Up until the last decade, the treatment options for metastatic renal cell carcinoma (mRCC) patients were limited. mRCC is resistant to systemic cytotoxic chemotherapy  and cytoRecent insights have linked responses to immune checkpoint blockade to mutation burden and the frequency of neo-antigens . VaccineWe conducted a phase II clinical trial with the primary objective of investigating whether the treatment with ATV, CpG-B and IFN-α was feasible and tolerable and resulted in higher clinical response rates than IFN-α alone . Secondary objectives were to assess progression-free survival and overall survival of treated patients compared to historical data. Here, we report on the biological and clinical efficacy of this experimental treatment.Patients with bi-dimensional measurable metastases of histologically proven RCC, and in whom progression before or after nephrectomy had been demonstrated, were eligible for this trial. Furthermore, a WHO performance status of 0 or 1 was required and patients were only eligible when sufficient numbers of tumor cells were available for the production of a minimum of three vaccines. Patients with a history of autoimmune- or antibody-associated disease, prior malignancy, patients who were using immune suppressive drugs, or who had undergone prior immunotherapy for metastatic disease  were excluded.During the first month of therapy, the patients were seen bi-weekly. Thereafter, follow-up visits started at E3 see Fig.  and wereThe primary endpoint of the study was tumor response compared to historical data. Secondary endpoints included toxicity, progression-free survival, overall survival, the relation between DTH responses against tumor cells and clinical responses, and the value of pre-vaccination tumor-specific T cell reactivity as a predictor of successful immunotherapy.Of note, IFN-α was the only treatment option for potentially eligible patients at the start of this trial. However, the enrolment of this study was halted in 2006 when sunitinib became available. Therefore, enrolment stopped at 15 patients instead of 41 as originally planned.6 viable cells per vial) and cryopreserved using a linear freezer. Vials were stored in liquid nitrogen until vaccination. Prior to vaccination, the frozen tumor cells were irradiated , thawed, counted and assessed for viability. For each patient, we aimed to produce as many vaccines as possible.Patients underwent a total nephrectomy or, if the kidney was already removed before inclusion, a metastasectomy. The tissue that was not used for pathologic diagnosis and staging was transferred to our vaccine production laboratory within 48 h of the surgery. The tumor tissue was then dissociated as previously described , 24. Bri7 viable autologous tumor, cells were used and 100 µg GM-CSF , 1 mg CpG-B  and 50 µg Keyhole Limpet Hemocyanine (KLH)  were added. Vaccines were administered intradermally. For individual evaluation of the injection sites, different locations were used for each administration as previously described for ATV in patients with colon carcinoma , at the day of the third vaccination [evaluation 2 (E2)] and 5–12 weeks after the third vaccination [evaluation 3 (E3)]  and monocytoid myeloid-derived suppressor cell (mMDSC) frequencies and activation status were assessed before and during treatment by four-color flow cytometry staining. Cell surface antibody staining of PBMC was performed in PBS/0.1% BSA/0.02% Sodium-Azide for 30 min at 4 °C. The following antibodies were used: FITC, PE, PerCP-Cy5.5 or APC-labeled Abs directed against human CD3, CD4, CD8, CD11c, CD14, CD15, CD16, CD19, CD25, CD27, CD33, CD45, CD45RO, CD45RA, CD56, CTLA4, CD123, HLA-DR, PD-1 , CD11b, FoxP3 , CD40 , Fab-M-FITC , and blood DC antigens BDCA1, BDCA2, BDCA3  and MDC8  and matching isotype control antibodies. Intracellular FoxP3 and CTLA-4 staining was conducted with the anti-human FoxP3 staining kit  according to the manufacturers’ protocol. Stained cells were analyzed on a FACScalibur (BD Biosciences) using Cell Quest software.+ or CD8+ T cells (Tn) were defined as CD27+CD45RO− cells, effector T cells (Teff) as CD27−CD45RO+, central-memory CD4+ T cells (Tcm) as CD27+CD45RO+ cells and effector-memory cells (Tem) as CD27−CD45RO+ [+CD4+CD25hi, and FoxP3+. As FoxP3 has also been described to be transiently up-regulated on dividing (activated) effector T cells [+CD25intermediate (CD4+CD25int) cells. For Treg gating procedures, we refer to Huijts et al. 2017 [Naive CD4−CD45RO+ . Tregs w T cells , we alsoal. 2017 .+CD14− BDCA3+ [hiCD19−CD14−BDCA1/CD1c+; non-classical monocytes [+CD14loMDC8+  andhiBDCA2+ . Classiclo cells . Activatlo cells , includil-glutamine  and 0.01 mM 2-mercapoethanol . Next, 50,000 ATC and 100,000 PBMC were cultured either alone or together for 20 h in complete medium in a 96-well round-bottom plate, after which supernatants were harvested and frozen. IFN-γ levels were determined by ELISA (sensitivity 1 pg/ml) according to manufacturer’s instructions . IFN-γ levels are given as the mean IFN-γ concentration in pg/ml per 1 × 10e6 PBMC/ml ± SD of triplicate wells. Responses were considered positive when the amount of IFN-γ produced by PBMC in response to ATC was at least twice the amount of the sum of IFN-γ detected in overnight unstimulated PBMC or ATC mono-cultures, and was at least 10 pg/ml. Mean ATC-specific IFN-γ concentration was calculated by subtracting the IFN-γ levels from ATC + PBMC alone from the IFN-γ levels from ATC:PBMC co-cultures.Tumor reactivity of T cells in peripheral blood before, during and after ATV was assessed by IFN-γ secretion. To this end, ATC suspensions were used as stimulator cells in an overnight stimulation assay. Tumor cell suspensions were thawed and resuspended in IMDM medium  supplemented with 10% FCS , 100 I.E./ml sodium penicillin , 100 µg/ml streptomycin sulphate , 2.0 mM 6 ATC and 5 µg KLH were injected intradermally into separate sites and 48 h later the DTH response was evaluated by measuring the induration by the “Sokal pen method”[ab) and given in mm2.Several studies have demonstrated that the size of a DTH response after autologous tumor cell vaccination strongly correlates with recurrence and survival of cancer , 14, 39.n method”. In brieα = 0.05) and 80% power, 41 evaluable patients had to be enrolled in the study. Abortion of the enrolment after 15 patients disabled us to perform a reliable response rate analysis. However, the sample size proved sufficient to obtain significance levels in the immune monitoring analyses.The response rate for IFN-α treatment, taken from historical data, was assumed to be 10% . A respop ≤ 0.05.Differences between immune parameters before (E1) and during treatment (E2 or E3) were analyzed with a two-tailed paired t test. A two-tailed unpaired t test was used for the analysis of the difference in induration area caused by ATC and KLH between patients with clinical benefit (CB)  and patients without clinical benefit (NCB) (PD) and for the analysis of the difference in tumor-specific T cell reactivity between CB and NCB before and during treatment. Microsoft Excel (version 2010) and GraphPad Prism (Version 6.02) were used for all graphs, tables and analyses. Differences were considered significant when n = 27) of the patients had sufficient tumor material for vaccine preparation. 15 patients were eventually enrolled and 12 patients were excluded due to death prior to the first vaccination (n = 5), additional malignancies (n = 3), the absence of progression or metastasis (n = 3) or poor renal function (n = 1). We refer to Table Between April 2004 and April 2006, 30 mRCC patients were assessed for eligibility to enter this single-centre, single-arm phase II trial. 90%  and has not shown any signs of disease since the experimental treatment and, therefore, never received any other form of therapy for RCC  were the most prominent grade three or four adverse events that might have been related to the experimental treatment. Furthermore, two cases of grade three or four anaemia were recorded and grade three or four melena, fatigue, dizziness, disturbed balance, sensory neuropathy, aphasia, hemiplegia were all recorded once  which were all considered to be unrelated to the experimental treatment . Common local toxicity consisted of induration and edema at the vaccination site. We did not observe any ulcerations at the vaccination sites.2 and to KLH was 201.1 mm2. Interestingly, we observed a significant difference in the size of the DTH response between patients with CB  and patients with NCB for ATC (p = 0.038), but not for KLH . We also found that the magnitude of the IFN-γ response was significantly higher in patients with CB at E1 (p = 0.046) and at E3 (p = 0.042) but, interestingly, not at E2 . However, ATV delivery induced activation of B cells and T cells, as reflected by significantly increased percentages of CD19+CD86+ B cells and of CD4+PD-1+, CD8+ PD-1+ and CD8+CTLA-4+ T cells following treatment  as early as 2 weeks after the first ATV.We found that ATC-specific IFN-ɣ production before ATV was related to clinical outcome Fig. b. IntereFurther immune monitoring revealed a decrease over treatment in cDC2, non-classical monocytes and pDC frequencies, which may also reflect recruitment of these myeloid effector subsets to effector sites. The frequencies of pDC (which are directly targeted by CpG-B via TLR-9) returned to baseline levels at E3, whereas cDC2 and non-classical monocytes frequencies further declined. Interestingly, the decline in non-classical monocytes and pDC frequencies was associated with a significant increase in their activation status (measured by CD40) at E3 which may be attributed to the bi-weekly CpG-B injections that started 1 week after E2. Also, mMDSC frequencies were significantly decreased by E3 in patients with CB.+ T cells, as well as enhanced tumor eradication and improved survival, as was previously shown in murine models [This study shows that i.d. administration of ATV has clinical activity in a subset of patients but it may be even more interesting to look at possible combinations of this vaccination approach with other treatment modalities for mRCC. It was found for example that sunitinib has the ability to modulate the anti-tumor immune response by reversing MDSC accumulation and Treg elevation in RCC . In a rae models , 44. In e models . This see models , 46, bute models . As depie models , and sine models . In contPatients received IFN-α after the third vaccination (E2) and since the clinical response evaluation (E3) was 12 weeks after the start of this active drug, it is possible that the clinical responses were (in part) the result of this treatment. Unfortunately we are unable to discriminate between the clinical effects of the ATV and the IFN-α in this trial. However, clinical responses in mRCC were previously demonstrated by others in vaccine-based clinical trials (without IFN-α) with cultured CD83 + blood DC loaded with autologous tumor cell lysates , DC pulsIn conclusion, our data show that our ATV approach combined with IFN-α in mRCC is feasible, well tolerated and clinically active. Moreover, this treatment approach induced DTH responses against ATC and systemic activation of circulating PBDC and T cells in mRCC patients. In addition, preexisting ATC responsiveness of circulating T cells may be predictive for clinical outcome following treatment. Based on our observations in these 15 patients, further investigation of our ATV approach and current treatment modalities is indicated to improve response rates in this patient group.Below is the link to the electronic supplementary material.Supplementary material 1 (PDF 276 KB)


Weight loss, especially fat mass reduction, helps to improve blood glucose control, insulin sensitivity, and β-cell function. This study aimed to compare the effect of exenatide and glargine on body composition in overweight and obese patients with type 2 diabetes (T2DM) who do not achieve adequate glycemic control with metformin.We performed a prospective, randomized study of 37 overweight or obese patients with T2DM who had inadequate glycemic control with metformin. The patients were treated with either exenatide or glargine for 16 weeks. Dual-energy X-ray absorptiometry was used to assess body composition.Post-intervention weight, body mass index (BMI), waist circumference, body mass, and fat mass were lower in patients treated with exenatide, while weight and BMI significantly increased with glargine. Reductions in weight, BMI, body fat mass, and percent fat mass (except for gynoid) were greater with exenatide than with glargine, and percent lean tissue (other than the limbs) increased with exenatide. In all body regions except for the limbs, fat mass decreased with exenatide to a greater extent than lean tissue. Glucose control, insulin resistance, and β-cell function were not different between the treatment groups.For overweight and obese patients whose T2DM was inadequately controlled with metformin, exenatide and glargine achieved similar improvements in glycemic control, insulin sensitivity, and β-cell function.However, exenatide produced better weight and fat mass reduction, which were beneficial for blood glucose control. Our findings may guide the selection of appropriate drugs for glycemic and weight control.NCT02325960, registered 25 December 2014. The prevalence of overweight and obesity has markedly increased worldwide , 2. ObesManagement of T2DM includes lifestyle modifications , 9 and uIt is not clear whether GLP-1 receptor agonists or basal insulin can achieve better control of body composition in overweight or obese T2DM patients who have inadequate glycemic control with metformin. There are no reports on the effects of exenatide or glargine on trunk and android body composition, which might be more associated with long-term glycemic control in patients with T2DM following metformin treatment. To fill this knowledge gap, we used dual-energy X-ray absorptiometry (DXA) to compare changes in body composition, including fat mass and lean tissue, after using exenatide or glargine in combination with metformin in overweight and obese T2DM patients with poor glycemic control. Our study aims to assist in selecting optimal therapies for glucose reduction and weight control, especially in overweight and obese patients with T2DM.2 (based on the China Obesity Task Force criteria) [Forty-five overweight and obese T2DM patients with poor glycemic control despite metformin monotherapy were randomized using arbitrary computer-generated numbers to receive exenatide or glargine treatments for 16 weeks in addition to their current metformin treatment. Block randomization was performed by third-party statisticians. The inclusion criteria were as follows: 1) patients with T2DM receiving a stable metformin dose of ≥1.5 g/d for > 8 weeks; 2) age between 18 and 70 years; 3) glycated hemoglobin (HbA1c) between 7.0 and 10.0%; and 4) body mass index (BMI) ≥24 kg/mriteria) . ExclusiThis prospective, randomized, and parallel design trial lasted 16 weeks. For participants assigned to receive exenatide, the initial dosage was 5 μg twice daily for 4 weeks followed by 10 μg twice daily for the remainder of the trial. For basal insulin glargine, the starting dose was 8 IU once daily, followed by a titrated dosage of ≥2 IU every 3 days based on fasting blood glucose (FBG) levels until the peripheral blood glucose level reached 6.1 mmol/L, following which a maintenance dose with fixed glargine was administered for the remaining 12 weeks  at baseline and at the end of the study. Body weight, waist circumference (WC), and blood pressure were measured at baseline and every four weeks.All participants were administered an identical 85 g carbohydrate-equivalent meal at baseline and at the end of the treatment period. Plasma glucose and insulin levels were measured at 0, 30, 60, and 120 min after the meal. Fasting serum HbA1c, ALT, AST, total cholesterol (TC), triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol (LDL-C), uric acid, and creatinine levels were measured at baseline and after 16 weeks of intervention.P < 0.05.The study was non-blinded, and all analyses were completed by an independent statistician using SPSS version 18.0 software . The a priori power calculations for sample size were previously described . A paireThirty-seven of the 45 randomized patients completed the study; three patients in the exenatide group and five in glargine were excluded for lack of DXA data Fig. . BaselinP < 0.001) significantly decreased after exenatide intervention, while body weight and BMI (both P = 0.048) significantly increased after glargine intervention (Table P = 0.012) in the exenatide group but did not change in the glargine group. The exenatide treatment group had greater decreases in body weight  and BMI  than the insulin glargine group (P < 0.001) , trunk , limb , android , and gynoid . However, no changes were observed after insulin glargine intervention , trunk fat , limb fat , android fat , and gynoid fat  decreased with exenatide, but were unchanged with glargine  ; this decreased was greater with exenatide than with glargine , trunk , limbs , android regions , and gynoid regions , while they were unchanged with glargine (Table P = 0.007), trunk , android regions , and gynoid regions  . Furthermore, there was an inverse correlation between△lean tissue percentage  and each of △FBG and △HbA1c  and that △android fat mass in terms of both mass and percent mass were correlated with △HbA1c .In the exenatide group, multiple regression analysis was used to evaluate variables independently associated with △ = − 2.0 mmol/L, P = 0.002; glargine, △ = − 2.1 mmol/L, P < 0.001) and HbA1c values . The reduction in FBG and HbA1c levels did not differ between the two groups. Blood glucose values at 30 min  and 60 min  also decreased with glargine treatment , the Matsuda insulin sensitivity index (ISIM) , disposition index 30 , and disposition index 120 . However, there were no statistical differences between the two groups. Moreover, the basal homeostasis model assessment of insulin secretion (HOMA-β), the early-phase ratio of the area under the curve of insulin to glucose during 0–30 min (InsAUC30/GluAUC30), and total-phase InsAUC120/GluAUC120 did not differ between the pre-treatment and post-treatment values in the two groups (Table Exenatide and glargine increased the 1/homeostasis model assessment of insulin resistance (1/HOMA-IR)  and LDL-C  significantly decreased in the exenatide group but were unchanged in the glargine group. Conversely, ALT  significantly decreased with glargine, but did not change with exenatide. The extent of reduction in TC, LDL-C, and ALT did not differ between the two groups ; they showed that exenatide markedly reduced both visceral and subcutaneous fat while premixed insulin did not . In contWe found that glargine treatment produced weight gain with little change in fat mass and lean tissue. In contrast, a 6-month study comparing glargine to detemir in insulin-naïve patients with T2DM who had inadequate disease control found that the former significantly increased body weight, total fat mass, trunk fat mass, fat mass, and lean mass in limbs, while the latter significantly decreased truncal lean mass . Unlike M, as well as similar improvements in insulin release and β-cell function as determined via the HOMA-β, InsAUC30/GluAUC30, InsAUC120/GluAUC120, and disposition index in response to exenatide or glargine. This may be a function of early insulin sensitivity and β-cell function improvement as a consequence of glycemic control, while the effect of weight loss on insulin sensitivity and β-cell function may require a longer time. Weight loss, especially android fat mass reduction, is related to insulin resistance improvement [Weight loss, especially adipose tissue reduction, typically improves blood glucose control in accordance with the degree of weight loss and also improves insulin sensitivity and β-cell function –42. Neverovement ; the posOur study had some limitations. Only 37 patients participated, which may negatively influence the statistical power; moreover, the 16-week period may not have been sufficient to evaluate the additional benefits of weight and fat mass reduction on insulin sensitivity and β-cell function, as has previously been reported , 43. A bOur results showed that exenatide and glargine produce similar improvements in glycemic control, insulin sensitivity, and β-cell function in overweight or obese patients with T2DM who are unable to achieve adequate glycemic control with metformin monotherapy. Our data showed that exenatide was superior to glargine in terms of reduced body weight, BMI, fat mass, and percent fat mass. Additionally, weight loss with exenatide was mainly a consequence of decreased fat mass rather than lean tissue mass, which may help improve glycemic and weight control.


N,N-dimethylformamide, dimethylacetamide, dimethyl sulfoxide, and N-methyl-2-pyrrolidone) were synthesized via a high-concentration solvent-based emulsion polymerization with very low amounts of SPAES as dopant (1% w/w with respect to EDOT monomer), characterized by different DS. The influence of solvents and of the adopted doping agent was studied on PEDOT_SPAESs analyzing (i) the chemical structure, comparing via X-ray diffraction (XRD) the crystalline structures of undoped and commercial PEDOTs with PEDOT_SPAES’ amorphous structure; (ii) solvatochromic behavior, observing UV absorption wavelength variation as solvents and SPAES’ DS change; and (iii) electrochemical properties: voltammetric peak heights of PEDOT_SPAES cast onto glassy carbon electrodes differ for each solvent and in general are better than the ones obtained for neat SPAES, PEDOTs, and glassy carbon.Despite the poor solubility in organic solvents, poly  (PEDOT) is one of the most successful conducting polymers. To improve PEDOT conductivity, the dopants commonly used are molecules/polymers carrying sulfonic functionalities. In addition to these species, sulfonated polyarylethersulfone (SPAES), obtained via homogeneous synthesis with different degrees of sulfonation (DS), can be used thanks to both the tight control over the DS and the charge separation present in SPAES structure. Here, PEDOTs having enhanced solubility in the chosen reaction solvents ( In recent years, the fabrication of conductive electrodes based on conductive polymer composites (CPCs) has gained an increasing academic and industrial research interest. Moreover, the use of conductive coatings has been growing for many different applications in smart fields, such as sensors, photovoltaics, and portable and flexible electronics; some examples of smart devices include pollutant detectors for industrial and civil waters, touchscreens displays, organic and inorganic solar cells, and capacitors .The fillMany research groups have fabricated conductive electrodes using different carbon-based fillers, such as carbon nanotubes (CNT) , graphenSeveral alternatives to metal nanoparticles, organic metal complexes, hybrids, CNTs, G, and GO, such as conducting polymers, have been developed: in fact, the synthesis and, therefore, the properties of a conducting polymer can be appropriately designed and tailored to meet the requirements for electrochemical applications In this context, poly  (PEDOT) is considered the most promising candidate to be used to manufacture highly flexible and printable electrodes due to a number of advantages, i.e., simplicity of manufacture, good mechanical stability, and high optical transmittance . On the 2-Naphthalenesulfonic acid (Naph), an organic molecule with one sulfonic moiety, is, in general, introduced in PEDOT formulations as a template to synthesize PEDOT_Naph water solutions with improved solubility in organic solvents, currently representing one of the most successful commercial products based on conductive polymers . PEDOT_NN,N-dimethylformamide (DMF), dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), and N-methyl-2-pyrrolidone (NMP) were successfully synthesized via a high-concentration solvent-based emulsion polymerization with ferric sulfate as the radical initiator. Furthermore, the oxidative polycondensation of 3,4-ethylenedioxythiophene (EDOT) monomer in the reaction solvents reported above and in the presence of a new kind of doping agent, sulfonated polyarylethersulfone (SPAES), that was synthesized on purpose, is illustrated.The present study aims to fill this technological gap by reporting, for the first time, the preparation of electrodes based on highly conductive and processable PEDOT coatings. For the above considerations, PEDOTs characterized by improved solubility in the chosen reaction solvents, i.e., Tg, up to 310 °C and low thermal expansion coefficient), optical properties, chemical stability, oxidation resistance, as well as by good mechanical properties and easy processability Cl3, 98%), distilled water Chromasolv® (≥99.9%), and dimethyl sulfoxide-d6  were supplied by Sigma Aldrich and used without purification.3,4-Ethylenedioxythiophene , 2-naphthalenesulfonic acid , ferric sulfate , Fe2(SO4)3·xH2O (5 g) as a radical initiator and reaction solvent (3 cm3). The polymerization mixture is stirred for 24 h at room temperature and under an air atmosphere. The obtained solutions , i.e., the polymerization products, are analyzed without purification. Furthermore, four PEDOT_Naph samples were synthesized as described in the case of PEDOT samples, adding Naph  as a doping agent.Four PEDOTs obtained using different reaction solvents, namely, DMF, DMAc, DMSO, and NMP, were synthesized. In a representative polymerization procedure, a 50 cm3−·g−1 of polymer—SPAES_0.5, SPAES_0.75, SPAES_1—were synthesized. The exact amount of the monomers used for the syntheses are reported in 2CO3—the latter used as a proton scavenger—are introduced into a 100 cm3 one neck round-bottom flask equipped with a modified Dean-Stark device, with magnetic stirring and under a nitrogen atmosphere. The flask, loaded with toluene and NMP in order to have a 10% w/v concentration of the monomers in the solution is put in an oil bath and the polymerization reaction is carried out under reflux for 6 h; the water formed during the reaction is removed as an azeotrope with toluene through the modified Dean-Stark device. After complete water removal, the polymerization temperature is gradually increased to 198 °C and then the reaction mixture is kept for 18 h at 198 °C. The dark-purple polymerization mixture obtained is precipitated into a large excess of cold water under magnetic stirring and a brown solid is obtained. The polymerization product is recovered via filtration and washed with cold water from residual monomers, solvents, and K2CO3. The difficult removal of NMP and toluene from SPAESs required repeating the purification procedure several times. After drying in a vacuum oven (about 4 mbar) at 200 °C for 24 h, the presence of residual solvents was checked via isothermal TGA analysis (2 h at 220 °C under nitrogen flow). Therefore, SPAESs were converted to the acid form by immersion of each sample in 50 cm3 of 1 M HCl solution for 24 h at 100 °C, followed by washing with water for 24 h at 25 °C and drying in a vacuum oven (about 4 mbar) at 150 °C for 24 h.Three SPAESs with increasing nominal degree of sulfonation (DS), expressed as meq R–SO3−·g−1 of polymer. PEDOT_SPAES samples were synthesized as described in the case of PEDOTs, adding SPAES (0.025 g) at 1% w/w with respect to EDOT monomer.The four syntheses of PEDOT polymers, as described previously in 1H NMR spectra were collected at 25 °C with a BRUKER 400 MHz spectrometer . Samples for the analyses were prepared dissolving 8–10 mg of polymer in 1 cm3 of DMSO-d6. The presence of R–SO3− groups in SPAES samples was quantitatively measured via 1H NMR spectroscopy calculating the integral ratios between the proton in ortho to the sulfonic group (g) of SHQ and the ones of BFPS (d), (f) and of BHP (a), as reported in 1H NMR spectrum of SPAES_0.5, using Equation (1) ; Id,f is the integral area of the peaks (d) and (f); Ia is sthe integral area of the peak (a); UrBFPS is the molecular weight of the BFPS repeat unit, i.e. 216.25 g·mol−1; UrBP is the molecular weight of the BP repeat unit, i.e. 184.21 g·mol−1; and UrSHQ is the molecular weight of the SHQ repeat unit, i.e. 226.26 g·mol−1).3− groups in SPAES samples in terms of ion exchange capacity (IEC) was also determined via potentiometric titration. A solution of benzoic acid (60 mg in 50 cm3 of water) was prepared. A 0.01 M NaOH solution was used for titration. A titration of pure water using the same NaOH solution (75 cm3 of water) was performed first, and the NaOH solution volume used for the titration is indicated as VH2O (cm3)  is the concentration of 0.01 M NaOH solution (mol·cm−3) and Mbenz is the molecular weight of benzoic acid (122.12 g·mol−1). SPAESs IEC was measured after soaking the protonated sample in 20 cm3 of 2 M NaCl solution for at least 24 h. The solution was then titrated with 0.01 M NaOH using a Titrino751 GPD automatic potentiometric titrator . IEC data can be calculated with Equation (3) ; x is the weight of the sample (g).The presence of R–SO2O (cm3) ; then, 1−1.SPAESs samples’ intrinsic viscosity was measured using an Ubbelohde viscometer in a thermostatic water bath at 25 °C. Samples were dissolved in DMAc and analyzed in the concentration range of 1 to 0.3 g·dL3·min−1 and 40 μL as injection volume. Samples were prepared by dissolving 40 mg of polymer in 1 cm3 of anhydrous DMF and then filtered with 0.45 μm filters. The calibration was built using the following monodispersed Polystyrene (PS) standards: Mp = 1,600,000 Da (D ≤ 1.13), Mp = 1,150,000 Da (D ≤ 1.09), Mp = 900,000 Da (D ≤ 1.06), Mp = 400,000 Da (D ≤ 1.06), Mp = 200,000 Da (D ≤ 1.05), Mp = 90,000 Da (D ≤ 1.04), Mp = 50,400 Da (D = 1.03), Mp = 30,000 Da (D = 1.06), Mp = 17,800 Da (D = 1.03), Mp = 9730 Da (D = 1.03), Mp = 5460 Da (D = 1.03), Mp = 2032 Da (D = 1.06), Mp = 1241 Da (D = 1.07), Mp = 906 Da (D = 1.12), Mp = 478 Da (D = 1.22); Ethyl benzene (molecular weight = 106 g·mol−1). For all analyses, 1,2-dichlorobenzene was used as internal reference.SPAESs molecular weight was evaluated via SEC analyses using a Waters 1515 Isocratic HPLC pump , three Waters Styragel columns set (HR3-HR4-HR5) and a refractive index (RI) detector Waters 2487. SEC analyses were performed at 25 °C, with a flow rate of 1 cm−1; (ii) 5 min of isotherm at 330 °C; (iii) cooling from 330 to 25 °C at 10 °C·min−1; (iv) 5 min isotherm at 25 °C; and (v) heating from 25 to 330 °C at 10 °C·min−1 (Tg was measured here). After drying in a vacuum oven (about 4 mbar) at 200 °C for 24 h, PEDOTs, PEDOT_Naphs, and PEDOT_SPAESs were analyzed with the following thermal program: (i) heating from 25 to 250 °C at 10 °C·min−1; (ii) 5 min of isotherm at 250 °C; (iii) cooling from 250 to 25 °C at 10 °C·min−1; (iv) 5 min isotherm at 25 °C; and (v) heating from 25 to 480 °C at 10 °C·min−1 .DSC analyses were conducted using a Mettler Toledo DSC 1 , under nitrogen atmosphere on samples of SPAESs, PEDOTs, PEDOT_Naphs and PEDOT_SPAESs weighing from 5 to 10 mg each. The temperature program for SPAESs is as follows: (i) heating from 25 to 330 °C at 10 °C·min−1. PEDOT, PEDOT_Naph and PEDOT_SPAES samples were analyzed after drying in a vacuum oven (about 4 mbar) at 200 °C for 24 h.TGA analyses were performed using a TGA 4000 Perkin Elmer instrument , under a nitrogen atmosphere, on samples weighing from 5 to 10 mg each and heating from 30 to 800 °C at 10 °C·min−1. The absorbance and absorption maximum wavelength of PEDOTs, PEDOT_Naphs, PEDOT_SPAESs and SPAESs in their solutions of DMF, DMAc, DMSO, and NMP (concentration of 0.1% w/w) were determined between in wavelength ranges of 1100–190 nm. Moreover, the absorbance and absorption maximum wavelength of DMF, DMAc, DMSO, and NMP solvents were measured in water solutions at 0.1% w/w of concentration.A Shimadzu UV-160 recording double-beam UV–visible spectrophotometer with a data processing system  was used. UV–visible spectra of sample solutions were recorded in 1 cm quartz cells at a scan speed of 40 nm·min2 beam. X-ray energy was 68 keV (wavelength = 0.18233 Å), allowing measurements in a range of momentum transfer (Q) up to Qmax = 20.0 Å−1. Diffraction images were recorded with a Dectris Pilatus 2 M CdTe during a total exposure time of 120 s per sample. A schematic representation of the high-energy X-ray diffraction setup used is reported in G(r) function using PDFgetX3 Cl3 in 0.1 M KCl as the supporting electrolyte. Prior to the modification, the glassy carbon electrode (3 mm of diameter) was polished with synthetic diamond powder (diameter 1 mm) on a Struers DP Nap wet cloth and rinsed in water. A solution 0.5 w/w of the chosen polymer was prepared with the selected solvent and 20 µL were deposited on the glassy carbon surface. The electrode was dried under controlled temperature  and pressure (250 mbar).Cyclic voltammetric experiments were performed with an Autolab PG-Stat204 potentiostat/galvanostat  in a conventional electrochemical cell, by using saturated calomel, a Pt wire, and a polymer-modified glassy carbon electrode as the reference, counter, and working electrodes, respectively. The potential was scanned from +0.3 V to −0.7 V with a scan rate of 0.1 V·s3− group of the doping agent and forming a stable PEDOT/dopant complex. Examples of commercial sulfonated PEDOT dopants are either organic molecules, for example, 2-naphtalene sulfonic acid (Naph) and para toluene sulfonic acid, or sulfonated polymers, i.e., sulfonated polystyrene (PSS) Cl3, able to interact with the negatively-charged moieties of the SPAES doping agent [Preliminary cyclic voltammetric experiments were performed using an electrochemical probe molecule with positive charges, [Ru were cast and dried onto the surface of glassy carbon electrodes in a range of temperature, pressure, and time of drying, and the heights of their voltammetric peaks compared. Previous results about SPAES  demonstrThe worst case in terms of film stability is DMSO , where aWhen DMF is the casting solvent , all theIn the case of DMAc , stable 3−·g−1 of polymer, showed peak currents double those of glassy carbon  than any of the PEDOT_SPAESs, but unlike PEDOT_SPAESs they fail to improve the electrochemical performance of PEDOT. The marked increase in peak currents, the solvatochromic effect, and the structural changes evidenced by XRD demonstrate that the doping with SPAES completely modifies the electrochemical response of PEDOT, which becomes solvent-dependent and exploits better than the Naph dopant in the presence of sulfonic groups. In further studies SPAES as a doping agent for PEDOT polymers will be investigated with other electrochemical probes.Moreover, it is important to consider the two blank samples, PEDOT and PEDOT_Naph (cast from DMF and NMP), whose voltammograms are reported in N,N-dimethylformamide (DMF), dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), and N-methyl-2-pyrrolidone (NMP).To overcome the well-known technical problems of poly  (PEDOT), i.e., difficult processability and patterning, due to its poor solubility in common organic and inorganic solvents, PEDOTs characterized by a full miscibility in the reaction solvents used were successfully synthesized by a high-concentration solvent-based emulsion polymerization reaction between ethylenedioxythiophene (EDOT) and an oxidant species, i.e., ferric sulfate, in four different organic reaction solvents: 3−·g−1 of polymer, and was performed for the first time, leading to new conducting materials, PEDOT_SPAES polymers, characterized by full solubility in the reaction solvents chosen.The oxidative polycondensation of EDOT in the presence of sulfonated polyarylethersulfone (SPAES) as a doping agent was characterized by three increasing degrees of sulfonation (DS), i.e., 0.5, 0.75 and 1.0 meq R–SOw/w with respect to the EDOT monomer, its influence on the PEDOT_SPAES structure was studied via X-ray diffraction (XRD) spectra comparing unmodified PEDOT and PEDOT doped with 2-naphthalenesulfonic acid (PEDOT_Naph), having crystalline structures, with PEDOT_SPAESs, that are characterized by amorphous structures.Despite the low amount of SPAES-based dopant used, only 1% Both SPAES and the reaction solvents used influenced the solvatochromic behavior and the electrochemical features were assessed; a UV absorption wavelength variation is clearly detectable as the reaction solvents polarity change and SPAES DS increases. Moreover, the voltammetric properties of PEDOT_SPAESs cast onto glassy carbon electrodes were investigated: it was found that the choice of reaction solvent and the casting solvent conditions adopted significantly influences the electrochemical behavior of the resulting electrode and, in the case of NMP, PEDOT_SPAESs’ voltammetric peak heights, in general, are better than the ones obtained for neat SPAES, PEDOT, PEDOT_Naph, and glassy carbon.Thanks to the unique properties deriving from the combined use of PEDOT with SPAES in different reaction solvents, a new kind of conducting polymers for electrodes and sensors could be available for several research fields. The work is continuing with an emphasis placed on the modification of SPAES macromolecular structure, i.e., from a linear structure to a branched one, with the subsequent study of its effect on PEDOT macromolecular and structural properties, and on the investigation of PEDOT_SPAES’ electrochemical properties with a lead-based probe.


Hieracolichus philippinensis sp. n. (Gabuciniidae) and Pseudalloptinus pithecophagae sp. n. (Pterolichidae) from the Great Philippine Eagle Pithecophaga jefferyi Ogilvie-Grant, 1896, and Pseudogabucinia nisaeti sp. n. (Kramerellidae) from the Mindanao Hawk-Eagle Nisaetus pinskeri Gould, 1863. The presence of H. philippinensis on P. jefferyi supports the recent finding that the Great Philippine Eagle belongs to the lineage of serpent eagles (Circaetinae) rather than to the Harpy and other eagles.Endangered species of hosts are coupled with endangered species of parasites, which share the risk of co-extinction. Conservation efforts sometimes include breeding of rare species in captivity. Data on parasites of captive populations of endangered species is scarce and the ability of small numbers of captive host individuals to support the biodiversity of native parasites is limited. Examination of ectosymbionts of the critically endangered Philippine eagles and the endangered Mindanao Hawk-Eagle kept at the Philippine Eagle Center, Philippines, revealed three feather mite species despite regular treatment with insecticide powder. No other ectosymbiont taxa were detected. Studies in morphology and molecular phylogeny of these feather mites based on mitochondrial and nuclear DNA markers indicate that species found were typical for Accipitridae. Three new pterolichoid feather mite species (Acari: Pterolichoidea) were described from two species of eagles (Accipitriformes: Accipitridae) endemic to the Philippines:   •Small captive groups of endangered birds can maintain native feather mite populations for decades despite regular antiparasitic treatment.•Our study is a first record of feather mites from endemic raptors or diurnal birds-of-prey in the Philippines, with three new species described.•Feather mites of the Great Philippine Eagle support the attribution of the host to the Circaetinae rather than to Harpiinae birds of prey lineages. Most collections of feather mites from raptors, especially rare species, have been made from museum skins . NowadayIn the present work, we studied the fauna of feather mites found on two eagles endemic to Philippines based on both morphology and molecular phylogenetic analysis . We provided descriptions of three species of pterolichoid feather mites and investigated whether these feather mite species likely represent native fauna of Philippine eagles as opposed to species recently acquired through prey-to-host transmission.2Pithecophaga jefferyi Ogilvie-Grant, 1896 and the Philippine Hawk-Eagle Nisaetus pinskeri Gould, 1863, during annual medical examination of birds by OOT in 2016. Parts of the feathers bearing mites were removed using forceps and a magnifying glass, placed in the tube with 96% ethanol and kept at 4 °C for subsequent studies.The mite material used in the present study was collected in the Philippine Eagle Center  from two species of endemic raptors, the Great Philippine Eagle 2.1Some of the collected mites were mounted on microslides in Hoyer's medium according to the standard techniques used for many groups of small acariform mites . Investi2.2Amerodectes turdinus (GenBank accession number KU203310) and Amerodectes sp. (GenBank accession numbers KU202819 and KU202968) were used as outgroups for phylogenetic reconstructions. The genus Amerodectes  was selected as an outgroup for the Pterolichoidea feather mites studied because this genus is well defined morphologically and represents another superfamily, Analgoidea, a sister lineage to all pterolichoidean mites used in our analysis. Taxa of feather mites used for phylogenetic analysis, their systematics and hosts are summarized in DNA was isolated from specimens fixed in 96% ethanol using Holterman's method  with addWe tested the congruence of operational taxonomic units (OTUs) by the application of two analytical methods: Generalized Mixed Yule Coalescent (GMYC)  and AutoDepositories of type material and voucher specimens used for molecular study are as follows: UMICHZ — Museum of Zoology of the University of Michigan, Ann Arbor, USA; ZISP — Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russia.33.1Superfamily Pterolichoidea Trouessart et Mégnin, 1984Family Gabuciniidae Gaud and Atyeo, 1975Hieracolichus Gaud and Atyeo, 1975Genus Pterolichus nisi Canestrini, 1878, by original designation.Type species: Hieracolichus, currently including nine species, are restricted to birds of the order Accipitriformes , THE PHILIPPINES, Agusan del Norte, Santiago, Mt. Mamajao near Lake Mainit, caught on April 1974, mite collector O.O. Tolstenkov. The bird was at least 42 years old in 2016 when the mites were sampled. Voucher specimen: paratype female ZISP 7434.Type material. Male holotype (ZISP 7412), 13 male and 9 female paratypes from Depository: holotype, 8 male and 5 female paratypes, including voucher – ZISP, remaining paratypes – UMICHZ.P. jefferyi, THE PHILIPPINES, Lanao del Sur, Wao, wild-caught on 25 April 2015, mite collector O.O. Tolstenkov. Voucher specimen: female ZISP 7454.Additional material. 3 males, 1 females from 3.2h3 on lobar apices 475 (465–490), greatest width at level of humeral setae 290 (270–290); length of hysterosoma 340 (330–350). Prodorsal shield: occupying almost entire prodorsum, Prodorsal shield: antero-lateral extensions protruding to margins of propodosoma between trochanters I and II and fused with epimerites Ia, antero-lateral margins heavily sclerotized, lateral margins with narrow and deep incisions encircling bases of setae se, posterior margin slightly sinuous, greatest length 135 (120–135), width at posterior margin 180 (170–180). Setae vi spiculiform, 70 (67–73) long, extending slightly beyond tips of palps. Setae si spiculiform, 57 (55–60) long. Distance between bases of scapular setae: se:se 87 (78–85), si:si 37 (28–35). Subhumeral setae c3 filiform, with lanceolate enlargement in basal 1/3, 100 (95–105) long. Hysteronotal shield: greatest length from anterior margins to bases of setae h3 330 (320–345), length along midline 230 (225–240), width at anterior margin 155 (150–160), anterior margin slightly concave, surface of anterior half with sparse transverse striation. Lateral bands distinct. Lobar areas of hysteronotal shield not separated from main body of hysteronotal shield. Supranal concavity small triangular. Setae c2 thin spiculiform, 70 (70–70) long, situated in anterior angles of hysteronotal shield, cupules ia immediately postero-mesal to their bases. Setae e1 situated at level of hysteronotal gland openings gl or slightly anterior to them. Length of terminal cleft from anterior end to lobar apices (setae h3) 93 (90–100), greatest width at level of setae h1 67 (65–75). Margin of anterior one third of terminal cleft heavily sclerotized, margin of remaining part membranous; this membranous margin strongly convex anterior to bases of setae h1, posterior ends of opisthosomal lobes with small semi-ovate extensions. Setae e2 spiculiform 52 (50–58) long, with apices extending slightly beyond level of setae h2; setae f2 narrowly lanceolate, 27 (27–32) long, situated at level of setae h2, setae h1 lanceolate with rounded apex, 23 (22–25) long, 3.5 (3.5–5) wide, situated posterior to level of setae h2. Distances between bases of dorsal setae and gland openings: c2:d2 120 (100–115), d2:e2 140 (140–150), e2:h3 60 (60–68), d2:gl 32 (29–35), h3:h3 95 (95–105), h2:h2 108 (100–115), d1:d2 37 (25–37), e1:e2 110 (105–115).MALE , Fig. 3D1a. Epimerites II slightly curved. Genital apparatus at level of trochanters IV, 23 (22–25) × 25 (25–30), aedeagus not extending to its base. Bases of setae 4a separated. Setae 4b are slightly posterior to level of setae 3a. Setae g at level of anterior pair of genital papillae. Distances between ventral setae: 4b:g 37 (35–42), g:4a 75 (67–75), 4a:ps3 37 (37–42), ps3:h3 93 (87–98), 4a: 4a 15 (13–16). Anal suckers 25 (22–25) in diameter, corolla with 18–19 rounded denticles.Epimerites I, II without inflated bases. Epimerites I with tips simple, not extending to bases of coxal setae cG of genu I spiculiform, 90 (85–90) long, slightly exceeding entire length of genu and tibia. Solenidion σ1 of genu I 8 (8–11) long, much longer than solenidion σ2. Solenidion σ of genu III situated in basal part of this segment. Solenidion φ of tibia IV s shorter than corresponding tarsus. Tarsus IV with seta d button like and seta e of minute spine-like. Legs IV with distal half of tarsus extending beyond level of lobar apices. Length of tarsi: I, II 22 (22–24), III, IV 24 (22–25). Ambulacral disc of tarsus I ovate and in longitudinal diameter noticeably longer than the more circular-shaped ambulacral discs of tarsi II–IV. Length of tarsi: I 45 (45–50), II 58 (56–59), III 62 (60–63), IV 68 (65–68). Length of solenidia: σ1I 18 (13–18), σII 6 (5–7), σIII 20 (15–20), ω1I 12 (11–14), ω1II 25 (24–26).Femora I, II without ventral crest. Seta vi spiculiform, 75–80 long, barely reaching tips of palps. Setae si spiculiform, 75–78 long. Distance between bases of scapular setae: se:se 105–120, si:si 35–38. Subhumeral setae c3 filiform, 115–125 long. Hysteronotal shield: main body with almost straight anterior margin, anterior angles acute, posterior end extending to midlevel between hysteronotal gland openings gl and setae e2, posterior margin with blunt-angular median extension and pair of shallow concavities, greatest length 360–370, width at anterior margin 270–280, surface with faint transverse striation. Setae c2 spiculiform, 92–105 long, situated off hysteronotal shield; cupules ia postero-mesal to them and also off this shield. Setae d2 short filiform, about 20 long. Setae e1 approximately at level of hysteronotal gland openings gl. Lateral bands well developed, longer than main body of hysteronotal shield, with posterior ends almost extending to cupules ip and slightly curved medially. Posterior one quarter of opisthosoma poorly sclerotized, with fine striation and, in some specimens, with barely distinct punctation. Setae e2 spiculiform, 115–125 long, setae f2 filiform 30–40 long, setae h1 short filiform, about 10 long; both pair situated on poorly sclerotized area of opisthosoma. Posterior end of opisthosoma with wide and rounded median extension bearing setae h2, h3 and ps1 and with strongly sclerotized margin. Distances between dorsal setae and gland openings: c2:d2 135–155, d2:e2 155–170, e2:h3 78–83, d2:gl 72–78, h1:h1 62–70, h2:h2 75–80. h3:h3 45–48.FEMALE E, F  in Africa , and the inner margins of opisthosomal lobes have a pair of noticeably convex membranes in the anterior part of the terminal cleft; in females, the hysteronotal shield is shaped as an inverted trapezium and the posterior one third of the opisthosoma is devoid of sclerotization except the posterior margin, and tarsus IV completely extends beyond the posterior margin of the opisthosoma. In both sexes of H. dobyi, setae c3 are narrowly lanceolate at base with filiform apex (80-90 μm long), and genual solenidion σ is situated at the midlength of genu III; in males, setae g are situated anterior to the level of genital papillae; setae h1 are narrowly lanceolate, curved and 30–35 μm long, and the inner margins of opisthosomal lobes are almost straight; in females, the hysteronotal shield is shaped as an inverted trapezium and the posterior one third of the opisthosoma is devoid of sclerotization except for the posterior margin, and tarsus IV slightly (by ¼ the length) extends beyond the posterior margin of the opisthosoma.n Africa  in havinEtymology. The specific epithet is derived from the country, where the mite was found.Family Pterolichidae Trouessart et Mégnin, 1884Subfamily Pterolichinae Trouessart et Mégnin, 1884Genus Pseudalloptinus Dubinin, 1956Type species: Pterolichus  aquilinus var. milvulinus Trouessart, 1884, by original designation.Pseudalloptinus originally included pterolichine mites associated with birds from the orders Accipitriformes, Falconiformes, Gruiformes, Ciconiiformes and Psittaciformes , 20 male and 20 female paratypes from Pithecophaga jefferyi Ogilvie-Grant, 1896 (Accipitridae), THE PHILIPPINES, Lanao del Sur, Wao, 25 April 2015, mite collector O.O. Tolstenkov. Voucher specimen: female paratype ZISP 7371.Depository. Holotype, 15 male and 15 female paratypes – ZISP, remaining paratypes – UMICHZ.P. jefferyi individuals originated from the following locations: 10 males, 10 females – THE PHILIPPINES, Agusan del Norte, Santiago, Mt. Mamajao near Lake Mainit, caught on April 1974; 5 males, 5 females, THE PHILIPPINES, Davao Oriental, Mati, Don Salvador, South Biasong, caught on 13 January 2011; 5 males, 5 females, THE PHILIPPINES, Davao City, Malagos, Philippine Eagle Center, 4 February 2002 (captive breed), mite collector O.O. Tolstenkov. Voucher specimens: female paratypes ZISP 7391, 7411.Additional material. 20 males, 20 females from 3 h3) 25 (325–350), greatest width at level of humeral setae 180 (180–195). Length of hysterosoma 215 (210–225). Prodorsal shield: occupying most part of prodorsum, antero-lateral extensions acute, lateral margins with deep and narrow extensions encircling bases of scapular setae se, posterior margin slightly concave, length along midline 98 (95–105), greatest width 102 (100–110). Setae vi filiform, 38 (28–38) long, not extending to palpal apices. Setae se separated by 65 (65–68). Setae si minute filiform, close to bases of corresponding setae se. Scapular and humeral shields present. Setae c2 filiform, 15 (12–15) long, situated on anterior margin of humeral shields. Subhumeral setae c3 lnceolate 20 (18–20) long, 4 (3.7–5) wide.MALE , 6A–C. (h3 212 (210–215), width at anterior margin 145 (140–150), anterior margin slightly concave, surface without ornamentation. Lateral bands distinct, narrow. Hysteronotal gland opening gl situated at level of trochanters IV. Setae d2 minute filiform about 10 (10–12) long; setae e2 filiform, 16 (15–18) long, situated at level of anterior end of supranal concavity, not extending to lobar apices. Opisthosomal lobes roughly triangular, at base slightly wider than long, rounded posteriorly. Terminal cleft roughly semi-ovate, 28 (25–30) long, 35 (34–38) in width at level of setae ps1. Supranal concavity open posteriorly into terminal cleft. Terminal cleft with narrow entire membrane forming semi-ovate terminal extensions on lobar apices, length of these extensions 10 (10–15) long, wide at base 18 (17–20). Setae ps2 long filiform, extending far beyond level of lobar apices; setae ps1 minute filiform, about 15 long, situated near bases of setae h2. Distances between dorsal setae: c2:d2 87 (80–88), d2:e2 75 (72–80), e2:h3 42 (40–45), ps1:ps1 40 (38–42), h2:h2 60 (60–65), h3:h3 50 (50–55), ps2:ps2 70 (70–75).Hysteronotal shield: greatest length from anterior margins to bases of setae 4b slightly posterior to level of setae 3a. Setae g equidistant from genital arch apex and level of setae 4b. Anterior genital papillae at level of genital arch apex. Epimerites IVa long, bearing bases of setae 4a near tips and flanking base of genital arch. Adanal apodemes with L-shaped inner ends flanking median area with bases of setae ps3 but not forming separate postegenital sclerite. Anal suckers 13 (13–15) in diameter, corolla without indentation, surrounding membrane very wide and extending laterally over lateral margins of opisthosoma. Distances between ventral setae: 4b:g 23 (22–25), g:4a 40 (40–47); 4a:ps3 30 (30–32), ps3:h3 67 (65–68).Epimerites I fused into a Y with short stem. Epimerites IIa present. Genital apparatus situated at level of anterior margin of trochanters IV, 14 (14–15) long, 14 (13–17) wide. Setae σ1 situated at its midlevel of genu I and 1.3–1.5 times longer than this segment. Genual setae cGI, cGII, mGI and mGII filiform, shorter than corresponding segments. Solenidion σ of genu III in distal part of segment. Legs IV with distal half of tarsus extending beyond level of lobar apices. Tarsus IV with claw-like apical extension, setae d and e minute are absent. Solenidion φ of tibia IV about 1.5 times longer than tarsus IV. Length of tarsi: I 35 (35–37), II 35 (35–38), III 38 (37–40), IV 33 (32–34). Length of solenidia: σ1I 40 (40–45), σII 8 (7.5–8), σIII 8 (8–10), ω1I 11 (10–03), ω1II 18 (16–18).Setae of tarsi I, II filiform. Solenidion se separated by 80–85; setae si minute filiform, situated closely to corresponding setae se. Scapular and humeral shields present. Setae c2 short filiform, 20 (18–20) long, situated in anterior margin of humeral shields. Subhumeral setae c3 lanceolate, 26–30 long, about half the length of humeral setae cp. Hysteronotal shield: entire, extending to posterior end of opisthosoma, anterior margin concave, 300–340 long, 210–220 wide at anterior margin surface without ornamentation, posterior end with desclerotized transverse area bearing setae e2. Setae c1 on hysteronotal shield near its anterior margin. Setae d2 situated approximately at midlevel between cupules ip and hysteronotal gland openings gl. Setae e2 filiform, about 10–12 long. Lateral bands present, poorly distinct. Posterior margin of opisthosoma with relatively wide terminal extension bearing setae h2, h3 and ps1. External copulatory tube minute, situated terminally about 2–3 long. Spermatheca and spermaducts as in e2 18–20, f2 8–10, ps1 5–6, ps2 15–18. Distances between dorsal setae and openings: c2:d2 175–190, d2:e2 80–95, d2:gl 34–36, h2:h3 40–52, h2:h2 35–38, h3:h3 17–18.FEMALE  G,H. Gna4b. Apodemes of oviporus narrow, barely sclerotized. Setae g situated approximately equidistant from levels of setae 4b and g. Distances between ventral setae: 4b:g 50–58, 4b:3a 52–65, g:4a 38–52.Epimerites I as in male. Epimerites IVa present. Epigynum semicircular, thin, 42–48 long, 65–80 wide, with tips extending to level of setae σ1I 68–80, σII 10–15, σIII 10–18, ω1I 12-14, ω1II 26–28.Legs I–III as in male. Solenidion σ of genu III in distal part of segment. Solenidion φ of tibia III slightly longer than corresponding tarsus; solenidion φ of tibia IV about 1/5 the corresponding tarsus. Legs IV with tarsus and distal half of tibia extending beyond posterior end of opisthosoma. Legs I, as in male. Length of tarsi: I 50–53, II 50–55, III 57–60, IV 78–80. Length of solenidia: Differential diagnosis. The new species, Pseudalloptinus pithecophagae sp. n. is most similar to P. africanusP. milvulinus  in having the following features: in both sexes, setae c3 are lanceolate; in males, opisthosomal lobes are well developed, with semi-ovate terminal membranes; and in females, the striated sejugal area is large and constitutes about 1/5th of the total length of the idiosoma. Pseudalloptinus pithecophagae sp. n. differs from these species by the following features: in males, the genital apparatus is situated at the level of the anterior margin of trochanters IV, epimerites IVa are long and almost extending to the genital arch, and setae e2 are filiform, situated at the level of the anterior end of supranal concavity and not do not extend to lobar apices; in females, the hysteronotal shield is entire, the epigynum is semicircular and extends to the level of setae 4b, setae c1 is situated on the hysteronotal shield, external copulatory tube is minute (only 2–3 μm long), and setae g are situated at the level of setae 3a. In males of P. africanus and P. milvulinus, the genital apparatus is situated at the level of the posterior margin of trochanters III, epimerites IVa are poorly developed, and setae e2 are spiculiform, situated posterior to the supranal concavity and extend beyond the lobar apices; in females, the hysteronotal shield is spit into a large anterior piece and a small pygidial fragment covering the very posterior end of the opisthosoma, the epigynum is bow-shaped and does not extend to the level of setae 4b, setae c1 are situated on striated tegument near the anterior margin of the hysteronotal shield, the external copulatory tube is about 15 μm long and curved ventrally, and setae g are situated posterior to the level of setae 3a.P. pithecophagae males, easily discriminating this species from all previously known Pseudalloptinus species, is the absence of the entire postgenital sclerite well separated from the adanal apodemes. In this species, L-shaped tips of adanal apodemes turned anteriorly and flank small median area with setae ps3, apparently corresponding to the lateral pieces of the postgenital sclerite of other species of this genus.The unique feature of Etymology. The specific epithet is derived from the generic name of the type host and is a noun in the genitive case.Family Kramerellidae Gaud et Mouchet, 1961Genus Pseudogabucinia Černy, 1961Type species: Pterolichus ciconiae Canestrini et Berlese, 1881, by monotypy.Pseudogabucinia has included only five species with hosts erratically distributed among non-passerine orders: Accipitriformes, Ciconiiformes, Falconiformes, Gruiformes, and Otidiformes  has been recorded from raptor birds of two orders: from falcons Falco , harriers Circus (Accipitriformes: Accipitridae) and buzzards Buteo . (Accipitridae), THE PHILIPPINES, Salaysay, Davao City, caught in 2005, mite collector O.O. Tolstenkov. Voucher specimen: female paratype ZISP 7312.Type material. Male holotype (ZISP 7307), 4 male and 1 female paratypes from Depository. Holotype, 3 male and 1 female paratypes – ZISP, 1 male paratype UMICHZ.h3) 270 (265–280), greatest width at level of humeral setae 175 (170–180); length of hysterosoma 195 (190–195). Prodorsal shield: occupying anterior part of prodorsum, roughly trapezoidal in shape, with slightly convex posterior margin and posterior angles slightly extending laterally, not extending to bases of scapular setae, length along midline 45 (45–48), greatest width 47 (45–50) (se separated by 57 (55–58). Setae si spiculiform, 35 (35–47) long, separated by 23 (22–25), approximately equidistant from midline and corresponding setae se. Scapular and humeral shields absent. Setae c2 spiculiform, 30 (27–32) long, situated in striated tegument. Subhumeral setae long filiform, nearly half the length of macrosetae cp. Hysteronotal shield: greatest length from anterior margins to bases of setae h3 185 (180–190), width at anterior margin 125 (115–125), anterior margin slightly concave, lateral margins almost straight, surface with fine longitudinal striae between levels of setae e1 and e2. Supranal concavity narrowed anteriorly and extending to level of setae e1. Hysteronotal gland opening gl situated approximately equidistant from levels of setae d2 and e2. Lateral bands poorly demarcated. Seta d2 minute filiform, about 5 long, setae e2 filiform 32 (27–33). Opisthosomal lobes roughly triangular, with rounded posterior ends, approximately as long as wide at base; apical and inner margins of lobes membranous. Terminal cleft wide triangular, with blunt anterior very end, 52 (52–55) long, 52 (50–55) in width at level of setae h3. Setae f2 narrowly lanceolate with short filiform apex 40 (32–40); setae ps2 blade-shaped 27 (25–27); setae h1 narrowly triangular, 15 (15–18) long thin, setae ps1 filiform, about 10 long, situated posterior to level of setae h1. Distances between dorsal setae: c2:d2 77 (70–80), d2:e2 57 (55–60), e2:h3 63 (57–63), d1:d2 37 (35–40), e1:e2 25 (22–28), f2:f2 112 (110–120), ps1:ps1 85 (82–88), h3:h3 72 (70–75), h2:h2 105 (100–105).MALE , 9A-E. ( (45–50) . Setae s3a and 4b situated at the same level. Setae g at level of apex of genital arch. Genital papillae situated lateral to anterior half of genital arch. Distances between ventral setae: 4b:g 17 (16–18), g:4a 23 (20–23); 4a:ps3 62 (60–64), ps3:h3 47 (47–50). Anal suckers 13 (12–14) in diameter, corolla with two rounded denticles. Small adanal sclerites presents between setae ps3 and anal suckers.Epimerites I free, slightly converging. Epimerites IIa present, barely distinct. Genital apparatus 15 (14–15) in length, 13 (13–17) in width, its base situated at midlevels of trochanters IV B. Setae σ1 of genu I approximately half the length of this segment. Setae mG of genu II much longer than of genu I. Setae cG of genua I and III filiform, slightly longer than corresponding segments. Solenidion φ of tibia IV slightly shorter than tarsus IV. Setae d and e of tarsi IV minute spine-like. Legs IV with ambulacral disc slightly extending beyond level of lobar apices. Length of tarsi: I 33 (32–34), II 42 (40–43), III 40 (37–40), IV 43 (40–43). Length of solenidia: σ1I 5 (5–6), σII4 (4–6), σIII 5 (5–6), ω1I 12 (12–14), ω1II 20 (18–20).Solenidion se separated by 68; setae si spiculiform, 45 long, separated by 30, situated approximately equidistant from midline and corresponding setae se. Scapular and humeral shields absent. Setae c2 thin spiculiform, 35 long, situated in anterior angles of humeral shields. Subhumeral setae c3 long filiform 37 long, about half the length of setae cp. Hysteronotal shield: length 180, width 125, anterior margin nearly straight, not extending to level of setae c2, surface without ornamentation, posterior margin with pair of narrow incision almost extending to level of setae e1 and wide semi-rounded extension between them. Setae d2 off hysteronotal shield. Lateral bands present, poorly demarcated. Spermatheca and spermaducts as in e2 38, f2 125, ps1 40, ps2 155, h1 10. Distances between dorsal setae: c2:d2 87, d2:e2 83, e1:e2 20, h1:h1 50, h2:h2 83, h3:h3 55, ps1:ps1 32.FEMALE . Gnathos4b:g 12, 4b:3a 15, g:4a 32. Legs I–III as in male. Solenidion φ of tibia III slightly longer than corresponding tarsus; solenidion φ of tibia IV about one third the corresponding tarsus. Legs IV with ambulacral disc extending beyond posterior end of the opisthosoma.Epimerites I as in male. Epigynum bow-shaped, situated between tips of epimerites II, 15 long, 40 wide. Apodemes of oviporus barely sclerotized. Setae g and 3a situated approximately at same level of setae. Distances between ventral setae: σ1I 10, σII 9, σIII 6, ω1I 12, ω1II 20.Length of tarsi: I 35, II 50, III 45, IV 50. Length of solenidia: Differential diagnosis. The new species, Pseudogabucinia nisaeti sp. n. is close to P. intermedia  known from falcons by in having, in both sexes, ambulacral discs of tarsi IV extending to or slightly beyond the posterior margin of the body, and setae c2 exceeding the distance between internal scapular setae si, and, in females, setae f2 and ps2 being equal to or exceeding the distance between their bases. Pseudogabucinia nisaeti sp. n. differs from that species by the following features: in both sexes, subhumeral setae c3 are long filiform and approximately half as long and humeral setae cp, solenidion ω1 of tarsus II does not extend to the apex of this segment; in males, the supranal concavity does not extend beyond the level of setae e1, setae 4a are situated posterior to the base of the genital arch; in females, the genital papillae are situated distinctly anterior to the level of setae g. In both sexes of P. intermedia, subhumeral setae c3 are about 1/3 the length of setae cp, solenidion ω1 of tarsus II extends to the apex of this segment; in males, the supranal concavity extend far beyond the level of setae e1, setae 4a are situated posterior at the of the level of genital arch base; in females, the genital papillae are situated at the level of setae g.Etymology. The specific epithet is derived from the generic name of the type host and is a noun in the genitive case.3.3P. pithecophagae, two specimens of H. philippinensis and one specimen of P. nisaeti (P. pithecophagae and H. philippinensis from Pithecophaga jefferyi while for P. nisaeti only the ABGD delimitation was significant, which can be explained by the presence of single specimen of the latter species available for analysis.We obtained sequences for the genes COI, EF-1α, 18S, 28S from four specimens of  nisaeti . Data on nisaeti . Therefo nisaeti . Althoug nisaeti . Operati4H. philippinensis. This fact, assuming this population of mites is endemic, suggests that these ectosymbionts have been able to survive 43 years in captivity.Most of the birds species host several groups of ectosymbionts, including obligatory feather mites and chewing lice species . HoweverHieracolichus philippinensis represents the original feather mite fauna on the Great Philippine Eagle rather than a recently acquired feather mite species.We describe for the first time feather mites of two endangered Philippine eagles, which, if they prove to be species-specific, are endangered species too. Based on the phylogenetic position of the species described herein and known reference data on associations of their genera and families, it is possible to drawn out very preliminary hypotheses on the origin of the examined feather mite species from eagles of the Philippines. Of 16 genera of the family Gabuciniidae, eight genera, including the genus Hieracolichus, are restricted to birds of the order Accipitriformes . Most reH. philippinensis, Pseudalloptinus pithecophagae also represents rather ancient and most likely the primary fauna on the Great Philippine Eagle.Currently the suprageneric system of the family Pterolichidae is not fully developed . Our attWide and mosaic distribution of the kramerellid genus Pseudogabucinia among birds orders and within Accipitriformes and Falconiformes  stronglyP. jefferyi indeed belongs to the lineage of serpent eagles Circaetinae, rather than derived lineages of typical eagles as Aquilinae and Harpiinae.The Great Philippine Eagles were historically placed in the subfamily Harpiinae related to other eagles but were recently moved to the family Circaetinae based on molecular studies . Althoug5We showed that a small captive group of endangered birds could maintain viable populations of native feather mites, demonstrating the utility of ectosymbiont examination for host individuals even after decades in captivity. We provided the first record of feather mites from endemic raptors or diurnal birds-of-prey in the Philippines, with three new feather mite species described, and revealed the native origin of the feather mites studied. Our work facilitated an understanding of biodiversity in the understudied family of feather mites Pterolichidae, although many more species should be sequenced before the relations in the family can be resolved clearly by molecular phylogeny.


This enlarges the range of free-flowing Al particle sizes to < 20 µm. Powder mixture homogeneity is examined optically in vibrated bulk powder and thinly spread layers. From various powder mixtures, solid samples are built layer by layer with the Additive Manufacturing (3D printing) technology Laser Beam Melting in metal powder bed (LBM). Chemical homogeneity of solids is evaluated via energy-dispersive X-ray spectroscopy, backscattered electron microscopy, metallographic analysis and tensile tests. Persistent homogeneity of Al-Cu powder mixtures and LBM solids is found only with particles < 20 µm dry coated with SiOx nanoparticles. Observed segregation phenomena are explained with a decrease in particle mobility at increasing local concentration and the decreasing effectiveness of mass in smaller particles. The main effects are based on geometry, so they are expected to be transferrable to other nanoparticles, alloying components and powder bed technologies, e.g., binder jetting.Up to now, minimizing segregation of free-flowing, microscale metal powder mixtures driven by different mass density is an open challenge. In this work, effects of particle size variation on homogeneity of Al-Cu mixtures, with a density ratio of 3.3, are examined. Dry coating Al particles with 0.3 wt% fumed silica SiO This cycle is repeated for each layer, until the product has been completely built. To ensure spreadability of powders in thin layers, typically spherical particles from atomization with inert gas are used. More details about the used LBM machine are explained in Additive Manufacturing (AM) or synonymously 3D printing increasingly pervades industrial production . An AM tIn many promising fields of application of AM, there is a need for more appropriate materials . CharactA/ρB, of the mixed components, A and B [rel [A/ρB, homogeneous distribution of alloying elements in LBM products is described as unsatisfactory [More recent publications on alloy creation in LBM from powder mixtures are summarized in  A and B ,24,25,26d B [rel ,21,22,25sfactory ,24,25. Hsfactory ,24. Locasfactory  and Elonsfactory , but notsfactory . ExaminaA generally applicable approach to avoiding segregation is avoiding the mixing of powders . In AM, Focusing on the powder material, segregation can be reduced by limiting relative mobility of powder components. One method following this approach is mechanical alloying by high energy ball milling before LBM ,30. ResuAnother universal approach to avoiding segregation is decreasing overall mobility of particles by increasing powder cohesion, e.g., by adding liquids ,38. ThisHowever, effects of interparticle attraction on segregation are not monotonous. Experiments with magnetic and non-magnetic particles of different density under varied strength of a magnetic field show segregation can be avoided, or the direction of segregation be inverted .Particle Size Distribution (PSD) is another influential factor in de-mixing phenomena ,41. In eNo publications describe segregation of free-flowing, metal, microparticles suitable for LBM.In a generalized way, mixing and de-mixing phenomena can be described independently of the experimental setup by the Fokker-Planck equation .(1)m(x)It describes the change of concentration, q, of a mixture component at a location, x, by balancing incoming and outgoing particles. It can be transformed to express local mass flow of a mixture component, ient, D* . If convient, D* . Convectient, D* . If incrMobility of particles depends on particle sizes, e.g., smaller particles can move through gaps between larger particles. Gravity and inertia also depend on particle size. With metal particles in the single-digit and double-digit micrometer scale, effects of particle mass can be pushed into the background by surface effects . One conx nanoparticles [The hypothesis put to experimental falsification in this work is: size variation of free-flowing metal microparticles does not allow stabilizing mixtures against segregation. The system of Al-Cu is chosen because of its challenging mass density ratio of 3.3, and because it attracts increasing scientific interest with regards to LBM ,58,59,60articles . Maximumrel, LBM experiments are conducted, as shown in The general methodology followed in this work is illustrated in x fumed silica nanoparticles Aerosil® R 106 by Evonik Industries AG . This is conducted by mixing in a Turbula® T2A shaker  on maximum speed for 1 h [Al powder atomized with N is sourced from Ecka Granules Germany GmbH , Cu atomized with N from TLS Technik GmbH & Co Spezialpulver KG , and a master alloy AlMg50 containing 50 wt% Mg atomized with N from Nanoval GmbH & Co. KG . Pure Mg powder is avoided due to flammability. Prealloyed Ti6Al4V atomized with Ar is sourced from TLS Technik GmbH & Co Spezialpulver KG . At delivery, powders are imaged with a Philips XL Scanning Electron Microscope (SEM) , and chemical composition is analyzed by X-ray fluorescence spectroscopy (XRF) on a SpectroMAXx . All powders are sieved under Ar with 20, 32, 40, 50 and 63 µm wide meshes. For all experiments, except establishment of the initial situation and some angle of repose measurements, Al powders are dry coated with 0.3 wt% SiO for 1 h ,63. No b for 1 h .x nanoparticles, as a reference point, and to demonstrate the challenges of in situ alloying in LBM, a powder mixture with eutectic composition 67.3 wt% Al and 32.7 wt% Cu is processed according to the current state of the art. Al and Cu are sieved to PSD 20–50 µm as established in LBM of Al [To establish the initial situation without SiOBM of Al ,66.® T2A  under Ar, in a half-filled 15 cm high container. Powders for vibration experiments are mixed for 16 h in the T2A under Ar, in half filled 10 cm high test tubes. 25 binary mixtures of 94.7 wt% Al, 5 wt% Cu, and 0.3 wt% SiOx are prepared by combining five particle size fractions < 20 µm, 20–32 µm, 32–40 µm, 40–50 µm and 50–63 µm. Based on the results, 81 quaternary powder mixtures are prepared. The chemical composition is chosen to approximate the high-strength and high-ductility alloy EN AW-2024 [x. The relatively high Ti content, still within the limits of EN AW-2024, is chosen to reduce crack tendency by grain refinement [x nanoparticles dissolves in the melt, it will not exceed the limit of 0.5 wt% for Si in EN AW-2024. Remaining O might form oxides with Ti, Al or Mg, which are more affine to O than Si [Components of powder mixtures are weighed on a scale with 0.1 mg accuracy. They are mixed for minimum 2 h in a Turbula AW-2024 , which i AW-2024 , with 3. AW-2024 . The foufinement ,69. If t than Si . 81 quat than Si .To examine segregation in bulk powder, test tubes are vibrated for 2.3 h with an amplitude of 0.5 mm at a frequency of 50 Hz. Tubes are mounted with a jig on a vibration sieve EML 200 digital plus . Segregation levels are categorized visually by the strong optical contrast between reddish Cu and grey Al particles. Because contrast between bright grey Al, dark grey Ti6Al4V, and dark grey AlMg50 is lower, duration of vibration is extended to 5.25 h for quaternary mixtures. Segregation of darker powders cannot be distinguished between Ti6Al4V and AlMg50. However, striations and separated dark bands at top and bottom of tubes are clearly visible against bright grey Al.® 50, by manufacturer ReaLizer GmbH . SLM 50 has a rotational wiper with two silicone rubber lips, working in turns to move powder back and forth over the build platform. It does not drop a portion of powder into an overflow vessel each layer. A constant rotational wiper velocity is set. It yields a minimum tangential velocity of 12.4 mm/s at the edge of the platform closest to the rotational axis of the wiper and a maximum of 21.8 mm/s. Potential effects of different wiper velocities can be detected along the radial direction marked with a violet arrow in z = 30 µm. Because of strong deviations in the first two imaged layers above the build platform, only the following 18 are analyzed. The first layers of LBM typically form support structures that are not part of the product. The Al build platforms are roughened by blasting with Al2O3 particles before use. Powder layer spreading and LBM experiments are conducted on a commercially available LBM machine, SLMx are analyzed with the setup in Three binary mixtures with 94.7 wt% Al, 5 wt% Cu, and 0.3 wt% SiOFor tangential and radial directions, nine different local means, ® 50  has a single mode Yb fiber laser YLM-100-AC from IPG Photonics  specified with 1070 nm wave length, linewidth of < 2.5 nm, maximum output power, P, of 100 W, random polarization, and a beam quality factor, M², of 1.05–1.1. It is operated in continuous wave mode. The laser beam is guided over the X, Y-plane with two galvanometer scanners from Cambridge Technology . Scan speed is set in the machine control software by point distance, dp, and exposure time, te. Actual v, which is determined as mean of 6 calibrated high-speed camera measurements made with a Phantom V1210  for each used combination of dp and te, is found to deviate up to 46% from theoretical scan speed vtheo, that is calculated, and the relative deviations are found to depend only on te.The LBM machine Realizer SLMdspot, of 66 µm, P = 100 W, and rotationally symmetrical Gaussian intensity distribution are confirmed by measurements with a MicroSpotMonitor . For LBM experiments, Ar is used as process gas to remove vapor and spatter from the interaction zone of the laser beam with the metal. Before the laser can be activated, the process chamber is flushed with Ar and the O content decreased below 1%. The build platform heating is set to the maximum 200 °C. Thermocouple measurements on the surface reveal local variations of the actual temperature from 112 to 124 °C across the platform, when a plateau has been reached ten minutes after heating has begun. The varied LBM parameters are illustrated in xy. Laser power, P = 100 W, spot diameter dspot = 66 µm, and layer thickness, Δz = 30 µm, are kept constant.In this work, only values for actual v, as confirmed by measurements, are mentioned. The laser beam is focused with an F-θ lens to ensure comparable intensity at different positions on the build platform. Constantly set spot diameter in the processing plane, v = 650 mm/s and Δxy = 25 µm. Alternating meanders are scanned, with double exposure of each layer.LBM parameters for establishment of initial situation in Evol, is calculated, equivalent to 410 J/mm³.From these parameters, volumetric energy density, rel in horizontal cylinders, maximum scan vector length is limited [LBM sample cubes have 5 mm edge length and 2 mm high supports designed in Magics V19.0.2.08 . All cubes are built with alternating meander scan strategy as illustrated in  limited ,72,73 byed [× 20 . Tensileed [× 20 .rel by thresholding and conversion to black and white following VDI 3405 Blatt 2 [2O, 0.8 mL HNO3, 0.25 mL HCl, and 0.75 mL HF. The sample from the initial situation in 2O, 2.6 mL HNO3, 0.9 mL HCl, and 0.2 mL HF. Grain sizes of in situ alloyed Al-Cu-Mg-Ti are determined from etched T4 samples in the style of ISO 643 [For micrographic preparation, LBM samples are embedded in resin, ground, and polished with subsequently finer grain size down to 1 µm. Samples made from the quaternary powder mixture are additionally lapped, to lay bare fine cracks by removing grinding debris with free-rolling abrasive 9 µm diamond particles. After lapping, polishing is continued down to 1 µm grain size. Analysis of stitched high-resolution optical microscope Aristomet  images covering full sections of LBM samples, with exception of the rough outer edges, delivers ρ Blatt 2 . Vickers Blatt 2 . For ana ISO 643 . Microgrrel in LBM of Al alloys [SEM images show the powders as procured before sieving. They have mostly spherical to slightly ovoidal particle shapes. That indicates atomization with inert gas and little O absorption, which would cause spattered particles ,79. Incrl alloys ,36. MostThe chemical analysis of powders listed in x nanoparticles, samples are built with powder and process parameters suitable for prealloyed Al-Cu. Results are illustrated in To exemplify challenges of in situ alloying Al-Cu powder mixtures in LBM without SiOrel of LBM samples [x, as shown in light green in x is observed with Al < 20 µm, falling below Ti6Al4V particles. The corresponding pile is shown in x nanoparticles can significantly improve flowability of Al particles < 20 µm, and thus enable the spreading of smooth layers and LBM with high ρrel [A visually tangible comparison of the flowability of different powder particle size fractions according to the angle of repose φ, in the style of ISO 4324, is shown in  samples ,81,85. T samples ,66. Al pigh ρrel . This exx.The results of test tube experiments with binary Al-Cu powder mixtures are summarized in qCu. qCu causes segregation [To explain these observations, the dispersive coefficient of Cu, regation . Four caqCu, as shown in x nanoparticles.With all particles < 20 µm, the experimental result of no segregation leads to the assumption that qCu increases chances that near gaps between larger Al particles are occupied by other Cu particles, which further decreases The second case of Al > 20 µm and Cu < 20 µm is illustrated in ibration ,40,42.qCu. This seems plausible, considering that the smaller Al particles, which are much more numerous than Cu particles in the investigated mixtures with 95 wt% Al, should be able to move into gaps between larger Cu particles. This way, they can separate Cu particles from each other. Since mass is expected to have significant effects at this size of Cu particles, it is apparently compensated by the smaller size of Al particles. A similar observation of stability under motion without convective transport is described for a mixture of 2.5 mm large glass balls with 3.5 mm steel balls, at a mass density ratio of 2.6 [In the case of Al < 20 µm and Cu > 40 µm, which is illustrated in o of 2.6 .qCu, but not sufficiently lower to cause strong de-mixing. Due to the smaller difference in particle size compared to qCu, which increases probability for Cu particles to collide with lighter Al particles of lower momentum.The fourth considered case is that of intermediate de-mixing observed for Al particles > 20 µm and Cu particles of the same or larger size, as illustrated in Results of test tube experiments with quaternary Al-Cu-Mg-Ti powder mixture are put together in Mixtures with all particles sieved < 20 µm are considered most promising for controllably homogeneous in situ alloying.The results of thin powder layer microscopy inside the LBM machine Realizer SLM 50 are refined in In In The comparison of Cu distribution between different layers spread subsequently at different vertical Z height over the build platform is illustrated in x. Segregation results in thin layers differ significantly from the results of vibrated test tubes. The mixture with larger Cu particles, which is stable under vibration, shows more segregation in thin layers than the mixture with larger Al particles, which is less stable under vibration. Conditions for de-mixing during spreading of thin layers differ significantly from vibrated bulk powder. They can be characterized by the moving wiper, that appears to selectively transport Cu by convection [x, as shown in In summary, the best homogeneity in vibration as well as spreading of thin layers is found in the mixture with all particles < 20 µm dry coated with SiOnvection . This sex in three different PSD are visualized in rel in colors shaded from red, for 90%, to dark green, for 100%. The dependence of Δxy is depicted along the horizontal and of v along the vertical axis. Other process parameters, P = 100 W, Δz = 30 µm, and dspot = 66 µm, are kept constant. The white lines in vol, as defined in equation (6). Pink contours mark ρrel = 99%. For each PSD, 32 cube samples of 5 mm edge length have been built on filigree supports and analyzed micrographically for ρrel and details of defects. In rel > 99.5% have been achieved. Examples of such cubes are shown in rel can be concluded. rel = 88.6% shows voids elongated in parallel to Z of approximately constant width and distance. The used Δxy = 154 µm must be too large for reliable coalescence. Evol = 193 J/mm³ alone cannot give a satisfactory explanation, as lower 179 J/mm³ suffices for higher ρrel = 96.6%, at Δxy = 56 µm and v = 334 mm/s. An example for too high Evol = 1135 J/mm³ resulting in less than optimal ρrel = 98.8% is shown in Experimentally defined process windows for binary Al-Cu powder mixtures with constant composition of 94.7 wt% Al, 5 wt% Cu, and 0.3 wt% SiOrel > 99 % of the mixture of Al and Cu particles <20 µm that proved most stable against de-mixing is smaller than of the other two mixtures and shifted towards higher Evol. The powder mixture with smaller Cu particles than Al has a smaller range of LBM parameters yielding ρrel > 99% than the one with larger Cu particles, shifted towards smaller Δxy. A potential approach for explaining this can be differences in density of powder layers caused by the different PSD [rel [(1)Al and Cu < 20 µm: 1.42 g/cm³(2)Al < 20 µm and Cu 32–40 µm: 1.45 g/cm³(3)Al 20–32 µm and Cu < 20 µm: 1.47 g/cm³The process window yielding ρrent PSD ,87, whicPSD [rel ,89. The PSD [rel , as meanrel. However, the one with the largest bulk density has a smaller process window than the one with intermediate bulk density. However, bulk powder density is not identical and not directly transferrable to density of thin powder layers [rel might extend further with smaller Δxy, which is prohibited by the LBM machine control software.The mixture with the smallest bulk density does show the smallest process window for high ρr layers ,90. The Another factor contributing to the rather small process window of the mixture with all particles < 20 µm may be a storage duration extended by several months before LBM compared to the two other mixtures. It may have increased oxidation. Surface oxide layers are known to impede melt coalescence in welding of Al . They sh(1)Al and Cu < 20 µm: 65.9 HV0.1 ± 6.2(2)Al < 20 µm and Cu 32–40 µm: 68.4 HV0.1 ± 7.5(3)Al 20–32 µm and Cu < 20 µm: 66.5 HV0.1 ± 5.6No significant differences are found in hardness of samples built from the three mixtures, tested at n = 27 indentations per mixture, and more than seven days after LBM, to ensure natural ageing as a potential outcome of the largely undefined thermal history of LBM has reached a plateau:x nanoparticles. In addition to the types of defects described above for the binary mixtures, e.g., increased gas porosity in samples with very high Evol, exemplified in rel = 99.2% confirming appropriateness of striped scanning [Results of experimental LBM parameter variation with the quaternary Al-Cu-Mg-Ti powder mixture are illustrated in scanning . For harCv in Cv are very close in the different samples. For Cu, the T4 in situ alloyed LBM sample reaches even lower Cv than the extruded sample. This means Cu distribution is more homogeneous than in the conventionally manufactured benchmark. For Mg and Si, Cv of the in situ alloyed samples is increased by T4 treatment, which might be caused by precipitation of intermetallic phases. The BSE images in Cv of Cu in The results of chemical homogeneity analysis of LBM samples alloyed in situ from powder mixtures, as built, and T4 heat-treated, are illustrated in The average elemental compositions of LBM sample and extruded sample are compared to the input from the powder mixture and the standard definition of EN AW-2024 in Stress-strain curves of the tensile tests are shown in rel = 99.2%. Fracture surfaces of horizontal Al-Cu-Mg-Ti samples reveal no increased occurrence of unmelted particles compared to the vertical samples as described in [Fracture surfaces of vertical T4 samples, shown in ribed in ,72.z [The etched micrographs of horizontal and vertical samples, shown in z . The graz . The graThe preferred orientation of grain boundaries in parallel to Z might be a major contributing factor to the lack of ductility in the horizontal samples. The accumulation of alloying elements and impurities in grain boundaries can promote intragranular fracture that may explain the cliffy shapes in x nanoparticles. Microparticle sizes > 20 µm result in pronounced de-mixing. Experimental segregation results with varied particle size fractions are contrasted to the theory that segregation in vibrated bulk powder is determined by the negative correlation of particle mobility with local concentration [It is shown that metal powder mixtures with density ratio of 3.3, as in Al-Cu, can be stabilized against segregation by using only particles < 20 µm that have been dry coated with SiOntration . The mosntration .A patent application resulted from the work reported in this manuscript .