The Sn and Sb incorporation boosts the electronic conductivity and lithium diffusivity of Si anodes, thereby reducing the stress due to lithium concentration gradient.The lithiation of modified electrode mimics isotropic solid solution reaction, rather than the original anisotropic two-phase reaction of Si, effectively weakening the stress concentration.−1 after 100 cycles at 0.1 A g−1 and maintain the excellent cyclic stability at 3 A g−1.The silicon-rich particles exhibit a capacity of over 1.9 Ah gThe online version contains supplementary material available at 10.1007/s40820-023-01190-7. 8.5Sn0.5Sb microparticles (mean particle size: 8.22 μm) show over 6000-fold and tenfold improvements in electronic conductivity and Li diffusivity than Si particles, respectively. The discharge capacities of the Si8.5Sn0.5Sb microparticle anode after 100 cycles at 1.0 and 3.0 A g−1 are 1.62 and 1.19 Ah g−1, respectively, corresponding to a retention rate of 94.2% and 99.6%, respectively, relative to the capacity of the first cycle after activation. Multicomponent microparticle anodes containing Si, Sn, Sb, Ge and Ag prepared using the same method yields an ultra-low capacity decay rate of 0.02% per cycle for 1000 cycles at 1 A g−1, corroborating the proposed mechanism. The stress regulation mechanism enabled by the industry-compatible fabrication methods opens up enormous opportunities for low-cost and high-energy–density Li-ion batteries.Si is a promising anode material for Li ion batteries because of its high specific capacity, abundant reserve, and low cost. However, its rate performance and cycling stability are poor due to the severe particle pulverization during the lithiation/delithiation process. The high stress induced by the Li concentration gradient and anisotropic deformation is the main reason for the fracture of Si particles. Here we present a new stress mitigation strategy by uniformly distributing small amounts of Sn and Sb in Si micron-sized particles, which reduces the Li concentration gradient and realizes an isotropic lithiation/delithiation process. The SiThe online version contains supplementary material available at 10.1007/s40820-023-01190-7. Li-iaterials –6. The cnodes 0.3 Ah g−1, ials [+) –10. Due  S cm−1) , the pulmentclasspt{minima S cm−1) . Further S cm−1) , 16.+ inventory in the formation of SEI. Finally, the complex and delicate fabrication process of nano-Si anodes greatly weakens the low-cost advantage of Si. These thorny flaws of nano-Si anodes have made micron-sized Si anode an increasingly important research direction.The volume change during lithiation/delithiation is found not to cause electrode fracture when the Si particle size is less than 150 nm , 15. NanFor micron-sized Si anodes, particle fracture during cycling is one of the most critical issues to be addressed. Preparation of robust protective layers in forms of binder –31, coat4 S cm−1) and Sb  [Doping or alloying Si with other elements are promising approaches to improve its electron/Li conductivity . Althoug S cm−1)  with a h S cm−1) –45. So f S cm−1)  or hot-p S cm−1) , to prep S cm−1) , to synt S cm−1)  or Sn na S cm−1) , and to  S cm−1) , 52. The8.5Sn0.5Sb alloy ingots through an electric arc furnace. High-energy ball milling was then used to pulverize the ingot and further promote the mixing of the three elements. The Si8.5Sn0.5Sb microparticle anode demonstrated tremendous improvements in electron/Li conductivity, mechanical integrity, and consequently the cycling performance compared to Si anode. After 100 cycles in conventional carbonate-based electrolyte, the Si8.5Sn0.5Sb microparticle anode exhibited discharge capacities of 1.62 and 1.19 Ah g−1 for the rate of 1 and 3 A g−1, respectively, and the capacity retention were both higher than 90%. The stress mitigation mechanism was substantiated by finite element simulation results and extended to quaternary and pentanary alloy systems, both of which yielded extraordinary cyclic stability.In this work, we propose a stress mitigation strategy to realize the stable cycling of Si-rich microparticle anodes via a simple two-step fabrication method. Bulk Si, Sn, Sb raw materials were first smelted into Si8.5Sn0.5Sb (at%) were prepared by arc-melting a mixture of raw Si, Sn, and Sb (purity > 99.9 wt%) under a Ti-gettered argon atmosphere. The melting was repeated at least four times to ensure chemical homogeneity. The alloy ingot was pre-crushed into half-centimeter-sized pieces and sealed into a 100-mL ball-milling jar under an argon atmosphere. The mass ratio of ball to alloy sample was approximately 20:1. The sample was milled (SPEX 8000) eight times for 30 min each at 15 min intervals. The ball-milled particles were sieved using a 300-mesh sieve and stored in a dry argon atmosphere. The arc-melted and ball-milled alloy particles were mixed with super P by ball milling for half an hour, and then mixed with vapor-grown carbon fibers and sodium carboxymethyl cellulose in deionized water for twelve hours. The mass ratio in sequence was 6.4:1.6:1:1. The mixture slurry was cast on a Cu foil (1.2 cm in diameter) and dried in a vacuum oven at 80 °C for 24 h. The mass loading of active materials per anode sheet was 0.75 ± 0.16 mg cm−2.Alloy ingots with a nominal composition of Si2O < 0.5 ppm, O2 < 0.5 ppm) using a piece of glass fiber  separator and Li foil as the counter electrode. The electrolyte was 1 M LiPF6 dissolved in ethylene carbonate -dimethyl carbonate  solution at the volume ratio of 1:1, with 5 vol% of fluoroethylene carbonate  as additive. Each coin cell contained 80 µL of electrolytes and was tested at 293 K. Electrochemical impedance spectroscopy (EIS) test was performed on an electrochemical workstation (Biologic SP150) between 100 kHz and 0.1 Hz with an amplitude of 5 mV. Cyclic voltammetry (CV) data were collected on an electrochemical workstation  at 0.05 mV S−1 between 0 and 1.5 V. Galvanostatic intermittent titration technique (GITT) tests were collected on an Arbin battery system at 0.1 A g−1 with a current pulse of 0.25 h and a relaxation time of 3 h. The batteries underwent 10 cycles at 1 A g−1 before the GITT test.Half-cells were assembled with CR2032 coin cells in an argon-filled glove box (Hk = d/(RA), where d and A is the thickness and cross area of the pallet, respectively, and R is the measured resistance. The influence of porosity in the pallet on the measured resistance was ignored here. In-situ XRD test was performed using a Swagelok cell equipped with a beryllium window. In-situ atomic force microscope  testing was performed in an argon-filled glove box using an instrument-equipped electrochemical cell to characterize the evolution of electrode morphology during cycling. For ex-situ characterizations  of the electrode after cycling, the cells were disassembled and the electrodes were rinsed with DMC in an argon-filled glove box to remove the electrolyte. For ex-situ TEM samples, the electrode material was scraped from the Cu foil and dispersed in DMC. A drop of the suspension was then deposited on the copper grid and dried in the argon-filled glove box. For ex-situ XRD samples, the electrode sheets were sealed in the homemade in-situ XRD cell in the argon-filled glove box before testing.The arc-melted alloy ingot was mechanically polished to 0.05 μm before examining its microstructure by using scanning electron microscopy . Particle samples for SEM measurements were prepared by sticking the alloy particles on the conductive glue of the sample stage with a toothpick. The size and element distribution of anode particles were evaluated by SEM. The transmission electron microscope  samples were prepared by dropping the particle-alcohol solution on a copper grid. The elemental distribution and microstructure within individual particles were characterized by energy-dispersive X-ray spectroscopy (EDS) and high-resolution transmission electron microscope (HRTEM) images. To test the electrical conductivity of prepared anode particles, they were first formed into pallets of 1 cm diameter under an isostatic pressure of 250 MPa. The thickness of the pallet was obtained by the vernier caliper. The pallet was then sandwiched between two stainless steel plates and sealed in a coin cell. Resistance was tested by EIS with amplitudes of 100 and 500 mV. The formula for calculating the electronical conductivity is The stress mitigation mechanism to stabilize Si-rich microparticle anode was achieved by combining arc melting and high-energy ball milling to uniformly distribute Sn and Sb in Si Fig. a. Bulk r8.5Sn0.5Sb-AMBM, where AMBM is short for “arc melting—ball milling”. As shown in Fig. 8.5Sn0.5Sb-AMBM microparticles has an over 6000-fold increase in electrical conductivity compared to Si powders.Due to the high toughness and ductility of Sn, if commercial Si, Sn, and Sb powders are directly mixed by high-energy ball milling, the so-called “cold welding” effect can lead to the self-agglomeration and growth of Sn powders . Pre-for8.5Sn0.5Sb-AMBM microparticles as LIB anode was evaluated in coin cell tests in a conventional carbonate-based electrolyte  were used to prepare the control group anode. Silicon anodes are usually cycled within a voltage window narrower than its theoretical one to avoid the severe pulverization [−1. Specifically, cells were cycled between 0.01 and 1.5 V at 0.1 A g−1. For other conditions, cells were first pre-cycled 3 times between 0.01 and 1.5 V at small current densities, and then cycled between 0.06 and 1 V at the targeted current densities.The performance of Siyte Fig. . A mixturization , 54. We 8.5Sn0.5Sb-AMBM and control group anodes tested at 0.1 A g−1 between 0.01 and 1.5 V (vs. Li/Li+) are shown in Figs. 8.5Sn0.5Sb-Mix anode exhibits a staircase shape, corresponding to multiple phase transition voltage plateaus for the three elements. The Si8.5Sn0.5Sb-AMBM anode presents completely different voltage curves compared with the above two samples. A short charging plateau corresponding to the lithiation of Sb appears at ~ 0.85 V [8.5Sn0.5Sb-AMBM anode indicates that its lithiation/delithiation process is closer to a solid solution reaction than a two-phase reaction [−1, the first discharge and charge capacities of the Si8.5Sn0.5Sb-AMBM anode are 2.78 and 2.30 Ah g−1, respectively, corresponding to the initial Coulombic efficiencies (CE) of 82.62%. The irreversible capacity may be caused by electrolyte decomposition to form SEI and incomplete delithiation. As shown in Fig. 8.5Sn0.5Sb-AMBM anode battery after 100 cycles remains 1.96 Ah g−1. Under the same test condition, the Si8.5Sn0.5Sb-Mix anode of the comparison group only retains a discharge capacity of 0.21 Ah g−1 . The cycling performance of the Si8.5Sn0.5Sb-AMBM anode is also superior when the mass loading is high. As shown in Fig. S8, a discharge capacity of 1.14 Ah g−1 is obtained after 100 cycles’ cycling at 0.5 A g−1 with a mass loading of 1.60 mg cm−2. Voltage profiles of the initial three cycles for Si~ 0.85 V , 57 in treaction . At a cu8.5Sn0.5Sb-AMBM anode at seven different current densities. The battery was first activated at 0.03 A g−1 for 3 cycles and then tested for 5 cycles at each subsequent rate [8.5Sn0.5Sb-AMBM anode at different current densities all exhibit a slop shape , indicating that the battery process is dominated by solid-solution reactions. The discharge capacities are 1.85, 1.73, 1.67, 1.61, 1.34 and 1.19 Ah g−1 for rates of 0.1, 0.25, 0.5, 1.0, 3.0 and 5.0 A g−1, respectively. After cycling at 5.0 A g−1, the battery is again cycled at 0.1 A g−1, recovering almost 100% of its capacity. These results indicate that the Si8.5Sn0.5Sb-AMBM anode has superior rate performance and cycling stability. Long-cycle performance of Si8.5Sn0.5Sb-AMBM and comparison group anodes at 1 and 3 A g−1 are presented in Figs. −1 may be due to the fact that at high current density, the relative large anode particles are more prone to breakage during the initial several cycles and generate new electrode surface that triggers the continuous SEI formation. The discharge capacities of Si and Si8.5Sn0.5Sb-Mix anodes  drop rapidly to 0.23 and 0.24 Ah g−1, respectively, after 100 cycles at 1 A g−1, corresponding to a 13% and 14% capacity retention with respect to the first discharge capacity after pre-cycling, respectively. In contrast, the Si8.5Sn0.5Sb-AMBM anode shows substantially improved cycling stability, whose discharge capacity is 1.62 (94.2% capacity retention) and 1.28 Ah g−1 (99.6% capacity retention) after 100 cycles at 1 and 3 A g−1, respectively. Furthermore, the reversible capacities of Si8.5Sn0.5Sb-AMBM anode after 300 cycles at 1 and 3 A g−1 remain at the values of 1.13 and 0.82 Ah g−1, respectively, corresponding to the capacity retention rates for each cycle of 99.86% and 99.88%, respectively.Figure ent rate , 54. The8.5Sn0.5Sb-AMBM anode performs particularly well in achieving high capacity and stable cycling over large-particle Si anodes.We summarize in Fig. 8.5Sn0.5Sb-AMBM and Si8.5Sn0.5Sb-Mix anodes after 3 cycles. The results of the charge transfer resistance (Rct) obtained after fitting the equivalent circuit [8.5Sn0.5Sb-AMBM anode has a much smaller Rct  [8.5Sn0.5Sb-AMBM anode is over 10 times higher than that of the Si8.5Sn0.5Sb-Mix anode . The EIS and GITT results imply that our proposed fabrication method can effectively enhance the electron/Li conductivity of Si microparticle anodes. The Sn and Sb network in Si provides pathways for electron and Li diffusion throughout the electrode surface and inside.The charge transfer resistances of anodes were assessed by EIS measurements. Figure S11a shows the Nyquist plots measured at open circuit potential for Si circuit  are summig. S12) , 67. For8.5Sn0.5Sb-AMBM  anodes to investigate the mechanisms of electrode reactions. Figure 8.5Sn0.5Sb-AMBM anode has peaks around 28.2 and 28.8 degree, corresponding to the crystalline Si and SnSb phases, respectively. As lithiation evolves, the SnSb peak rapidly decreases in the subsequent XRD patterns, disappears completely from the fourth XRD curve (around 0.6 V), and does not reappear at the end of delithiation. The crystalline Si peak also greatly reduces with the progress of lithiation and is not recovered during delithiation [8.5Sn0.5Sb-AMBM anode exhibits a similar lithiation/delithiation behavior to amorphous Si from the second cycle onward. When pure Si is discharged to ~ 0.05 V, amorphous lithiated Si undergoes a two-phase transformation process to the Li15Si4 crystalline phase. To avoid stress concentrations caused by the phase transition process, a discharge limit (0.07 to 0.06 V) is usually set for LIBs with Si anodes [8.5Sn0.5Sb-AMBM anode is discharged to 0.01 V, no peaks corresponding to the Li15Si4 crystalline phase appear [15Si4 phase were also not observed in the first CV curve . One possible reason is that the lattice distortion induced by the inclusion of Sb and Sn atoms in the Si matrix effectively hinders the formation of long-range ordered structures and suppresses the two-phase reaction. The faint presence of the peak for Si in Fig. In-situ XRD measurements were carried out on the first cycle of SiMBM Fig. a and Si8thiation , 68, 69.i anodes , 54. But8.5Sn0.5Sb-AMBM and Si8.5Sn0.5Sb-Mix anodes after 3 cycles at 0.1 A g−1 are shown in Figs. S16 and S17, and the chemical compositions of the SEI formed on these two electrodes are very similar. The mechanical properties of SEI including Young’s modulus and elastic strain limit are evaluated by a two-step nanoindentation test based on the atomic force microscope [8.5Sn0.5Sb-AMBM electrode are close to those of the SEI formed on Si8.5Sn0.5Sb-Mix electrode but the range of data distribution is much smaller. These results indicate that by uniformly distributing Sn and Sb within the Si microparticle does not modify the chemical compositions of SEI but facilitates a more homogeneous SEI than just mix the Si, Sn and Sb particles together.The XPS results for the Sicroscope –72. The 8.5Sn0.5Sb-AMBM anodes after 3 pre-cycles at 0.1 A g−1 and 47 cycles at 1 A g−1 are shown in Fig. S19a, b, respectively. The electrode morphologies are similar in these two images. In marked contrast to Si8.5Sn0.5Sb-Mix anode , EDS images for Si8.5Sn0.5Sb-AMBM anode taken after 3 pre-cycles at 0.1 A g−1  also indicate uniform distributions of Sb and Sn in Si. In addition, the HRTEM and diffraction results  were performed on the dimension change, Li concentration distribution, and stress state of individual anode particle during lithiation. In the discharge cycle, lithiation starts from the surface of anode and proceeds inward. A Li concentration gradient forms from the particle surface to the centre, resulting in varying degrees of volume change and stress [−1 was applied to the outer surface of the sphere .To reveal the mechanism of the improved mechanical stability of Sid stress –76. Simud stress , 78. Thid stress . A generd stress –83. In s8.5Sn0.5Sb-AMBM anodes at different DODs , respectively. For all DODs, the Li concentration gradient in the Si anode , as well as quaternary and pentanary alloy-type anodes for LIBs using the same technique. The discharge capacities of the Si4Sn2.8Sb-AMBM microparticle anode after 300 cycles at 1.0 and 3.0 A g−1 are 0.81 and 0.68 Ah g−1, respectively, corresponding to a retention rate of 80.6% and 86.2%, respectively, relative to the capacity of the first cycle after activation. The quaternary system includes Si, Sb, Sn and Ge and the pentanary system adds Ag on this basis. These two anodes are referred to as SiSnSbGe-AMBM and SiSnSbGeAg-AMBM, respectively. In both systems, the mole fraction of each element is equal. The microparticle SiSnSbGe-AMBM and SiSnSbGeAg-AMBM anodes demonstrate uniform element distribution  and extraordinary cycling stability. Figure −1 and 47 cycles at 1 A g−1 also show that the distribution of the various elements in the electrodes remains uniform.To test the applicability of the stress mitigation mechanism, we prepared micron-scale Si-based ternary alloy anode with a composition of Si−1 are shown in Figs. −1 are 1.02 and 0.78 Ah g−1, respectively. In addition, after 1000 cycles at 1 A g−1, the capacity retention of the SiSnSbGeAg-AMBM anode is as high as 79.43% relative to the first cycle after activation, corresponding to an extremely small capacity decay rate of 0.02% per cycle. The superior cycling stability of SiSnSbGe-AMBM and SiSnSbGeAg-AMBM microparticle anodes proves that the arc melting together with high-energy ball milling is a promising route to prepare microparticle alloy-type anodes for LIBs.The long-cycle performance of SiSnSbGe-AMBM and SiSnSbGeAg-AMBM anodes at 0.1 and 1 A g8.5Sn0.5Sb microparticle anode has a much higher electronic conductivity and Li diffusivity than Si, which facilitates the reduction of stresses caused by Li concentration gradients. Moreover, the electrode process of Si8.5Sn0.5Sb microparticle anode is similar to isotropic solid solution reactions, which greatly avoids the stress concentration caused by the two-phase reactions. Under these mechanisms, the discharge capacities of Si8.5Sn0.5Sb microparticle anode after 100 cycles at 1.0 and 3.0 A g−1 are 1.62 and 1.19 Ah g−1, respectively, corresponding to a retention rate of 94.2% and 99.6%, respectively, relative to the capacity of the first cycle after activation. We believe that this stress regulating mechanism enabled by the simple industry-compatible fabrication method combining arc melting and high-energy ball milling could present tremendous opportunities for the impending low-cost and high-energy density Li ion batteries.For micron-sized Si anodes, severe particle pulverization during cycling hinders their practical application in LIBs. Our work demonstrates that eliminating high stressors that lead to particle fracture can effectively improve the cycling stability of Si-based microparticle anodes. To maintain the competitive advantages of high specific capacity and low cost of Si anode, Sn and Sb with the same advantages are used as modifiers. Combining arc melting and high-energy ball milling, Sn and Sb are uniformly distributed inside the Si microparticles. The prepared SiSupplementary file1 (PDF 3597 kb)Below is the link to the electronic supplementary material.


Methods: We examined whether immune cell profiles differ between healthy women (n = 38) and breast cancer survivors (n = 27) within 2 years of treatment, and whether any group-differences were influenced by age, cytomegalovirus infection, cardiorespiratory fitness and body composition. Using flow cytometry, CD4+ and CD8+ T cell subsets, including naïve (NA), central memory (CM) and effector cells (EM and EMRA) were identified using CD27/CD45RA. Activation was measured by HLA-DR expression. Stem cell-like memory T cells (TSCMs) were identified using CD95/CD127. B cells, including plasmablasts, memory, immature and naïve cells were identified using CD19/CD27/CD38/CD10. Effector and regulatory Natural Killer cells were identified using CD56/CD16.Results: Compared to healthy women, CD4+ CM were +Δ21% higher among survivors (p = 0.028) and CD8+ NA were −Δ25% lower (p = 0.034). Across CD4+ and CD8+ subsets, the proportion of activated (HLA-DR+) cells was +Δ31% higher among survivors: CD4+ CM (+Δ25%), CD4+ EM (+Δ32%) and CD4+ EMRA (+Δ43%), total CD8+ (+Δ30%), CD8+ EM (+Δ30%) and CD8+ EMRA (+Δ25%) (p < 0.046). The counts of immature B cells, NK cells and CD16+ NK effector cells were higher among survivors . Subsequent analyses examined whether statistically significant differences in participant characteristics, influenced immunological differences between groups. Compared to healthy women, survivors were older (56 ± 6 y vs. 45 ± 11 y), had lower cardiorespiratory fitness (−1 min−1: 28.8 ± 5.0 vs. 36.2 ± 8.5), lower lean mass (42.3 ± 5.0 kg vs. 48.4 ± 15.8 kg), higher body fat (36.3% ± 5.3% vs. 32.7% ± 6.4%) and higher fat mass index (FMI kg/m2: 9.5 ± 2.2 vs. 8.1 ± 2.7) . Analysis of covariance revealed divergent moderating effects of age, CMV serostatus, cardiorespiratory fitness and body composition on the differences in immune cell profiles between groups, depending on the cell type examined. Moreover, across all participants, fat mass index was positively associated with the proportion of HLA-DR+ CD4+ EMRA and CD8+ EM/EMRA T cells . The association between fat mass index and HLA-DR+ CD8+ EMRA T cells withstood statistical adjustment for all variables, including age, CMV serostatus, lean mass and cardiorespiratory fitness, potentially implicating these cells as contributors to inflammatory/immune-dysfunction in overweight/obesity. The numbers and characteristics of leukocyte subtypes assessed in blood prior to cancer therapy have been linked to treatment responses, disease progression, mortality and recurrence . For exaThe numbers and characteristics of leukocyte subtypes in blood also change during cancer therapy and with disease progression. The mechanisms are thought to include direct effects of chemotherapeutic drugs and alterations to haematopoietic stem cells, the bone marrow niche and the thymus, in combination with inflammation and chronic stimulation by tumour antigens . Overallboth increases and decreases in cell counts and frequencies following cancer treatment is partly because not all studies employ detailed immunophenotyping of major leukocyte sub-types. In doing so, cells with specific functions can be quantified, providing a more detailed understanding and nuanced interpretation of the direction of changes to cell counts. However, a contributing explanation for different results between studies is that age and lifestyle factors–which are known to affect the numbers and characteristics of leukocyte sub-types in blood–are often not considered . For inclusion, these patients were aged 25–69 years, had adequate renal, liver and bone marrow function, no significant cardiac comorbidity, a World Health Organisation performance status of 0–1 and were due to receive neoadjuvant chemotherapy for approximately 6 months prior to surgery. Thus, the group of patients help to initially isolate possible effects of (1) the formation and presence of a tumour; in the absence of long-term exposure to cancer cells or treatment. All participants provided informed consent and the studies were approved by NHS research ethics committees, reference numbers: 15/SW/0004 and 18/WA/0314.Given that possible differences between groups could be driven by; 1) the past formation and presence of a tumour; 2) previous disease progression and chronic stimulation by tumour antigens and tumour-derived inflammation; or 3) the effects anti-cancer treatments can have on the immune system, we also recruited recently diagnosed female patients with biopsy-proven non-metastatic non-bilateral stage I–III invasive breast cancer, who had not received any form of treatment, as part of a preliminary analysis (n = 27), 22.2% of the participants had been diagnosed with ductal carcinoma in situ (DCIS) and 7.4% had a multifocal carcinoma. 74.1% participants were positive for hormone receptors (ER/PR+) and 7.4% were positive for HER2 . Adjuvant therapies included radiotherapy (81.5%), endocrine therapy (66.7%), chemotherapy (33.3%) and/or anti-HER2+ therapy (7.4%). Endocrine therapy included treatment with letrozole, anastrozole or tamoxifen, and chemotherapy included treatment with either FEC-T , FEC-TH  or AC-paclitaxel . At recruitment, the average of time since diagnosis was 14 ± 6 months and the average time since treatment was 12 ± 6 months.Diagnostic and treatment history is shown in n = 5), the majority (80%) had grade three tumours, involved lymph nodes (60%), and were ER negative (60%) and HER2 negative (80%). The chemotherapy combination patients were due to receive was either FEC-T , FEC-TPH (with pertuzumab and trastuzumab added for cycles 4–6) or FEC-paclitaxel .Among the patients scheduled to undergo treatment  using the dominant arm among healthy women, or the contralateral arm to the affected breast among survivors and patients. Height was assessed with a wall mounted stadiometer  and body mass was measured without shoes using electronic scales . Waist and hip circumferences were measured (in centimetres) with a tape . Dual energy X-ray absorptiometry  was used for body composition analysis. Cardiorespiratory fitness (sing age .3–EDTA) tube  and an anticoagulant-free serum separation tube for preparation of plasma and serum respectively. The tube for serum was left to clot for 30 mins at room temperature. Tubes for serum and plasma were centrifuged for 10 min at 2,000 × g and 4°C, and the supernatant was collected and stored at −80°C until analysis. To isolate PBMCs, blood was diluted 1:1 with sterile RPMI , layered onto Ficoll-Paque™ plus  and centrifuged at 500 × g at 21°C for 25 min. PBMCs were washed in RPMI by centrifuging at 400 × g at 21°C for 10 min and washed again in RPMI at 300 × g at 21°C for 7 min. Platelets were removed by washing in RPMI at 200 × g at 21°C for 7 min, and then cells were counted with Trypan blue (1.5% acetic acid) using a haemocytometer. PBMCs were resuspended in freezing mix , and frozen at −1°C/min in a freezing container  in a −80°C freezer.After a 10-min seated rest, approximately 40 mL of blood was collected into a syringe containing sodium heparin (4 IU/mL) for preparation of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using density gradient centrifugation. An additional 10 mL of blood was collected into a potassium–ethylenediaminetetraacetic acid  by centrifuging at 300 × g at 21°C for 7 min. If clumping was visible, 0.3 µL of benzonase nuclease  was added to the pellet and incubated for 5 min before washing with media and centrifuging at 300 × g at 21°C for 7 min. Cells were counted and rested overnight in media, at a concentration of 2 million cells per mL in tubes that enable gas exchange, at 37°C and 5% COTwo antibody panels were prepared per sample . A subsequent control panel prepared with cells from each participant included isotype controls for assessment of T cell activation. Cells were stained with a Fixable Viability Stain (FVS) conjugated to v450  and 400,000 cells were added to tubes for each panel. The T cell panel included anti-CD3—PERCP clone SK7, anti-CD4—PE-Cy7 clone L200, anti-CD8—APC clone SK1, anti-CD45RA—FITC clone HI100, anti-CD127—APC-Cy7 clone A019D5, anti-HLA-DR—V500 clone G46-6, anti-CD27—PE clone M-T271 and anti-CD95—BV605 clone DX2. The B cell/NK cell panel included anti-CD3—PE-Cy7 clone SK7, anti-CD19—PERCP clone 4G7, anti-CD10—APC-Cy7 clone HI10a, anti-CD27—APC clone L128, anti-CD38—FITC clone HB7, anti-CD16—V500 clone 3G8 and anti-CD56—PE clone B159. The isotype control panel included HLA DR—V500 isotype control  and anti-CD3—PERCP clone SK7. Antibodies were purchased from BD Biosciences  with the exception of anti-CD127—APC-Cy7, anti-CD95—BV605 and anti-CD10—APC-Cy7 .Samples were analysed within 2 h of preparation using a FACSAria III flow cytometer . Voltages were optimised and maintained for all samples and all participants. Compensation was performed each day using single-stained tubes and positive and negative compensation beads  and parameters calculated automatically . Acquisition flow rate was maintained across samples. Approximately 70,000–100,000 events (T cell panel), 50,000–75,000 events (B cell panel) and 15,000 events (isotype control tube) were recorded from the lymphocyte gate.3–EDTA blood on the day of sampling . The CD4+/CD8+ ratio was calculated to examine inverted or high ratios (<1 or >2.5), which have been linked to immunosenescence and chronic inflammation . After doublets were excluded, an initial plot was created with SSC (side scatter) versus FSC (forward scatter) , panel Bammation . Normal ammation .CMV-specific IgG was assessed in serum using enzyme-linked immunosorbent assays (ELISAs) according to manufacturer instructions . Absorbance at 450 nm was determined using a SPECTROstar Nano plate reader . Values were blank corrected, and concentrations were calculated using a 4-parameter curve. CMV+ was considered to be >1.2 IU/mL, and CMV− was <0.8 IU/mL. Inter and intra assay variation was 1.74% and <5.2% respectively.n = 38) and survivors (n = 27). Given the small sample size of patients (n = 5), data were included for qualitative comparisons. Normal distribution was assessed using descriptive statistics and Shapiro Wilk and Kolmogorov-Smirnov tests. Most data were not normally distributed and therefore all data were log10 transformed and compared between groups using univariate analyses of variance (ANOVA). Levene’s tests were interpreted for homogeneity of variance between groups (p > 0.05). When participant characteristics  were significantly different between groups, these variables were examined as covariates using analyses of covariance (ANCOVA). Effect sizes were reported as eta squared (ƞ2), where, ƞ2 = 0.01 is a small effect, ƞ2 = 0.06 is a medium effect and ƞ2 = 0.14 is a large effect size . Figures were created with GraphPad Prism v9.0.0 for Windows .Statistical analyses were conducted between healthy women  < 0.630, p > 0.430, ƞ2<0.01]  = 140.743, p < 0.001, ƞ2 = 0.740, Survivors: F = 159.808, p < 0.001, ƞ2 = 0.822] and CD8+ cells  (p < 0.01). Breast cancer patients exhibited a similar pattern to breast cancer survivors and healthy women  = 38.674, p < 0.001, ƞ2 = 0.468, Survivors: F = 28.862, p < 0.001, ƞ2 = 0.474] and the CD8+ T cell subsets  (p < 0.05). A similar pattern was shown among breast cancer patients (data not shown).Both healthy women and breast cancer survivors exhibited statistically significant differences in the proportion of activated HLA-DR+ cells across the CD4+ T cell subsets [ANOVA: Healthy: F= 0.270] . For botpatients . Analysi = 5.039, p = 0.028, ƞ2 = 0.074] and CD8+ NA T cells were lower  among survivors compared to healthy women <1.6, p > 0.211, ƞ2 < 0.025]  < 2.018, p > 0.160, ƞ2 < 0.031]  > 4.160, p < 0.046, ƞ2 > 0.068]  > 4.131, p < 0.047, ƞ2 > 0.068]  < 3.992, p > 0.051, ƞ2 < 0.065] (data not shown).The proportion of cells expressing the activation marker HLA-DR was significantly higher among survivors compared to healthy women for CD4+ CM, CD4+ EM and CD4+ EMRA [Summary of ANOVAs: F> 0.068]  and for > 0.068] . There w< 0.065] . Among b = 2.945, p = 0.091, ƞ2 = 0.045] (p = 0.138). Among breast cancer patients, 40% exhibited a normal CD4+/CD8+ ratio, 20% had an inverted ratio, and 40% had a high ratio.The CD4+/CD8+ ratio tended to be higher among survivors (3.7 ± 2.7) compared to healthy women (2.8 ± 1.5) but this difference was not statistically significant [ANOVA: F= 0.045] . Overall < 3.531, p > 0.065, ƞ2 < 0.053] but cell numbers among survivors were approximately half of those exhibited by healthy women  . Breast  = 4.417, p = 0.040, ƞ2 = 0.070] and a tendency for higher counts in the other B cell subtypes  < 0.133, p > 0.717, ƞ2 < 0.002]  = 11.403, p = 0.001, ƞ2 = 0.162] and CD16+ NK effector cells  compared to healthy women  = 1.840, p = 0.180, ƞ2 = 0.030]. Breast cancer patients tended to have NK cell and NK cell subset counts that were closer to the counts exhibited by survivors and higher than healthy women.Survivors exhibited a greater number of total NK cells   < 2.668, p > 0.107, ƞ2 < 0.041; Compared to healthy women, breast cancer survivors were older and shorter, exhibited lower cardiorespiratory fitness  = 5.039, p = 0.028, ƞ2 = 0.074] was lost when controlling for age and  < 3.503 p > 0.066 ƞ2 < 0.053] but it was maintained when controlling for body fat percentage, lean mass, fat mass index and CMV status [Summary of ANCOVAs: F > 4.008 p < 0.050 ƞ2 > 0.061]  = 4.690, p = 0.034, ƞ2 = 0.069) was lost when controlling for age,  < 3.793, p > 0.056, ƞ2 < 0.058] but it was maintained when controlling for lean mass or CMV status   = 4.160, p = 0.046, ƞ2 = 0.068] was lost when controlling for age, lean mass and fat mass index [Summary of ANCOVAs: F < 3.642 p > 0.061 ƞ2 < 0.061] but it was maintained when controlling for  > 4.211, p < 0.045, ƞ2 > 0.070]. The statistically significantly difference in the proportion of HLA-DR+ CD4+ EM and EMRA T cells which was higher among survivors  was maintained when controlling for all variables . = 6.408, p = 0.014, ƞ2 = 0.101], was lost when controlling for age and fat mass index [Summary of ANCOVAs: F < 3.168 p > 0.081 ƞ2 < 0.054] but it was maintained when controlling for  > 4.162 p < 0.046 ƞ2 > 0.069]. The statistically significant difference in the proportion of HLA-DR+ CD8+ EM T cells, which was higher among survivors  was maintained when controlling all variables [Summary of ANCOVAs: F > 6.556 p < 0.013 ƞ2 > 0.105]. Finally, the statistically significant difference in the proportion of CD8+ EMRA T cells which was higher among survivors  was lost when controlling for all variables [Summary of ANCOVAs: F < 3.688 p > 0.060 ƞ2 < 0.062] except for body fat percentage and CMV status [Summary of ANCOVAs: F > 4.022 p < 0.050 ƞ2 > 0.067].For CD8+ T cells, the statistically significant difference in the proportion of HLA-DR+ CD8+ T cells, which was higher among survivors compared to healthy women [ANOVA: F = 2.945 p = 0.091 ƞ2 = 0.045], controlling for CMV serostatus strengthened the relationship [ANCOVA: F = 3.293 p = 0.074 ƞ2 = 0.050]. The CD4+/CD8+ ratio was not influenced by other variables [Summary of ANCOVAs: F < 2.663 p > 0.108 ƞ2 < 0.041)  . = 0.473 p = 0.494 ƞ2 = 0.007. CD8+ TSCMs: F = 3.531 p = 0.065 ƞ2 = 0.053] and there were no effects of controlling for other variables   = 3.687 p = 0.059 ƞ2 = 0.056].There were no differences in the counts of CD4+ and CD8+ TSCMs between survivors and healthy women [ANOVA: CD4+ TSCMs: F< 0.056] . Althoug = 4.417 p = 0.040 ƞ2 = 0.070] was lost when controlling for age and fat mass index [Summary of ANCOVAs: F < 3.148 p > 0.081 ƞ2 < 0.051] but it was maintained when controlling for  > 4.457 p < 0.039 ƞ2 > 0.071]  < 0.133 p > 0.717 ƞ2 < 0.002] and these patterns were maintained when controlling for all variables [Summary of ANCOVAs: F < 0.521 p > 0.473 ƞ2 < 0.009].The statistically significant difference in immature B cells which were higher among survivors compared to healthy women [ANOVA: F> 0.071] . There w > 11.403 p < 0.001 ƞ2 > 0.162] was maintained when controlling all variables [Summary of ANCOVAs: F > 8.272 p < 0.006 ƞ2 > 0.125]  = 1.840 p = 0.180 ƞ2 = 0.030] and this pattern was maintained when controlling for all variables [Summary ANCOVAs: F < 2.115 p > 0.151 ƞ2 < 0.035].The statistically significant difference in NK cells and Effector NK cells which were higher among survivors compared to healthy women [Summary of ANOVAs: F> 0.125] . There wr > 0.305, p < 0.019)  . In gener > 0.305, p < 0.019) was lost when controlling for  < 3.957, p > 0.052, R < 0.310, R2 < 0.096 R2Adjusted < 0.064 R2Change < 0.064], but was maintained when controlling for age, lean mass and CMV status   was maintained when controlling for all variables [adjusted linear regression: F > 4.622 p < 0.036 R > 0.320 R2 > 0.102 R2Adjusted > 0.070 R2Change > 0.073]. Finally, although there was no significant correlation between HLA-DR+ CD4+EM and fat mass index , a statistically significant relationship was shown when controlling for lean mass [adjusted linear regression: F = 4.594 p = 0.036 R = 0.316 R2 = 0.100 R2Adjusted = 0.067 R2Change = 0.074].Regression analyses showed that the positive association between fat mass index and the percentage of both HLA-DR+ CD4+ EMRA and HLA-DR+ CD8+ EM  of the breast cancer survivors included, that their T cells recognize tumour-associated antigens  . High T cell activation 6–12 months after treatment has been reported by analysis of serial blood samples from patients with brain cancer, sarcoma, Non-Hodgkin lymphoma and breast cancer, but has not been compared to healthy women . We showaglobin) .Other data from this study could also be interpreted as reflecting a previous anti-tumour response. For example, the lower CD8+ naïve T cell counts and higher CD4+ central memory cell counts among breast cancer survivors, might reflect a previous expansion of effector cells and concomitant shrinking of the naïve T cell pool. These changes are typically followed by a subsequent contraction of effector cells, and maintenance of central memory cells, to provide long-lived memory once tumour cells have been eliminated . Indeed,Furthermore, the present study found a trend for higher counts of total B cells and their subtypes among breast cancer survivors, whereas immature B cells were significantly higher than in healthy women. There is controversy over whether B cells contribute to pro- or anti-tumour responses, and this relationship is influenced by the frequencies of functionally distinct B cell subtypes . ExperimAlthough one interpretation of differences in leukocyte sub-types among breast cancer survivors compared to healthy women relates to a potentially favourable immune response to cancer, another interpretation could be that the observations made in this study reflect immunosenescence . Hallmarn = 5) who had not undergone treatment, show that activation of CD4+ and CD8+ EM and EMRA T cells was closer to the levels exhibited by healthy women.It is well established that two strong drivers of immunosenescence are age and CMV infection. Initial analyses in the present study identified differences in the counts and activation levels of several cell types when comparing breast cancer survivors to healthy women. Further analyses were undertaken to understand whether age and CMV contribute towards these group differences. In summary, the counts of CD4+ CM T cells were higher and CD8+ NA T cells lower among breast cancer survivors. However, this statistically significant difference was lost when controlling for age but maintained when controlling for CMV serostatus. Likewise, the greater proportion of activated CD4+ CM T cells, total CD8+ and CD8+ EMRA T cells among survivors was also lost when controlling or age, but not CMV serostatus. These findings imply that age may have a stronger influence on the characteristics of the T cell pool than CMV among breast cancer survivors. Thus, the influence that cancer and its treatment might have on the T cell pool, could be interpreted as being less dramatic than the effects of age. However, for other measurements, including the proportion of activated CD4+ EM and EMRA and CD8+ EM T cells, which were higher among survivors than healthy women, remained statistically significant when controlling for both age and CMV . Thus, an interpretation of these latter findings is that cancer treatment might have a stronger influence than age and CMV on some T cell sub-types compared to others. In support, our preliminary analysis of recently diagnosed patients (p = 0.091). However, the percentage of survivors with an inverted (<1.0) ratio or higher ratio (>2.5) was greater than among healthy women , which could be a combined effect of cancer and inflammation  higher counts of all leukocytes compared to both healthy women and survivors, 2) similar T cell profile and T cell subset activation patterns to both groups, 3) similar counts of CD8+ NA T cells to both groups, 4) similar counts of CD4+ CM cells to survivors but higher counts than healthy women, 5) lower proportion of activated CD4+ and CD8+ T cells and subsets than survivors but an activation profile more closely aligned with healthy women, 6) lower CD4+ TSCM counts compared to both groups, 7) higher CD8+ TSCM counts compared to survivors but similar to healthy women, 8) similar total B/NK cell and subset counts to survivors but higher than healthy women and 9) the percentage of patients with an inverted or higher CD4+/CD8+ ratio (60%) was higher than healthy women (47.3%). These preliminary qualitative observations indicate that when immune profiles are assessed prior to treatment, the counts and distribution of cell types are generally aligned with breast cancer survivors . However, there was large inter-individual variation among patients which could influence prognosis and/or response to treatment. In support, studies have linked baseline pre-treatment levels of some immune cells in blood with clinical outcomes among patients with breast cancer. For example, higher blood lymphocyte counts have been linked with pathological complete responses after neoadjuvant chemotherapy in primary breast cancer , and witn = 27, 35–69 years) compared to healthy women . Statistical analyses conducted after removing n = 10 healthy women under the age of 35 years are presented in the n = 24 pre-menopausal and n = 14 post-menopausal women, except for very slightly higher plasmablasts and immature B cells among pre-menopausal women (data not shown).In this manuscript, breast cancer survivors and healthy women were recruited as part of separate studies and some inclusion criteria were different. For example, there was a narrower age range for breast cancer survivors (In conclusion, this study has shown that compared to healthy women, breast cancer survivors exhibited higher levels of CD4+ central memory T cells, lower levels of CD8+ naïve T cells and higher activation levels of CD4+ and CD8+ effector-memory T cells. Immature B cells, total NK cells and CD16+ effector cells were also significantly higher among survivors. Uniquely, this study also shows that fat mass index is positively associated with the activation levels of CD4+ and CD8+ effector memory T cells across healthy women and breast cancer survivors. Further, the relationship between fat mass index and CD8+ EMRA T cell counts withstood statistical adjustment for all variables, including age, CMV serostatus, cardiorespiratory fitness and other measures of body composition, such as lean mass. This finding shows a potentially important relationship between adiposity and a subset of T cells that could contribute to inflammation and immune dysfunction in overweight and obesity. Overall, our results emphasise that age, CMV serostatus, cardiorespiratory fitness and in particular, body composition, should be taken into consideration, when profiling leukocytes in blood in humans. Future studies may reveal similar associations with other participant characteristics, such a habitual physical activity level and diet.


In this study, a sensitive and selective sensor design for OTA detection was successfully performed by utilizing the selectivity and catalysis properties of MIP and the synthesized MnCO3NS/CF material at the same time. MnCO3 nanostructures incorporated into carbon fibers were first characterized by using various analytical techniques. The sensor revealed a linearity towards OTA in the range of 1.0 × 10−11–1.0 × 10−9 mol L−1 with a detection limit (LOD) of 2.0 × 10−12 mol L−1. The improved electrochemical signal strategy was achieved by high electrical conductivity on the electrode surface, providing fast electron transportation. In particular, the analysis process could be finished in less than 5.0 min without complex and expensive equipment. Lastly, the molecular imprinted electrochemical sensor also revealed superior stability, repeatability and reproducibility. A novel electrochemical sensor based on MnCO Aspergillus and Penicillium genus, is structurally an isocoumarin derivative. OTA is a colorless, crystalline compound, soluble in sodium bicarbonate solutions, recrystallized with weak xylene, with low water solubility and acidic properties. It is a fungal compound that threatens the health of people in every climate due to its greater chemically stable and half-life compared to other derivatives and its immunosuppressant properties that affect organs, such as the liver, kidney and thymus, including carcinogen . Th. Th−1 OT3NS/CF/GCE, platinum wire and Ag/AgCl/KCl(sat) were used for the working electrode, counter electrode and reference electrode, respectively, for the analysis. Analytical applications were carried out from the calibration graph obtained by plotting the current values of the peaks occurring at +0.5 V in the +0.2/+1.0 V potential range against the OTA concentration. Apple juice samples taken from the local supermarket were first kept in the refrigerator at +4 °C for 1 day and then transferred to a test tube. After adding ethanol (5.0 mL) to this test tube, it was exposed to a shake bath system for 15 min. After the centrifugation for 5 min at 1000 rpm, the upper phase was spiked with a fine syringe . Then, t3 (50%) for 45 min, MnCO3NS was interconnected onto the CF surface by hydrothermal process. The surface characterizations of CF and MnCO3NS were explored by SEM 6]3−/4− 6]3−/4− . Electro)6]3−/4− A. Howeveb of 3NS ,40. Afte−1) (R2 = 0.9991), the obtained linearity indicated that the oxidation phenomenon was diffusion-controlled [In addition, when CV anodic peak currents were plotted against the square root of the scan rate (10–1000 mV sntrolled . 3NS/CF/GCE were shown on the inset of ct), the solution resistance (Rs) and the constant phase element (CPE). Rct values were studied to be 45 ohm for bare GCE, 35 ohm for MnCO3NS/GCE, and 25 ohm for MnCO3NS/CF/GCE, confirming the accuracy of CV results.EIS measurements were applied to confirm the CV results B. Experi3NS/CF/GCE  = 9.8744x  − 0.0246, (R2 = 0.9989) was given in −11 mol L−1, and 2.0 × 10−12 mol L−1, respectively . The electrochemical signals (µA) were obtained for 1.0 nmol L−1 OTA, 100.0 nmol L−1 OTB, 100.0 nmol L−1 AFB1, 100.0 nmol L−1 AFB2, 100.0 nmol L−1 CIT, 100.0 nmol L−1 CAT and 100.0 nmol L−1 RES on MIP/MnCO3NS/CF/GCE and NIP/MnCO3NS/CF/GCE  value was obtained as 0.19% for 45 consecutive measurements. Hence, the high repeatability of one MIP/MnCO−1 OTA. RSD of 0.77% was obtained for 20 different measurements, providing a high reproducibility.Finally, the reproducibility test of 20 different OTA imprinted sensors was investigated, and 20 different OTA imprinted sensors were applied to 0.5 nmol L3NS/CF was presented for OTA detection. MnCO3NS/CF composite improved the electrochemical performances in OTA analysis. This result can be explained by using these reasons: (i) MnCO3NS, including interconnected structures, provided high electrical conductivity during the electrochemical process, ensuring rapid electron transportation. (ii) MnCO3NS was incorporated into the CF surface through Mn-C bonds, providing electrode stability. According to analytical data, the OTA imprinted electrode indicated linearity (1.0 × 10−11–1.0 × 10−9 mol L−1) with a low LOD of 2.0 × 10−12 mol L−1, ensuring excellent sensitivity and selectivity. The fact that the developed sensor also has an environmentally friendly feature will lead to the emergence of new perspectives for the detection and analysis of other mycotoxins in the future. In addition, OTA imprinted electrode’s ability to make rapid analysis in a short time can cause more reliable food consumption.In this study, a sensitive and selective molecularly imprinted electrochemical sensor based on MnCO


Burkholderia cepacia complex (BCC) contamination. Therefore, minimizing the frequency of outbreaks may be conducive to the development of a quick and sensitive approach that can distinguish between live and dead loads of BCC. We have assessed an exo probe-based recombinase polymerase amplification (RPA) with 10 µM propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in various concentrations of antiseptics  and benzalkonium chloride (BZK) solutions) after 24 h. The optimized assay conducted using a set of primer–probes targeting gbpT was performed at 40 °C for 20 min and shows a detection limit of 10 pg/µL of genomic DNA from B. cenocepacia J2315, equivalent to 104 colony-forming units (CFU/mL). The specificity of a newly designed primer and probe was 80% (20 negatives out of 25). The readings for total cells  from 200 µg/mL CHX using PMAxx-RPA exo assay was 310 relative fluorescence units (RFU), compared to 129 RFU with PMAxx . Furthermore, in 50–500 µg/mL BZK-treated cells, a difference in the detection rate was observed between the PMAxx-RPA exo assay in live cells (130.4–459.3 RFU) and total cells (207.82–684.5 RFU). This study shows that the PMAxx-RPA exo assay appears to be a valid tool for the simple, rapid and presumptive detection of live BCC cells in antiseptics, thereby ensuring the quality and safety of pharmaceutical products.Both sterile and non-sterile pharmaceutical products, which include antiseptics, have been recalled due to  Burkholderia comprises over 120 species of Gram-negative bacteria found in a variety of ecological niches [Burkholderia cepacia complex, or BCC, consist of a group of bacteria that have recently been linked to numerous outbreaks. This resulted in contamination of disinfectants, intravenous solutions, nebulizer solutions, mouthwash products, medical devices, respiratory treatment equipment as well as antiseptics  and benzalkonium chloride (BZK)) [Burkholderia spp. was the most common cause of non-sterile drug recalls , followed by Ralstonia pickettii , and Salmonella spp.  [Burkholderia cepacia complex [The genus l niches . Specifie (BZK)) . Accordiecalls 10 recalls,ickettii 5 recalls complex , which cAlthough molecular techniques—based on polymerase chain reaction (PCR)—have been widely used to detect BCC, their reliance on complex heat cycling devices, trained workers and a time-consuming workflow has limited their application for on-site testing or in resource-constrained situations ,8. A proEscherichia coli recombinase A (recA) and single-stranded DNA binding protein (SSB) to match primers to their target on the template DNA [Staphylococcus aureus (Sau) polymerase extends the primer and displaces the native strand. Real-time monitoring detection of fluorescence DNA amplification can be achieved using exo probes . The reaction is rapidly carried out in a fluorometer with repeated cycles resulting in the exponential amplification of the target sequence [Burkholderia spp., including Burkholderia pseudomallei [Burkholderia mallei [Burkholderia species, 16S rRNA gene analysis has limited taxonomic resolution. For instance, it was established that multilocus sequence typing (MLST)  as a means to identify BCC at the species level surpassed 16S rRNA [RPA reactions are relatively easy to perform with a simple, portable, battery-powered device, and do not require complex equipment or extensive training. The mechanism involves two major proteins to substitute for the traditional heat denaturation step in PCR: the late DNA ,11. Thensequence ,11,13. Tsequence ,11,13. Rdomallei ,15,16, Ba mallei , and BCCa mallei . More rea mallei  reported16S rRNA ,19. Ther16S rRNA .Finally, we emphasize the development of technology that can rapidly detect and differentiate live from dead BCC cells, which is necessary to reduce the frequency of outbreaks caused by BCC contamination. However, RPA, as so many other isothermal amplification techniques, is unable to separate DNA originating from live or dead cells. Dead bacteria do not cause serious diseases; rather, their presence leads to false positive results and inflated BCC counts . TherefoBurkholderia [gbpT) with three conserved sequence regions of ≥26 bp (www.twistdx.co.uk/docs/default-source/RPA-assay-design (accessed on 3 April 2023)) [www.ncbi.nlm.nih.gov). The probe contained a basic nucleotide analog, which replaced a nucleotide in the target sequence flanked by a dT-fluorophore and a corresponding dT-quencher group. These probes were blocked from any potential polymerase extension by a suitable 30–modification group. In a double-stranded context, the tetrahydrofuran (THF) residue presented a substrate for the DNA-repair enzyme exonuclease III, which cleaved the probe at the THF position, thereby separating the fluorophore and the quencher, thus generating a fluorescent signal  of 266 complete genomes  of the genus holderia ,20,21. If ≥26 bp . The pril 2023)) . The staB. cenocepacia J2315 for the PMAxx-RPA exo assay. Briefly, 20 mM PMAxx  was diluted in high purity water, creating a 1 mM PMAxx stock solution. Then, 10 µL of 1 mM PMAxx was added to 990 µL of a suspension containing live and dead B. cenocepacia J2315 (1.5 × 108 colony forming units (CFU)/mL) cells, to a final concentration of 10 μM. Dead cells were obtained from heating 1 mL of suspended cells at 100 °C for 10 min [PMAxx was utilized as described previously . Among tr 10 min ,12,20.The PMAxx-RPA exo assay was performed in a 50 µL volume using enzyme pellets from the TwistAmp exo kit . The reaction consisted of 29.5 µL rehydration buffer, 2.1 µL forward and reverse primers (10 μM), a 0.6 µL probe (10 μM) and 9.2 µL nuclease-free water. The mixture was briefly vortexed, spun and transferred into a PCR reaction tube. Then, 2.5 µL of magnesium acetate (280 mM) and 4 µL of a DNA template were pipetted into the tube lid. The lid was then closed, the tube was vortexed, spun shortly and immediately incubated under set conditions. The PMAxx-RPA exo assay was performed in a CFX96™ qPCR instrument  with fluorescence detection in the 6-carboxy-fluorescein (FAM) channel for 2 min at 40 °C followed by 30 cycles for 17 s (20 min). RPA products were then directly analyzed with both a CFX96™ qPCR instrument (relative fluorescence units (RFU)) and 2% agarose gel electrophoresis. Before electrophoresis, exonuclease within the RPA product was inactivated at 95 °C for 5 min . B. cenocepacia J2315 at different temperatures, namely 35, 37, 40, 42, and 45 °C for a period of 20 min, per manufacturer recommendations. Following optimal temperature selection, the assay was performed for 10, 15, 20 and 25 min reaction times (10–50 cycles in a CFX96™ qPCR instrument) to monitor amplification kinetics. Furthermore, 12, 14 and 16 mM magnesium acetate (280 mM stock solution) was utilized in the optimization process. To determine the optimal amplification temperature, the PMAxx-RPA exo assay was carried out with 4 µL of approximately 1 ng/µL genomic DNA of Burkholderia species. The University of Michigan’s Burkholderia cepacia Research Laboratory and Repository provided 36 strains of Burkholderia spp. and two strains of Caballeronia spp. All strains were cultivated as described previously [To evaluate the primer/probe specificity, we used 56 bacterial strains (38 species), which included 13 BCC, 8 non-BCC and 17 non-eviously .B. cenocepacia J2315 (1.5 × 108 CFU/mL) was used as templates for the RPA to determine the limit of detection (LOD). A serial dilution (4 μL) of the DNA, ranging from approximately 10 ng/µL to 100 fg/µL , corresponding to approximately 107–102 CFU/mL, was amplified by the PMAxx-RPA exo assay to determine the LOD of the assay. All assays were performed in triplicate.Approximately 94.3 ng/µL of genomic DNA from B. multivorans HI2229 (1.0 × 109 CFU/mL), either PMAxx-treated or untreated. For cross-species validation of our PMAxx-RPA exo assay, we chose in the present study to use B. multivorans HI2229, which was susceptible at the lowest CHX and BZK concentration (50 µg/mL CHX and 50 µg/mL BZK) [To investigate the effect of CHX and BZK on the PMAxx-RPA exo assay, DNA was extracted from cells of /mL BZK) . After DB. multivorans HI2229 cells, OD600nm = 0.2 equivalent to 109 CFU/mL, were spiked with 0.4 mL of 100, 1000 and 2000 μg/mL CHX to achieve final concentrations of 10, 100 and 200 μg/mL CHX, respectively. The treated cells were not incubated, but genomic DNA was extracted from cells with or without 10 µM PMAxx-treatment. Following the 10 µM PMAxx-treatment, the cells were washed in 1 × PBS buffer, followed by centrifugation at 15,000 rpm for 10 min. The supernatant was then removed, and the cells were resuspended in 500 μL nuclease-free water. A total of 100 µL was transferred to a clean 1.5 mL Eppendorf microcentrifuge tube and boiled for 10 min at 100 °C followed by spinning at 1000 rpm using an Eppendorf ThermoMixer® C to extract genomic DNA [To determine the most efficient DNA for live/dead cell discrimination with RPA, we compared cell lysates to pure genomic DNA using the boiling method (cell lysates) and a commercially available kit (pure genomic DNA). Briefly, 4 mL of omic DNA . The rem9 CFU/mL of B. multivorans HI2229  were treated with 100, 1000, 2000 µg/mL CHX and 500, 2000, 5000 µg/mL BZK to achieve final concentrations of 10, 100, 200 µg/mL CHX and 50, 200, 500 µg/mL BZK, respectively. The treated cells were incubated in the dark at room temperature for 24 h. One milliliter of each bacterial suspension was subjected to the PMAxx treatment as described above. The cells were then centrifuged, and the resulting pellet was resuspended in 1× PBS and centrifuged at 15,000 rpm for 10 min at 4 °C. Pellets were resuspended in 500 µL nuclease-free water and 100 µL of that suspension was used for DNA extraction by the boiling method. For control purposes, DNA was also extracted from cells treated with CHX and BZK without PMAxx treatment. Next, 10 ng/µL DNA of both PMAxx-treated and untreated sample, in triplicate, were used for the PMAxx-RPA exo analysis. Statistical analysis was performed via Student’s t-test using SigmaPlot vs. 13.0 software .To validate our PMAxx-RPA exo assay, 10B. cenocepacia J2315. As shown in The PMAxx-RPA exo assay was carried out in accordance with the manufacturer’s instruction with consideration of parameters such as temperature, reaction time and magnesium acetate concentration in order to optimize the assay. The RPA exo assay with gbpT primer and probe successfully amplified 13 BCC species. To optimize the reaction temperature, five different temperatures  were evaluated on 0.9 ng/µL genomic DNA of B. cenocepacia J2315 (1.5 × 108 CFU/mL) cells in nuclease-free water with 10 µM PMAxx. Total cells were not treated with PMAxx on live BCC cells and were compared to live cells for examining any possible interference of PMAxx. All the DNA samples were normalized to approximately 1 ng/μL. No statistical difference was observed between total cells  and live cells . No RFU values were detected from dead cells treated with PMAxx (1.41 ± 0.268 RFU)  . TherefoBurkholderia species were tested using the RPA exo assay. The result shows that all 13 BCC (24 BCC strains) yielded RFU , whereas non-Burkholderia strains did not yield any RFU / + TN) × 100; / + 20 (TN)) × 100 = 80))To investigate the specificity of the newly designed primer–probe, genomic DNA from 13 BCC species, 8 non-BCC, and 17 non- any RFU . Two preB. cenocepacia J2315 (approx. 10 ng/µL (corresponding to 107 CFU/mL), 1 ng/µL, 100 pg/µL, 10 pg/µL, 1 pg/µL, 100 fg/µL). gbpT gene could detect 10 pg/µL DNA molecules. Analysis by gel electrophoresis showed a band for the PMAxx-RPA exo assay products (lanes with ≥10 pg/µL) (gbpT primers showed an LOD of approx. 10 pg/μL (50 ± 8.6 RFU), corresponding to 104 CFU/mL. The LOD of the PMAxx-RPA exo assay was evaluated by assessing a 10-fold serially diluted genomic DNA of 0 pg/µL) . However0 pg/µL) . TherefoB. multivorans HI2229 (1.0 × 109 CFU/mL) cells was used in a 50 μL PMAxx-RPA exo assay. Without CHX in DNA, as a positive control, total cells  yielded higher RFU values compared to live cells . Similarly, when DNA was spiked with 10 µg/mL CHX, for total cells (without PMAxx) and live cells (with PMAxx), we recorded an average RFU of 398.683 ± 4.85 and 143.15 ± 20.99, respectively. No RFU was detected in 100 and 200 µg/mL CHX, in either PMAxx-treated or untreated cells  in various concentrations of CHX. Although the boiling method showed lower RFU compared to the commercial kit method, the boiling method had sufficient detection capability of PMAxx-RPA exo assay for total or live cells (>50 RFU).We compared two DNA extraction methods, pure genomic DNA  and cell lysates (boiling method) without purification procedure to determine the most suitable and efficient procedure for live/dead differentiation with the PMAxx-RPA exo assay. In total cells  with various CHX concentrations a, RFU va9 CFU/mL of B. multivorans HI2229 cultured for 24 h with 0, 10, 100 and 200 µg/mL CHX and 0, 50, 200 and 500 µg/mL BZK. Using the boiling method for DNA extraction, cell lysates were templates for the PMAxx-RPA exo assay. When not treated with CHX, total B. multivorans HI2229  showed 297.89 ± 93.88 RFU while the live control  yielded 382 ± 206.05 RFU. With 10, 100 and 200 µg/mL CHX, total cells showed no differences with an average of 297.04 ± 52.65 RFU, 418.79 ± 139.91 RFU, and 310.16 ± 45.77 RFU, respectively. Similar results were also obtained from the live cells  within the range of 374.54 ± 177.12 RFU, 464.46 ± 116.74 RFU in 10–100 µg/mL CHX. However, when the 200 µg/mL CHX were assayed, total cells showed 310.16 ± 45.77 RFU and live cells decreased to 129.71 ± 70.01 RFU  from 0 µg/mL BZK (596.48 ± 204.48 RFU) and 50 µg/mL BZK (684.53 ± 30.45 RFU) were notably different from those that received 200 µg/mL and 500 µg/mL BZK (281.61 ± 111.87 RFU and 207.82 ± 57.08 RFU). However, the live cells  from 0 µg/mL BZK (347.15 ± 148.62 RFU) and 50 µg/mL BZK (459.27 ± 103.24 RFU) displayed similar RFU values of total cells . With 200 and 500 µg/mL BZK, RFU values of live cells (130.40 ± 43.08 RFU and 191.47 ± 117.52 RFU) were lower than from total cells (281.61 ± 111.87 RFU and 207.82 ± 57.08 RFU) b. The PMgbpT)-based colorimetric PMAxx-RPA exo assay described here can be performed within about 1 h without any enrichment at 40 °C. We observed that after optimization, this assay was able to detect BCC in nuclease-free water and various concentrations of CHX and BZK solutions. Rapid and accurate detection of live BCC cells is important for regulatory purposes and essential in protecting public health. To detect BCC cells, conventional PCR, real-time quantitative PCR (qPCR) ,7,8, drogbpT) that has three conserved sequence regions of at least 26 bp [Burkholderia species resulting in 80% specificity. This represents a major obstacle in the correct identification of BCC using one molecular target from closely related non-BCC species as reported previously [For the successful detection of BCC using a PMAxx-RPA exo assay, the primers and probe must meet the standard parameters for isothermal amplification. They must also have sufficient biological specificity and sensitivity to allow detection of BCC. For the molecular-based detection of BCC, we previously used two gene clusters encoding a 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1.99.12)/GTP cyclohydrolase II for LAMP and PMAxx-ddPCR assays  and an ist 26 bp . These teviously ,25,26,274 CFU/mL. Similar results were obtained from artificially spiked human blood and urine samples, where the RPA-lateral flow (LF) assay’s detection limit was 4.8 × 104 and 4.95 × 104 CFU/mL of B. pseudomallei, respectively [Burkholderia cepacia complex in non-sterile pharmaceutical products [The selected primers were suitable for the PMAxx-RPA exo assay as they successfully amplified selected target genes with amplification products around 383 bp verified via agarose gel electrophoresis and RFU. Since exo probes can result in the exonuclease mediated degradation of DNA, thus digesting RPA reaction final product , in thisectively . Alternaproducts . AlthougB. mallei, B. pseudomallei and BCC [Salmonella from spiked milk samples [Amplification of DNA from dead cells would provide an unreliable indication of human health risk assessment posed by the presence of pathogens, since the copy number would be falsely increased and thus, would lead to overestimating bacterial counts . Althoug and BCC ,16,17,18 samples . In this samples . PMAxx cv/v), which had no discernible effect on the reaction [v/v), urine (10% v/v), and selenite cysteine inhibited RPA [Several PCR inhibitors have been shown to be tolerable by RPA, including hemoglobin 20–50 g/L), heparin (0.5 U), urine (up to 5%), and ethanol  exposed to CHX and BZK [Legionella in biofilm displayed large variability between replicates [BZK and CHX are present in a variety of everyday nonsterile products  ,24. Typi and BZK , consequplicates . Therefoplicates . Howeverplicates . Furtherplicates . DespiteBurkholderia cells. This assay is quick, simple and offers a new potential way of detecting BCC. The PMAxx-RPA exo assay does not require any sophisticated instrumentations and could be carried out using common lab equipment, such as a simple water bath or portable thermal equipment. The direct boiling method could be used for non-sterilize pharmaceutical samples at a significantly reduced cost to process samples. Compared with the traditional detection method of BCC, the PMAxx-RPA exo assay has the advantages of short detection time and simple operation, making it useful as a presumptive test to detect BCC.Here, we reported an RPA exo assay combined with PMAxx for the detection of live


Resting membrane potential is a bioelectric property of all cells. Multiple players govern this property, the ion channels being the most important. Ion channel dysfunction can affect cells’ resting membrane potential and could be associated with numerous diseases. Therefore, the drug discovery focus on ion channels has increased yearly. In addition to patch-clamp, cell-based fluorescent assays have shown a rapid and reliable method for searching new ion channel modulators. Here, we used a cell-based membrane potential assay to search for new blockers of the Kv10.1, a potassium channel strongly associated with cancer progression and a promising target in anticancer therapy. We found that fluoxetine and miconazole can inhibit the Kv10.1 channel in the micromolar range. In contrast, BL-1249 potentiates Kv10.1 currents in a dose-dependent manner, becoming the first molecule described as an activator of the channel. These results demonstrate that cell-based membrane potential assay can accelerate the discovery of new Kv10.1 modulators. This cerization .Since 1999, the voltage-gated potassium channel Kv10.1, also named Eag1, has emerged as an exciting target in cancer therapy . Kv10.1 ® membrane potential (FMP) for searching new Kv10.1 modulators. Most of the molecules tested here were supplied from Alomone labs’ ion channel modulator explorer kits, which were selected based on their know effect on other potassium channels, e.g., the Kv11.1. Thus, we found that fluoxetine, miconazole nitrate (MN), and BL-1249 (BL) modulate the fluorescents signals associated with Kv10.1 activity. Patch-clamp recordings corroborate FMP results and showed that the three molecules had dose-dependent effects on Kv10.1 channels, with fluoxetine and MN having inhibitory and BL opener activity. These results demonstrate that FMP can produce reliable results and accelerate the discovery of new Kv10.1 modulators.Here, we validate a primary screening using the cell-based FLIPR2 incubator.HEK293 wild-type cells  and HEK293 cells stably expressing the human Kv10.1 potassium channel (HEK-Kv10.1)  were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  containing 10% fetal bovine serum  and 1% Penicillin/Streptomycin . HEK-Kv10.1 were supplemented with Zeocin (30 μg/mL)  as a selection antibiotic. All cells were cultured at 37°C in a 5% CO® membrane potential assay BLUE  was performed following the manufacturer’s protocols. Flat-bottom 96-well plates  were plated with HEK-WT or HEK293-Kv10.1 at a density of 20,000 cells/well in 100 µL of supplemented DMEM and incubated for 24 h at 37°C in a 5% CO2 incubator. After 24 h, 100 µL of the FMP buffer was added for 30 min at 37°C in a 5% CO2 incubator; at this point, the molecules of interest were mixed in the FMP buffer and pre-incubated in the selected wells. All the molecules were tested at 100 μM; except astemizole, which was tested at 50 µM and DMSO (0.1%). After pre-incubation, 96-well plates were transferred to a FlexStation3 microplate reader (Molecular Devices) controlled by the SoftMax Pro 7 software. FLIPR dye was excited at 530 nm, and the emitted fluorescence was recorded at 565 nm. Data were acquired at 0.5 Hz for 120 s: the first 20 s represent the basal fluorescence, and then, 50 µL (5x) of the high potassium solution was added , and the fluorescence signal was recorded for another 100 s. The fluorescence responses were normalized by F=F/F0, where F represents the fluorescence at any given time, and F0 is the mean basal fluorescence obtained in the first 20 s of recording. Once normalized, the amplitude was calculated as the difference between the basal fluorescence and the maximal amplitude at 120 s. The maximal rate of fluorescence increase is obtained on the first derivative of the fluorescence signal.FLIPR2, 1.3 MgCl2, 10 HEPES, and 10 D-glucose . Intracellular patch-pipettes had resistances of 2–3 MΩ and were filled with an internal solution containing (in mM): 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES . Ionic currents were recorded with a setup composed of Multiclamp 700B/Digidata 1,550/pCamp10 , filtered at 5 kHz, and digitally sampled at 10 kHz. 40%–60% of the series resistance was electronically compensated. Drugs tested on Kv10.1 currents were evaluated from a holding voltage (Vh) of −70 mV, and currents were evoked with 250 ms duration voltage steps from −70 to +30 mV (for BL-1249) or +50 mV (for fluoxetine and miconazole) applied every 5 s. I-V curves were constructed using a protocol of 250 ms voltage steps from −100 mV to +50 mV in 10 mV increments and a Vh of −70 mV; the amplitude of Kv10.1 currents was measured at the end of voltage steps (average of the last 3 ms) and plotted versus voltage. Conductance was assessed as G = IK/(V-VK), where VK is the reversal potential for K+ flux, IK is the Kv10.1 current, and V is the membrane potential. The corresponding normalized G (G/Gmax) is plotted versus voltage; data were fit in Origin 2019 with a single Boltzmann distribution: G/Gmax = 1/(1+e−(V−V1/2)/s), where V1/2 and s are the arithmetic means of half-maximal activation potentials and slope factors, respectively.Cells were plated on 18 mm circular glass coverslips previously treated with poly-l-lysine for patch-clamp recordings. Coverslips were transferred to a recording chamber , and whole-cell voltage- and current-clamp recordings were performed at room temperature and under continuous perfusion (at a flow rate of 2 mL/min) with a standard bath solution. The standard bath solution contained (in mM): 137 NaCl, 5.4 KCl, 2 CaClWe performed whole-cell current clamp recording in the gap-free mode to test the effect of BL-1249 on the resting membrane potential of HEK-WT and HEK-Kv10.1 cells. Cells were held without current injection (0 pA), and the protocol consisted of 60 s in the standard bath solution, followed by 60 s in BL-1249 or DMSO, and 60 s for washout.n/(IC50n + cn), where Bmax is the maximum block, IC50 is the concentration of half-maximal inhibition, c is the concentration of the molecules, and n is the Hill coefficient.Concentration-inhibitory curves were fitted with the Hill equation using the Levenberg–Marquardt method implemented in Origin 2019 software: Inhibition = Bmax*c(LogEC50−X)), where EC50 is the concentration of half-maximal potentiation. This model assumes data is normalized, forcing the curve to run from 0% to 100%.For BL-1249, the concentration-effect curve was fitting using the method implemented in GraphPad Prism 8 software: Potentiation = 100/(1 + 10Amitriptyline (A-155), bupivacaine hydrochloride (B-125), celecoxib (C-190), citalopram (C-195), flupirtine maleate (F-150), fluoxetine hydrochloride (F-155), ICA-11081 (I-160), L-ascorbic acid (L-140), linopiridine dihydrochloride (L-156), loperamide hydrochloride (L-100), miconazole nitrate (M-206), rosiglitazone (R-125) and 2,5-dimethylcelecoxib (D-150) were selected of a screening package for ion channel from Alomone Labs. BL-1249 (B2186), astemizole (A2861), and DMSO (D2650) were purchased from Sigma-Aldrich. Most stock solutions were prepared in 100% DMSO or water at a concentration of 100 mM. All molecules were diluted in a standard bath solution before starting every experiment. In patch-clamp experiments, drugs were delivered using a perfusion system VC-6 coupled to a fast-step solution charger .p < 0.05) was assessed by Student’s t-test. For multiple comparisons, statistical significance (p < 0.05) was assessed by 1-way analysis of variance using the Dunnet post hoc test. Significant differences are expressed in figures: *p < 0.05, **p < 0.01, ***p < 0.001.All data were analyzed using Origin 2019  and GraphPad Prism 8 . Results are presented as mean ± SEM of at least three independent experiments. When two means were compared, statistical significance . Using the first derivative of the fluorescence responses, we observed that the maximum rate of change in HEK-Kv10.1 cells was around three times faster than in HEK-WT . HEK-Kv10.1 cells that were not incubated with the FMP dye did not show a fluorescence signal either during the basal recording or the stimulation with a high potassium solution .First, we tested the fluorescence responses to depolarization from HEK-WT and HEK-Kv10.1 cells using the FMP BLUE formulation. Adding a high Ksolution . These rp = 0.0394, 0.0054, and 0.0192, respectively, 1-way ANOVA). DMSO (0.1%) did not produce any change in the responses . These results confirmed the method’s reliability. Next, we evaluated 12 small molecules and found that fluoxetine and miconazole nitrate significantly decreased the responses by 21.7% and 27.7%, respectively . Interestingly, BL-1249 seems to increase the Kv10.1-mediated responses by 30% (p = 0.0189), suggesting BL could have activity on Kv10.1 channels.To validate the FMP assay, we pre-incubated HEK-Kv10.1 with previously described channel blockers, including astemizole, loperamide, and amitriptyline. As expected, astemizole, loperamide, and amitriptyline significantly decreased the fluorescence responses induced by the high potassium depolarization by 34.8%, 31.9%, and 25.5%, respectively    . The inh t-test) .+ channels . Since we could not test higher concentrations of BL, the maximal potentiating concentration is unknown. However, we decided to perform a trend analysis to evaluate whether the dose-dependent effect followed a direct relationship . Posterior, the dose-concentration fitting yielded an EC50 of 36.5 µM and a hill slope of 1.4. Analysis of Kv10.1 conductance showed that BL shifted their V1/2 activation by 44 mV to the left, from 0.6 ± 1.1 mV in control to −44.9 ± 6.2 mV in the presence of 100 µM BL (n = 6). These results suggest for the first time the potentiation effect of a molecule on Kv10.1 channels.BL-1249 has been described as a negatively charged activator of different types of Kchannels . Here, wp = 0.345; paired t-test). In contrast, BL (100 µM) hyperpolarized the RMP of HEK-Kv10.1 cells by 20 mV, from −57.3 ± 1.9 mV in control to −77.2 ± 1.2 mV in the presence of BL . Interestingly, during the washout, the RMP suffered a transitory rebound to more depolarized values.Next, we are interested in evaluating whether BL could hyperpolarize the resting membrane potential (RMP) of HEK-Kv10.1 cells. We performed current-clamp recordings to measure the RMP without current injection. HEK-Kv10.1 cell had a RMP of −56.8 ± 3.1 mV (n = 11). In the presence of DMSO (applying for 1 min), the RMP did not show any statistically significant change, from −56.1 ± 8.7 mv in control to −56.3 ± 8.9 mV with DMSO     . Likewis t-test) .p = 0.0029, paired t-test) in the Kv10.1 currents measured at +30 mV. Surprisingly, the combined application of fluoxetine plus BL (100 µM) potentiated the inhibitory effect of fluoxetine to 93% . The inhibitory effect of fluoxetine was reversible when the molecule was retired from the bath solution. Then, BL tested alone still could potentiate the Kv10.1 currents . These results open new possibilities for pharmacological studies on the Kv10.1 channel.We tested next whether it was possible to counteract the inhibitory effect of some of the new antagonists described here with BL. As is shown in Ion channel pharmacology is essential to test and validate ion channel function in specific cells and tissues. In this way, ion channel research has been enriched with the increasing number of fluorescent dyes and the development of high-throughput platforms. Thus, cell-based fluorescence assays have become helpful for addressing ion channel research questions regarding the pharmacological characterization and discovery of known or unknown modulators, respectively .Potassium channels coordinate multiple cellular phenomena and can be the origin of diverse pathologies . ConsequHere, we studied whether it is possible to find new Kv10.1 modulators through a fluorescence assay associated with changes in membrane potential. The FMP reliability has been confirmed using small molecules previously described by our group as Kv10.1-inhibitors . Here we®, is used as an antidepressant by millions worldwide. This drug is a selective serotonin reuptake inhibitor , so it is thought that their pharmacological activation may follow common mechanisms. In the case of negatively charged activators, such as BL-1249, it has been reported that these molecules bind below the selectivity filter, where the charge of the molecules promotes K+ binding to the pore cavity, which causes an increase in K+ occupying the selectivity filter, which finally increases ion permeation and channel-open probability  was applied to wild-type HEK293 cells, indicating a direct consequence of BL-1249 on Kv10.1 channels. BL-1249 is a non-steroidal anti-inflammatory, which has been described as an activator of Kectively . Here, tbability . This phbability  and becabability , we can The potassium channels’ activity is associated with excitability processes in neurons and myocytes , while iIn this sense, the Kv10.1 channel shows an increased expression in various types of tumors , and thi


Oligonychus Berlese  is an agriculturally important and the largest genus of spider mites, comprised of 211 species (including new species), two subgenera, four species groups, and 11 species subgroups. The present study comprehensively addressed the morphotaxonomic-based identification of world species of the subgenus Reckiella Tuttle and Baker. Five diagnostic keys were developed for identifying Oligonychus (Reckiella) species belonging to five species subgroups: iseilemae, pritchardi, biharensis, gossypii, and exsiccator. Taxonomic notes are provided on intraspecific variations and some closely related Oligonychus (Reckiella) species representing six species complexes, viz. the afrasiaticus complex, the litchii complex, the pratensis complex, the plegas complex, the sacchari complex, and the tylus complex. One new spider mite species, Oligonychus bahaensis sp. nov., is described and illustrated from Grasses (Poaceae) under the subgenus Reckiella. Oligonychus Berlese  are economically important pests of various agronomic crops2. Such as the date palm mite O. afrasiaticus (McGregor), the tea red spider mite O. coffeae (Neitner), and the banks grass mite O. pratensis (Banks) are considered key agricultural pests4.Spider mites belonging to the genus Oligonychus is the largest genus in the family Tetranychidae Donnadieu, consisting of two subgenera: Oligonychus Berlese and Reckiella Tuttle and Baker. It comprises 210 spider mite species6, excluding two recently synonymized species, viz. O. mangiferus (Rahman and Sapra) and O. vitis Zaher and Shehata7. However, the subgenus Reckiella contains 119 species6.The Oligonychus species are very challenging to identify due to morphological similarities in females4, limited numbers of potential diagnostic characters, and intraspecific variations8. Furthermore, precise differentiation among Oligonychus species must have specimens of both sexes9. Moreover, most Oligonychus species have only been accurately identified based on differences in shape and orientation of male aedeagus10. In addition, various species complexes have been highlighted in this genus12. Such complexes require a comprehensive taxonomic revision to be resolved7. Indeed, no species-level diagnostic key has yet been developed to identify world Oligonychus species, except for some regionally prepared keys17. However, a world diagnostic key was constructed to identify subgenera, species groups, and species subgroups of the genus Oligonychus5.Oligonychus species of the subgenus Reckiella with taxonomic notes on intraspecific variations and some closely related Oligonychus (Reckiella) species. In addition, one new species O. bahaensis sp. nov. is described under the subgenus Reckiella.This study provides five diagnostic keys to the world Oligonychus species belonging to the subgenus Reckiella was collected through personal communication with acarologists in different countries, using various search engines/websites as well as the websites of different acarological/entomological research journals. The published Oligonychus literature, i.e., original and subsequent descriptions of all species and regionally prepared diagnostic keys, was critically reviewed to develop a key for identifying Oligonychus (Reckiella) species.All the published taxonomic literature of world O. bahaensis sp. nov.) described in the present study were collected in 2019/2021 from grasses (Poaceae) in the Baha province of Saudi Arabia (SA). A sample of O. pratensis was also received from the country (USA) of its type locality, which was sent by our colleagues18 at our request. Slide-mounted male and female specimens were observed under a BX51 phase-contrast microscope , and the generic level identification was performed following the key of Bolland, et al.3. Whereas the subgenera, species groups, and subgroups were identified using the diagnostic key of Mushtaq et al.5, and species-level identification was performed following the key developed in the present study. The nomenclature of Lindquist19 was followed for leg chaetotaxy and other terminologies, and Grandjean20 and Grandjean21 for body setae. Furthermore, following Mushtaq et al.6, different traits of the male aedeagus were measured to identify new species and for differentiation among closely related species belonging to the Reckiella . As defined by Mushtaq, et al.5.SubgenusReckiella Tuttle and Baker.Type speciesTetranychus pratensis Banks, 1912: 97.Diagnosis . As defined by Mushtaq, et al.5.Diagnosis —Male: Empodium I with two claws, proximoventral claw subequal or slightly shorter than main dorsal claw; aedeagus turns dorsad with distinct knob, knob with narrowly rounded anterior projection and acute posterior projection, shaft axis forming strong acute angle with knob axis. Female: Tibia I with nine and tibia II with seven tactile setae; peritremes straight, with bulbous end. Opisthosoma medially with transverse striae, except longitudinal/irregular longitudinal or slightly oblique striae with/without forming V-shaped pattern in-between setae f1 and f2, dorsal setae slender, serrate, not set on tubercles and longer than longitudinal distances to the bases of setae next in-line.Female  310 (282–310); width (c3–c3) 225 (221–225).le Figs. ; n = 5—LDorsum , sc1 92 (84–92), sc2 67 (67–72), c1 79 (79–81), c2 82 (74–82), c3 72 (72–79), d1 75 (74–75), d2 75 (75–80), e1 74 (74–80), e2 82 (75–82), f1 74 (74–76), f2 71 (69–71), h2 46 (43–46). Distances between dorsal setae: v2–v2 62 (59–62), sc1–sc1 83 (79–83), sc2–sc2 172 (172–175), c1–c1 69 (67–69), d1–d1 71 (71–75), e1–e1 48 (47–49), f1–f1 39 (32–39), h2–h2 26 (26–31), c1–d1 51 (49–51), d1–e1 51 (46–51), e1–f1 49 (49–53), f1–h2 54 (51–54).um Figs. A,B: PropVenter , 1b 51 (50–52), 1c 52 (52–56), 2b 52 (50–52), 2c 67 (62–67), 3a 38 (36–38), 3b 54 (50–54), 4a 46 (45–48), 4b 51 (51–53), ag 52 (50–52), g1 34 (34–36), g2 38 (37–38), ps1 16 (16–17), ps2 16 (15–16), h3 33 (29–33).ter Fig. C: CoxistGnathosoma . Palp spinneret suξ length 4.5 (4–4.5), width 2.8 (2.4–2.7), solenidion ω length 4 (4–5) -9(1ω)-9(1ω). Tarsus I with four tactile setae proximal to proximal duplex setae  187; width (c3–c3) 140.le Figs. ; n = 1: Dorsum  slender, serrate, not set on tubercles and longer than longitudinal distances to the bases of setae next in-line. Lengths of dorsal setae: v2 47, sc1 65, sc2 48, c1 52, c2 51, c3 53, d1 52, d2 62, e1 55, e2 60, f1 52, f2 45, h2 26. Distances between dorsal setae: v2–v2 46, sc1–sc1 53, sc2–sc2 120, c1–c1 54, d1–d1 53, e1–e1 33, f1–f1 25, h2–h2 16, c1–d1 37, d1–e1 33, e1–f1 28, f1–h2 30.sum Fig. A: PropodVenter , width 2 (2–2.1), solenidion ω length 4 (4–5) -9(1ω)-9/10(1ω). Tarsus I with four tactile setae proximal to proximal duplex setae  of Saudi Arabia, from which the new species was collected.Type material: Holotype male and paratypes twenty females and four males, grasses (Poaceae), Bani Saad, Baha province, November/September 2019/2021, 20° 00′ 39.4″ N, 41° 25′ 34.7″ E, coll. HMS Mushtaq M Kamran, EM Khan, and N Elgoni.Remarks: Oligonychus bahaensis. sp. nov. closely resembles O. pratensis (Banks), because both have female palp spinneret maximum two times longer than its width, only male empodium I with proximoventral claw, female/male peritremes straight with normal length, not hooked distally, female tibia I with 9 and II with 7 tactile setae, and various similar aedeagal characteristics, e.g., aedeagus with the distinct terminal knob, knob with rounded anterior and acute posterior projections, the axis of aedeagal knob forming an acute angle with the shaft axis. However, the new species differs from O. pratensis by male aedeagus knob with posterior projection about sub-equal or slightly longer than anterior projection (vs. 1.5 times longer), shaft dorsal margin maximum 2.5 the length of knob (vs. about three times longer) in O. pratensis  and O. pratensis (GenBank accession number: OQ185450)22, aligned with the previous findings of interspecific divergences among various tetranychid species23.sis Fig. . Additio24, this new species O. bahaensis sp. nov. is observed to inhabit grasses in only one locality in Baha province.Although we comprehensively explored the spider mites’ fauna from numerous localities/hosts in almost the whole of SA since 2018O. anneke Baker and Pritchard could not include in key to species of the subgroup exsiccator due to the unavailability of morphological information regarding the number of tactile setae on tibia II in females.*O. saccharoides Baker and Tuttle could not include in key to species of the subgroup exsiccator due to the unavailability of male morphological information about proximoventral hair/spur on empodium II.*Oligonychus5. In the present study, five dichotomous keys are developed using male/female morphological characters to identify the world Oligonychus species belonging to five Reckiella subgroups , except the subgroup comptus that having only one species5. However, five Oligonychus (Reckiella) species, namely, O. annonicus (McGregor), O. anneke, O. bruneri (Livschitz), O. stenoperitrematus (Ugarov and Nikolskii), and O. saccharoides could not be included in species-level keys due to the lack of required key morphological information in the published literature29. Among them, O. annonicus and O. stenoperitrematus did not even assign to any species group due to the unavailability of required female morphological characters. Also, O. bruneri was only assigned to the level of species group exsiccator5.A diagnostic key has been erected for distinguishing the subgenera, species groups, and species subgroups of the genus exsiccator is the most diverse grouping in Reckiella5. In this subgroup, we found some intraspecific variations in few species, namely, O. formosanus, O. indicus, O. modestus, O. pennisetum, and O. pratensis. In some specimens of O. pennisetum, the female striae pattern was observed as irregular instead of having transverse between setae e1-e18. Also, the shape of male aedeagus in one reported population of O. indicus is totally different 4 from original or other described populations 30. Likewise, one aedeagal illustration of O. formosanus looks somewhat different  was found to be variable among various populations, reported from different geographical localities39. Indeed, the Chinese population of O. pratensis was detected as a cryptic species that even did not belong to the subgroup exsiccator12. Furthermore, Meyer10 synonymized O. monsarrati Gutierrez with O. grypus and also agreed by the later authors5. However, still, O. monsarrati has been considered a valid species40, but no study proved/discussed its validity.The subgroup Oligonychus (Reckiella) species viz. O. aquilinus, O. afrasiaticus, O. keiferi, and O. menezesi represent the afrasiaticus complex; O. litchii and O. taiwanicus represent the litchii complex; O. araneum, O. orthius, O. plegas, O. sayedi, and O. velascoi representing the plegas complex; O. etiennei, O. senegalensis and O. tylus represent the tylus complex; O. sacchari and O. saccharinus represent the sacchari complex; and, O. krantzi, O. pratensis, O. shinkajii, and O. virens represent the pratensis complex41 are morphologically very closed to each other. However, some of these species are distinguished in the present study using some characteristics, e.g., the number of tactile/sensory setae on male tibia I, tactile setae behind to proximal duplex on male tarsus I, and the shape of male aedeagus. Still, some species  are difficult to differentiate using their available morphological information because key characters of aedeagus are variable in their expression among/within reported populations41. Therefore, we brought the specimens of O. pratensis from USA  and observed the key diagnostic character of male aedeagus to differentiate between O. pratensis  species. We suggest using molecular data with morphology to validate the identification of any newly described or closely related Oligonychus species. Integrative taxonomic approaches are crucial to resolving the issues of species complexes in the subgenus Reckiella.In conclusion, we developed five diagnostic keys based on the published morphotaxonomic literature to facilitate the identification of world