size and OS within the pT1 subgroup  [Our results in pT1N0 BC patients contradict those of previous studies. Narod et al. observed no significant difference in OS between 67–2.90) . SimilarBRCA1-associated BC patients suggested that patients who did not receive chemotherapy appeared to have worse overall survival (OS) than patients who did receive chemotherapy [BRCA1 mutation carriers with pT1N0 BC appear to benefit more from chemotherapy. One of the explanations for this might be that BC in BRCA1 mutation carriers more often is of the triple-negative or basal-like phenotype, and of higher histologic grade [BRCA1-associated BC could benefit from chemotherapy than previously assumed, even when detected by screening in an early stage. While we cannot demonstrate a direct benefit of chemotherapy, we did see that those treated with chemotherapy had better OS than those not treated with chemotherapy, with the exception of women with a pT1a tumor. Considering the small proportion of pT1aN0 patients receiving chemotherapy, this finding may simply be due to chance. Caution is warranted however, as we do not know the reason why physicians chose to advise adjuvant chemotherapy for some, but not all patients. Consequently, the chemotherapy and non-chemotherapy groups may not be comparable in terms of prognosis.Systemic therapy can improve outcome of pT1 sporadic BC, although only of selected subgroups, and the effect might be small , 19. A sotherapy . While tic grade , 21. TheBRCA-associated breast tumors show a different biological background than tumors among the general population .Despite screening, still 8.6% of pT1a, 18.8% of pT1b, and 24.9% of pT1c BC patients had positive nodes at diagnosis. This appears to be similar to the general population –24, but o et al. ). Indeedpatients . This maative BC –29. In oBRCA1/2-associated BC patients, with a relatively long follow-up. Furthermore, studies directly investigating the impact of screening usually have to deal with length–time bias and lead-time bias [One of the strengths of our study was the ability to assemble a large cohort of ime bias . BecauseBRCA1/2-associated and sporadic BC. It is currently unclear whether overall survival is worse for BRCA mutation carriers, but if assumed to be the case, this may be largely due to the higher incidence of triple-negative BC (TNBC) within the BRCA1 population as well as the potential for survival bias [BRCA1 mutation carriers may result in another complication. We used left-truncation to minimize survival bias from BC patients who were tested for BRCA mutation after their BC diagnosis. However, because the hazard for death is especially high in the first few years after diagnosis of TNBC, selection of favorable TNBC cases may still occur when the interval between BC diagnosis and testing is long. While this did not seem to be the case for our cohort , one can never truly know which patients are missing due to selection bias, and the survival rates reported here may be an overestimation. However, comparing survival of only those who were tested for a BRCA mutation before breast cancer diagnosis , we found that the 10-year survival rates are 100% for pT1aN0, 98.3% for pT1b, and 89.2% for pT1c. All are higher than what we found for the whole cohort  between val bias –34. FurtBRCA1 and BRCA2 mutation carriers should ideally be analyzed separately, due to their tumor’s biological differences. However, despite having a much larger population of BRCA mutation carriers than any study before, this would still result in subgroups too small to draw reasonable conclusions. Instead, we opted to adjust the multivariable models for these biological differences, as well mutation status . A more important limitation was the small number of pT1a patients, making it difficult to draw useful conclusions about their prognosis. Combining the pT1a and pT1b groups into a 0.1–1.0 cm category could improve power, but would only allow for a generalized clinical application. When combined, we find 10-year survival rates of 91.9% for pT ≤ 1 cm (T1aN0/T1bN0) and 86.2% for pT > 1 cm (p = 0.02). For chemotherapy recipients, 10-year survival rates were 96.0% for pT ≤ 1 cm and 91.6% for pT > 1 cm. Among patients not receiving chemotherapy, 10-year survival was 91.0% for pT ≤ 1 cm and 77.1% for pT > 1 cm (p = 0.006), adjusted hazard ratio 0.40 (0.16–0.99).This also suggests that Another limitation arose as a result from the median year of BC diagnosis in our cohort being in the early 2000s, with hormone receptor status and especially HER2-status missing for a substantial proportion of cases. Therefore, we could not use TNBC as a variable for stratification. Instead, we used the imputed variables ER-status and HER2-status for adjustment. Further, the cause of death was unknown for a large proportion of cases, making BCSS analyses impossible. However, because we had a relatively young cohort with a median age of 42.3 years at BC diagnosis, and we censored at diagnosis of another cancer (including ovarian cancer), we can assume that the majority of the unknown causes of death are BC related. Finally, although a complete screening variable would be preferred, we expect our practical solution of a DNA-test result-based imputation provides a close enough approximation for our analysis on lymph node involvement where screening was taken into account.BRCA1/2 mutation, under the assumption that screening indeed leads to finding BC when the tumor is small and before lymph node involvement occurs.Ultimately, we observed (1) better prognosis with a smaller tumor size at diagnosis, (2) possibly improved survival after adjuvant chemotherapy treatment for those with a pT1bN0 or pT1cN0 tumor, and (3) less lymph node involvement at diagnosis for those with a smaller tumor size. These findings confirm several potential benefits from intensive screening for women at high risk of developing BC due to a BRCA1/2-associated breast cancer patients is better when they are diagnosed with a smaller tumor size within the pT1 category. Lymph node involvement is a frequent occurrence in BRCA1/2-associated BC and increases with larger tumor size. The results support current intensive screening strategies in BRCA1/2 mutation carriers, aiming to detect preferentially pT1a/b BC to improve survival and reduce the need for systemic therapy. To achieve early detection more often, research into further optimization of imaging techniques may be warranted.In conclusion, overall survival of Electronic supplementary material 1 (DOC 36 kb)Below is the link to the electronic supplementary material.