The proposed SHG detection scheme is not only interface specific but also expected to be widely applicable, which, in principle, undertakes a nanometer-scale spatial resolution across interfaces.Observing interfacial molecular adsorption and desorption dynamics in a label-free manner is fundamentally important for understanding spatiotemporal transports of matter and energy across interfaces. Here, we report a label-free real-time sensing technique utilizing strong optical second harmonic generation of monolayer 2D semiconductors. BSA molecule adsorption and desorption dynamics on the surface of monolayer MoS Biomolecular activities at interfaces are fundamental phenomena of lives. Interpreting the interfacial dynamics of biomolecules is important for constructing accurate disease models ,2,3, perSurface plasmon resonance microscopy, elegantly utilizing the localized interactions between interfacial electromagnetic fields and biomolecules, possesses molecular-level sensitivity and interfacial spatial resolutions beyond the optical diffraction limit. Inspired by the physical scheme of surface plasmon resonance microscopy, we intend to develop a complementary optical spectrum sensing technique which can realize interfacial biosensing specificity for microfluidic chips in a label-free manner. In our opinion, interfacial second harmonic generation (SHG), a second-order nonlinear optical effect induced by broken inversion symmetry of an interface, is promising to fulfill this goal. Unfortunately, second-order susceptibility of biomolecule interfaces is usually rather small, resulting in a weak SHG signal. In practice, a PMT of a single pixel is usually required to magnify the weak SHG signals. The consequence is that it is difficult to monitor the biomolecular dynamics of an interface in real-time. An alternative way to overcome such difficulty is to significantly increase the power of a fundamental femtosecond laser, which imposes a high risk of damage to the biomaterials and biostructures.2, with broken inversion symmetry, possessing large second-order nonlinear optical susceptibility, can produce strong SHG signals under femtosecond laser pulse excitations, which have been comprehensively investigated in the recent decade [Two dimensional (2D) semiconducting monolayers, such as monolayer MoSt decade ,17,18. I2 through time-resolved SHG. Chitosan nanoclusters and bovine serum albumin (BSA) molecules have been used to form effective Coulomb attractions with the negatively charged surface of monolayer MoS2. By measuring the SHG intensity changes as a function of time, we realize the label-free real-time sensing of biomolecular adsorption and desorption processes all-optically in liquid environments. Our results open new avenues of label-free interfacial biosensing, taking advantage of the strong optical SHG of monolayer 2D semiconductors.To establish progress, we report a label-free interfacial biomolecular sensing technique by monitoring biomolecular adsorption and desorption processes on the surface of monolayer MoS2 is an ideal platform to construct biosensors for interfacial molecule adsorption and desorption processes, considering that the monolayer MoS2 lattice undertakes a sub-nanometer thickness with broken inversion symmetry. Inspired by the pioneering work of strong SHG observations in monolayer MoS2 in 2013 [2 can be considered as a sub-nanometer thick nonlinear optical source emitting SHG with extremely space-confined dipole moments, which can facilitate the interfacial sensing and imaging of ultrahigh spatial resolution across interfaces. Moreover, large specific surface areas and the abundant binding sites of monolayer MoS2 can further enable effective interaction with biomolecules. To be specific, negatively charged surfaces of monolayer MoS2 samples grown by chemical vapor deposition (CVD) [Monolayer MoS in 2013 , monolayon (CVD) ,22,23 ar2 embedded in a microfluidic chip. The wavelength of a fundamental laser is centered at 780 nm, with a pulse width of about 60 fs and a repetition frequency of about 80 MHz. The fundamental laser is reflected by the dichroic mirror and then focused on the MoS2 monolayer by the objective lens (NA = 0.55). SHG of the monolayer MoS2 is collected by the same objective lens. A short-pass filter is deployed behind a dichroic mirror to eliminate the reflected fundamental residual. The SHG signals are simultaneously sent to a spectrometer and a CCD by a beam splitter. In our measurements, the power of the fundamental laser was low enough to avoid optical damage of monolayer MoS2. The fine structure of the microfluidic chip is illustrated in 2 is attached to the optical glass window. The microfluidic channel enables unidirectional flow of liquid solutions of biomolecules to form a laminar flow. As a result, a homogeneous 3D fluid–2D solid interaction is constructed to facilitate adsorption and desorption of biomolecules on the surface of monolayer MoS2. Monolayer MoS2 grown by CVD on double-sided polished sapphire substrates  were used as received. To confirm the monolayer nature of these samples, optical characterizations were carried out before microfluidic experiments. 2. Lorentzian fitting of the PL spectrum points to a resonant peak centered at about 669 nm, which agrees well with the A-exciton resonance peak of the optical absorption spectrum [2, as indicated by the purple squares and red dots in 2 samples are monolayers, and strong SHG can be readily recorded by our experimental setup equipped with a regular spectrometer.spectrum ,25. Empl2, we employed positively charged chitosan to interact with monolayer MoS2. Chitosan was dissolved into a water solution of acetic acid, configuring a solution with a mass fraction of 0.8 mg/mL. Before adding the chitosan solution, a pixel-to-pixel SHG mapping of a monolayer MoS2 sample in the air was taken by scanning a 2D translation stage . The SHG image of this sample was presented in 2 region, strong SHG leads to a distribution of triangle reflecting the spatial profile of monolayer MoS2 lattice. For the substrate region, the absence of SHG points to a black background. Subsequently, 1µL of chitosan solution (one drop) was added onto the same monolayer MoS2 sample. When the solution completely evaporated at room temperature, pixel-to-pixel SHG mapping of the monolayer MoS2 sample was repeated. The obtained SHG image was presented in 2 sample covered by two and three drops of chitosan solutions, respectively. We anticipate that the electrostatic adsorption process can be initiated by the Coulomb forces between the negatively charged monolayer and chitosan. Accumulated amounts of chitosan are expected to form chitosan nanoclusters on the surface of monolayer MoS2, as illustrated by the schematic diagram in 2 can enhance SHG’s intensity. To directly reveal these differences, we subtracted the SHG image in 2. Rather, the differential SHG intensity shows an increasing trend from 2, SHG enhancement effects turn out to be stronger, suggesting that the edge region with more charged active sites tends to facilitate chitosan adsorptions [To justify the electrostatic adsorption effect of monolayer MoSorptions .2, we measured the AFM image after dropping and drying a chitosan solution for monolayer MoS2 samples on the sapphire substrate. As shown in 2 (triangle region) form many white dots. Furthermore, on the sapphire substrate, some chitosan nanoclusters can still be absorbed, but their density of distribution as well as size are smaller than the case of monolayer MoS2. The physical reason is that positively charged chitosan molecules tend to be more efficiently adsorbed by the negatively charged monolayer MoS2 through attractive Coulomb forces [2 (distance range: from 1.4 to 3.5 µm) could be as large as about 13 nm. The measured thickness of monolayer MoS2 is less than 1 nm (about 0.9 nm) according to 2 and on the sapphire substrate were analyzed, as shown in 2, the averaged diameter of adsorbed chitosan nanoclusters is about 78 nm, validating that Coulomb attraction forces between monolayer MoS2 and chitosan nanoclusters enhance the adsorption processes. The size FWHM of adsorbed chitosan nanoclusters on monolayer MoS2 is about 42 nm, which is about twice that (about 22 nm) on the substrate. At this point, we can conclude that chitosan nanoclusters can be effectively adsorbed on monolayer MoS2 by electrostatic attractions and mediate SHG intensity after drying. However, whether such an interfacial adsorption effect can induce enough SHG intensity change for biomolecules flowing in a liquid environment is still unknown.To visualize the adsorbed chitosan nanoclusters on the surface of monolayer MoSb forces ,21,22,232 tends to apply attractive Coulomb forces. Then, by controlling the pH of the liquid environment, protonated BSA molecules can gain electrons, and positive charges of adsorbed BSA molecules will be neutralized. Laminar flow in the microfluidic channel will take away these interfacial BSA molecules and trigger a desorption process. Before placing the monolayer MoS2, the microfluidic chip and tubes were carefully cleaned. The BSA solution (1 μg/mL) was configured in PBS buffer solution (pH = 3.6) using BSA . The power of the fundamental laser was fixed at 8 mW. By focusing the fundamental laser tightly on the center of a monolayer MoS2 sample by a 50× objective lens (NA = 0.55), we ensured that the size of the focal spot (1.7 µm) was much smaller than the lateral size of the monolayer MoS2 sample (about 15 µm). By finely tuning the axial position of monolayer MoS2 sample, we maximized the intensity of SHG spectrum recorded by the spectrometer. A computer program was coded to record the SHG spectra every 500 ms, which integrated all non-zero SHG counts between 370 nm and 410 nm.To demonstrate the feasibility of our SHG technique towards real-time sensing for flowing biomolecules in liquid environments, we selected bovine serum albumin (BSA) molecules and performed time-resolved SHG spectra measurements employing the experimental setup in 2 became stable in flowing conditions, as indicated by the time-resolved SHG signals (purple spectra) before 90 s in 2. Then, at 480 s, the PBS solution (pH = 7.4) was sent to trigger a BSA molecule desorption process. Interestingly, the intensity of SHG signals started to decrease and, eventually, recovered to a constant magnitude at about 850 s, which is equal to the scenario when no BSA was added (before the 90 s mark). Furthermore, a control experiment was performed by shifting the fundamental laser focus onto a nearby monolayer MoS2 sample and replacing the BSA solution with a PBS solution without BSA molecules, while other experimental conditions were kept the same. As indicated by the lower panel of 2 remained a constant. This result strongly validates that ions or other molecule components in the PBS solutions would not induce a detectable intensity change of monolayer MoS2 SHG signals for our experimental setup. The baseline decrease is attributed to the lattice orientation difference in CVD MoS2 from sample to sample. To evaluate the contributions of adsorbed BSA molecules to the refractive index change as well as intensity change of SHG signals, we focused the fundamental laser on the sapphire substrate. By replacing the short-pass optical filters in front of the spectrometer, a small portion of the fundamental laser (780 nm) was allowed to pass. Hence, we can measure fundamental and SHG signals at the same time. The BSA adsorption experiments were repeated by adding BSA solutions at 200 s. As shown in 2. The low SHG conversion efficiency of sub-nanometer thick monolayer MoS2 and high noise-level of our spectrometer lead to a relatively low signal to noise ratio. This issue can be further improved by increasing the integration time of each SHG spectrum and optimizing the design of microfluidic chips.In our measurements, a fluid pump sent solutions into the microfluidic channel at a constant flowing rate of 22 μL/s. In the beginning, we flushed the microfluidic chip with a PBS solution (pH = 7.4) until the SHG signal of monolayer MoSχ(2) and χ(3) are the second-order optical susceptibility of monolayer MoS2 and third-order optical susceptibility of the interface, respectively. In our case, we believe that the spatial distribution of adsorbed BSA molecules on the surface monolayer MoS2 is random. Specifically, since the initial charge distribution of monolayer MoS2 is inhomogeneous in a 2D plane defined by the flat substrate, the orientations and 3D stacking orders of adsorbed BSA molecules are expected to be highly random. As a result, directions of interfacial electric fields between the monolayer MoS2 and adsorbed BSA molecules in disorder will no longer be strictly perpendicular to the 2D plane. Therefore, the angle between the wave vector of the fundamental laser and the direction of interfacial electric fields will not be zero, so that the 2 and interfacial electric fields will depend on their initial phase difference.To address the physical mechanism of observed SHG signal changes accompanying the BSA adsorption process, we can consider the second-order polarization of the interface with the follow model ,29: E2ω∝2 can be utilized for label-free biomolecule sensing. Through static SHG mapping experiments, we show that the intensity of SHG in monolayer MoS2/adsorbed chitosan nanocluster heterostructures can be mediated due to electrostatic attractions. With a time-resolved SHG measuring system equipped with a microfluidic chip, we further realize label-free sensing of BSA adsorption and desorption dynamics in real time through the SHG intensity change of monolayer MoS2 in liquid environments, which has been tailored by Coulomb interactions between BSA molecules and monolayer MoS2. Our work provides a complementary mean of label-free interfacial biomolecule sensing, which, in principle, undertakes molecular-level spatial resolution across the interfaces for applications including, but not limited to, in vitro medicine evaluation.We have comprehensively demonstrated that the interfacial SHG of monolayer MoS


P < .001).Incomplete documentation of β-lactam reactions often leads to inappropriate antibiotic prescribing. The objective of this study was to evaluate the impact of a structured interview on the quality of β-lactam reaction documentation. After 203 interviews, documentation of the core components of a β-lactam reaction improved (48% vs 1%;  These self-reported β-lactam allergies have downstream consequences, including receipt of inappropriate antibiotic therapy and poor clinical outcomes. Incomplete documentation to describe the reaction contributes to the prescribing of less effective and more toxic antibiotics, such as fluoroquinolones and aminoglycosides. Improving the quality of β-lactam reaction documentation can be a powerful tool for stewarding resources, particularly at centers without allergy consultation services and/or the ability to perform penicillin skin testing.Beta-lactam antibiotics are often first-line therapy for the prevention and treatment of infection. Unfortunately, self-reported β-lactam allergies are common: ∼10% of people in the United States have a documented β-lactam allergy. With the appropriate information, the reaction type can be classified and the risk of cross reactivity to other β-lactam antibiotics can be determined. Complete documentation in the electronic health record (EHR) can provide important information regarding the ability to challenge with the same or alternative β-lactam antibiotic. In November 2018, we developed a questionnaire to guide β-lactam reaction interviews in the inpatient setting. In this study, we evaluated the impact of structured interviews on the quality of β-lactam reaction documentation.Structured interviews have demonstrated the ability to improve antibiotic use among patients with β-lactam allergies even in the absence of penicillin skin testing.This single-center, prospective, quasi-experimental study was conducted among inpatients with a β-lactam allergy label. Hospitalized adult patients with a β-lactam allergy label who underwent an interview using the questionnaire between November 1, 2018, to November 31, 2019, were included. The quality of β-lactam reaction documentation was evaluated based on 3 core components: time of the reaction, timing in relation to initiation of the β-lactam course, and a description of the reaction. Documentation of interventions received in response to the reaction and tolerance of other β-lactams were also evaluated. Secondary outcomes included the number of β-lactam allergy labels removed (delabeled) and antibiotic use 1 year before versus 1 year after the interview among the included patients. Antibiotic use was assessed using a continuous measure of days of therapy (DOT) per inpatient day and a nominal measure of receipt or absence of antibiotic doses. Antibiotic DOT and inpatient days were obtained using our hospital’s charge master.In November 2018, members of the Antimicrobial Stewardship Program (ASP), Section of Infectious Diseases and Global Health, and Department of Allergy and Immunology developed a questionnaire to guide β-lactam reaction interviews . The questionnaire was available on the internal ASP website and was not embedded into the EHR. Patients with a β-lactam allergy label were identified for potential interview using a report in the EHR. Patients were randomly selected from this list by pharmacists, pharmacy residents, and pharmacy students during their ASP rotation experience (∼3 interviews per week). All patients with a beta-lactam allergy label were eligible to be interviewed, regardless of the need for antibiotics during the index admission, but patients receiving ciprofloxacin and aztreonam were prioritized. After the interview, pharmacists updated the documentation in the allergy field in the patient’s medical record. If a reaction was eligible for deletion , the patient was provided verbal education and asked whether the allergy could be removed from the chart. Throughout the study period, inpatient allergy consultation and penicillin skin testing were not available.P ≤ .05 was considered statistically significant. Data were analyzed using SPSS Statistics version 26 predictive analytics software .We compared paired data among the same patients before and after the intervention. Nominal variables were compared using the McNemar test. Ordinal or nonparametric data were compared using the Wilcoxon signed-rank test. Comparisons were based on valid data for each mediation type listed above. Tests of significance were 2-tailed and During the study period, 203 adult patients with a β-lactam allergy label underwent a structured interview. The median time to complete an interview was 10 minutes . Prior to the interview, the most recent documentation was completed by a nurse (47%), physician (23%), pharmacist (15%), medical assistant (12%), or other healthcare worker (3%). The median age of the study population was 62 years old , and 93% of these patients were female (93%). Patients reported reactions to a penicillin (78%) and/or cephalosporin (29%). In total, 26 patients (13%) reported having a reaction to multiple β-lactam classes.P < .001). Upon interview, most patients were able to recall the reaction (94%) and how long ago it had occurred (92%), but only half of patients could recall the timing of the reaction in relation to the initiation of the medication  β-lactam allergy labels were removed from patient records. None of these patients had their allergy relabeled in the year after the interviews. Of the remaining patients, documentation of all 3 core components of the β-lactam reaction significantly improved after the interviews .Non–β-lactam gram-negative antibiotic use was lower in the postintervention period (63.2 vs 28.9 DOT per inpatient day). Individually, aztreonam (17 vs 6.8 DOT per inpatient day), aminoglycoside (6.8 vs 1.7 DOT per inpatient day), and fluoroquinolone (39.4 vs 20.3 DOT per inpatient day) use were also lower in the postintervention period. After the interviews, patients were less likely to receive non–β-lactam gram-negative antibiotics in the following year . This intervention can be particularly useful in resource-limited settings when inpatient allergy consultation or penicillin skin testing is not available. In a national US survey including 121 respondents, fewer than half of hospitals had access to inpatient allergy specialist consultations (44%) and inpatient penicillin skin testing (39%). our study quantifies the specific components of the history and better describes the quality of documentation. Based solely on an interview, we were able to delabel 7% of patients. In a similar study of 175 inpatients, only 1% of labels were delabeled. We also collected antibiotic use data beyond the index admission among the same patients, whereas previous studies have focused on antibiotic use during the admission in which the reaction history was obtained compared to a historic control group.Although many studies have evaluated the impact of β-lactam allergy interviews, our study is unique in several ways. While several studies have reported rates (34%–93%) of patients requiring any change in allergy documentation,Our study has several limitations. As a single-center study, similar results may not be observed at centers that provide guidance or have programs to optimize allergy documentation. We are also unable to attribute the observed changes in antibiotic use directly to the intervention. For example, evaluating antibiotic use in the same patients before and after the intervention introduces survivor bias, and antibiotic use would be affected by antibiotic discontinuation (if the patient no longer needs antibiotics).Our study demonstrates important improvements in the quality of β-lactam reaction documentation as a result of a structured interview. These improvements may be linked to antibiotic prescribing and have the potential to translate to improved patient outcomes.


The prognosis of children with refractory acute myeloid leukemia (AML) is poor. Complete remission (CR) is not always achieved with current salvage chemotherapy regimens before transplantation, and some patients have no chance of transplantation. Here, we aimed to describe a new regimen of conventional chemotherapy drugs ) for refractory AML and its mechanism in vitro.  We retrospectively collected the clinical data of 5 children with primary refractory AML using HCA as reinduction chemotherapy, and CR rates, adverse reactions, and disease-free survival (DFS) were analyzed. The effects of homoharringtonine, cladribine, and aclarubicin alone or in combination on the proliferation of HL60 and THP1 cells were analyzed by CCK-8 assay. Furthermore, CCK-8 was used to determine the effects of HCA, alone or in combination with apoptosis inhibitors, necroptosis inhibitors, ferroptosis inhibitors, or autophagy inhibitors, on the proliferation of HL60 and THP1 cells and to screen for possible HCA-mediated death pathways in AML cells. The pathway of HCA-mediated AML cell death was further verified by Hoechst/PI staining, flow cytometry, and Western blotting.  After 2 cycles of conventional chemotherapy, none of the 5 children with AML achieved CR and were then treated with the HCA regimen for two cycles, 4 of 5 achieved CR, and another child achieved CR with incomplete hematological recovery (CRi). After CR, 3 children underwent hematopoietic stem cell transplantation (HSCT), and only 2 of them received consolidation therapy. As of the last follow-up, all 5 patients had been in DFS for a range of 23 to 28 months. The inhibition rate of homoharringtonine, cladribine, and aclarubicin in combination on HL60 and THP1 cells was significantly greater than that of a single drug or a combination of two drugs. We found that inhibitors of apoptosis and necroptosis were able to inhibit HCA-mediated cell death but not ferroptosis or autophagy inhibitors. Compared with the control group, the number of apoptotic cells in the HCA group was significantly increased and could be reduced by an apoptosis inhibitor. Western blot results showed that PARP, caspase-3, and caspase-8 proteins were activated and cleaved in the HCA group, the expression of Bax was upregulated and that of Bcl-2 was downregulated. The expression of apoptosis-related proteins could be reversed by apoptosis inhibition. Compared with the control group, the expression levels of the necroptosis-related proteins RIP1, RIP3, and MLKL were downregulated in the HCA group but were not phosphorylated. The necroptosis inhibitor increased the expression of RIP1 but caused no significant changes in RIP3 and MLKL, and none were phosphorylated.  HCA, as a new regimen of conventional drugs, was a safe and efficacious reinduction salvage strategy in children with refractory AML before HSCT. HCA exhibits the synergistic growth inhibition of AML cells and induces cell death mainly through apoptosis. The outcomes of children with acute myeloid leukemia (AML) have been significantly improved in recent years because of the application of hematopoietic stem cell transplantation (HSCT), the combination of multiple drugs with chemotherapy, and the rise of novel targeted drugs. However, children with refractory AML still have poor outcomes. The main reason for the formation of refractory AML is the resistance of leukemia cells to chemotherapeutic drugs. The treatment options for refractory leukemia mainly include the use of drugs without cross-resistance, medium and high doses of cytarabine (Ara-C), HSCT, new targeted therapy drugs or biological therapy. Among these treatment regimens, HSCT may be the best choice for refractory patients. It is important to achieve complete remission (CR) before HSCT and longer disease-free survival (DFS) for patients who have no chance of transplantation.Although there has been significant development of targeted drugs, traditional chemotherapy drugs were mature, stable, and economical. We should not give up the optimization of traditional chemotherapy regimens. Common chemotherapeutic agents for AML include purine analogues, Ara-C, homoharringtonine, and anthracyclines. However, among these chemotherapeutic agents, increasing anthracycline and Ara-C doses may not improve response rates in AML with relapse or nonresponse . RegimenIn this study, we described a new regimen of conventional chemotherapy drugs ) for refractory AML. The patients were followed up for 23-28 months, and the CR rate and DFS were 100%. Moreover, we examined the effects of HCA on AML cell line proliferation and identified the cell death pathway of these drugs.After the study was approved by the IRB of the Third Xiangya Hospital of Central South University, we retrospectively collected the basic data, clinical indicators, and laboratory indicators of the children treated with the HCA regimen in the Xiangya Hospital or in the Third Xiangya Hospital. All five children enrolled in this study were treated at Xiangya Hospital .2/day as a continuous infusion for 24 hours on days 1–4,aclarubicin at 14 mg/m2/day as an intravenous infusion over 2 hours on days 1–4, homoharringtonine at 1 mg/m2/day as an intravenous infusion over 3 hours on days 1–7.The HCA regimen was as follows: cladribine at 5 mg/mBone marrow (BM) aspirates were collected before each course of HCA and posttreatment on day 28 (±7 days) after HCA to assess the response. Response was assessed by bone marrow aspirates and MRD. Cytogenetic analysis can also be used to assess response status. Discontinue treatment in the event of a serious adverse reaction. Provide supportive care according to local institutional guidelines.9/L, platelet count > 80 × 109/L). Reaching CR with incomplete hematological recovery was defined as a CRi. Refractory acute myelogenous leukemia was defined as patients who had not achieved CR after at least two courses of previous-line induction chemotherapy [CR was defined as less than 5% blasts on BM morphology examination and satisfactory hematological recovery . RPMI-1640 medium was used to culture HL60 and THP1 cells. DMEM was used to culture Panc-1 cells. All cells were cultured at 37°C with 5% COAntibodies against GAPDH , PARP , caspase-3 , cleaved caspase-3 , caspase-8 , cleaved caspase-8 , Bax , Bcl-2 , p-RIP1 , RIP3 , anti-rabbit IgG, HRP-linked antibody , and anti-mouse IgG, HRP-linked antibody  were obtained from Cell Signaling Technology . Antibodies against MLKL , p-MLKL , RIP1 , and p-RIP3  were purchased from Abcam . Cladribine, homoharringtonine, Z-VAD-FMK, cycloheximide, chloroquine, ferrostatin-1, and necrostatin-1 were purchased from TargetMol . Aclarubicin was obtained from APExBIO . A stock solution containing cladribine, homoharringtonine, and aclarubicin was prepared in DMSO and kept at -80°C. TNF-alpha was purchased from ABclonal .5/mL) were seeded in 96-well plates with different concentrations of homoharringtonine, cladribine, and aclarubicin. 10 μL of CCK-8 solution was added to each well after 24 or 48 hours in culture. The 96-well plates were incubated in a cell incubator in the dark for 4-6 hours, and then, the absorbance at 450 nm was measured using a microplate reader . The cell line experiments were repeated at least three times with three replicates for each trial.The inhibition of HL60 or THP1 cell growth was assessed with CCK-8 . Cells  after 15-minutes of incubation at room temperature in the dark. Early apoptotic cells showed Annexin V-FITC+/PI−, and late apoptotic cells showed Annexin V-FITC+/PI+.Cells were treated with different combinations of reagents for 48 hours, collected in EP tubes, washed twice with PBS, and resuspended by adding binding buffer. Apoptotic cells were stained using an Annexin V-FITC/PI Apoptosis Detecton kit . Then, 5 μg/mL, incubated at 37°C for 10 minutes, harvested, and washed with PBS. Then, the cells were resuspended in PBS containing PI (10 μg/mL), incubated at 4°C in the dark for 15 minutes, and washed with PBS. The cell death rate was calculated by determining the Hoechst+/PI+ ratio.HL60 and THP1 cells were treated with different combinations of reagents for 24 hours, Hoechst 33342 was added to the culture medium at a final concentration of 2 Cells were collected into EP tubes, washed twice with PBS, lysed using RIPA buffer containing protease and phosphatase inhibitors, incubated on ice for 30 minutes, subjected to high-speed centrifugation and collected from the protein solution. The protein concentration was measured with a BCA (Thermo Fisher Scientific). The calculated protein solution was mixed with 4× SDS sample buffer, boiled at 100 degrees for 10 minutes, and then separated by electrophoresis. After electrophoresis, proteins were transferred to PVDF membranes, blocked with 5% nonfat milk for 1 hour on a shaker, and incubated with primary antibodies overnight at 4°C. Primary antibodies were washed with TBST and then incubated with secondary antibodies for 2 hours at room temperature (RT) on a shaker. Finally, the membranes were washed with TBST, and the proteins were visualized using an ECL kit .The membranes were scanned and analyzed using a gel imager .Between June 2019 and December 2019, a total of 5 AML patients treated with the HCA regimen were reviewed. Patient baseline characteristics are summarized in 2 cladribine on days 1-4, 1 mg/m2 homoharringtonine on days 1-7, and 14 mg/m2 aclarubicin on days 1-4.Five children with AML who failed to achieve CR after two to three courses of conventional chemotherapy received the HCA regimen for two cycles. The regimen for HCA treatment was as follows: 5 mg/mAll patients received the HCA regimen, and the details of the responses and evaluation of hematological can be seen in Among the five patients, one child received 2 courses, and one child received 1 course of consolidation chemotherapy without HSCT and was followed up without other treatment. One child received 1 course of consolidation chemotherapy and underwent allogeneic stem cell transplantation (Allo-SCT) 3 months later. The remaining 2 children did not receive consolidation chemotherapy and underwent Allo-SCT at the 6th and 2nd months after achieving CR using the HCA regimen. As of the last follow-up, all 5 patients were in DFS, ranging from 23 to 28 months.9/L was 15 days, and that for platelet recovery > 20 × 109/L was 6 days. The most common grade 3 to 4 nonhematologic toxicities were febrile neutropenia, pneumonia, sepsis, and intestinal infection. There were treatment-related deaths in this study. The toxicity of the HCA regimen in the children with AML is shown in Five patients received a total of 10 cycles of the HCA regimen. All patients developed severe bone marrow suppression due to their disease and chemotherapy. The median time for neutrophil recovery > 0.5 × 10μmol/L and 0.17 μmol/L, 69.59 nmol/L and 11.8 nmol/L, and 1.69 μmol/L and 0.61 μmol/L, respectively (n = 3). The IC50 values of cladribine, homoharringtonine, and aclarubicin for THP1 at 24 and 48 hours were 3.57 μmol/L and 0.22 μmol/L, 110.2 nmol/L and 18.22 nmol/L, and 0.41 μmol/L and 0.33 μmol/L, respectively (n = 3). The cytotoxicity of the three drugs to HL60 and THP1 cells was measured with CCK-8 assays. The inhibition of HL60 or THP1 cell growth was concentration dependent. Our data show that HCA has a significantly higher inhibitory effect on AML cells than any monotherapy or two-drug combination . The combination index (CI) values when HCA was applied to AML cells were analyzed using CompuSyn software, and the results showed a synergistic effect , homoharringtonine , and aclarubicin . The half-maximal inhibitor concentration (IC50) values of cladribine, homoharringtonine, and aclarubicin for HL60 at 24 and 48 hours were 1.18  Figures  , such as nucleoside analogs , tyrosine kinase inhibitors (sorafenib), proteasome inhibitors (bortezomib), epigenetic therapies (decitabine), and immunotherapies (gemtuzumab ozogamicin), have been used in children with RR-AML . The fluCladribine, the first-generation deoxyadenosine analog, has unique antileukemic properties. In the treatment of adult AML, the addition of cladribine to the standard regimen has a better efficacy . For AMLStreptomyces galilaeus that exhibits major chemical differences from the conventional anthracyclines daunorubicin (DNR) and adriamycin (ADM). Aclarubicin can not only inhibit the replication and repair of DNA and the synthesis of RNA and proteins [Aclarubicin is an anthracycline antibiotic isolated from proteins  but it cproteins . In addiproteins . Aclarubproteins –33, whicproteins .β pathway components, and NF-κB repressing factor [Homoharringtonine is a plant alkaloid that is widely used to treat adults with initially diagnosed or RR-AML. Homoharringtonine exerts antitumor effects by inhibiting the synthesis of target proteins, such as phospho-eIF4E, SP1/TET1/5hmC, Smad3, and TGF-g factor –38, and g factor –43. Ma eg factor . Satisfag factor , 46. Theg factor .2 cladribine on days 1-4 [2 homoharringtonine on days 1-7, and 14 mg/m2 aclarubicin on days 1-4 as the reinduction protocol for children with primary refractory AML. All five children who did not respond to conventional chemotherapy achieved CR or CRi after two cycles of the HCA regimen, and the CR/CRi rate was 100%. The most common events were severe myelosuppression, febrile neutropenia, and intestinal infection. All of the toxicities were tolerable and acceptable. No neurological or cardiac events or treatment-related deaths occurred during treatment. The median time for platelet recovery > 20 × 109/L was 6 days, which was shorter than that reported with the CLAG regimen (13 days), and the median time for neutrophil recovery > 0.5 × 109/L was 15 days, which was comparable to that reported in a previous study [Identifying new salvage regimens that produce a relatively high CR rate and low toxicity in children with RR-AML is still an area of investigation. For the selection of a treatment regimen for RR-AML, resistance to individual prior therapies and their associated toxicity must be considered. An increased dose of a conventional chemotherapeutic agent, such as Ara-C or daunomycin, is not optimal for pediatric RR-AML because of dose-related toxicities and non-obvious improvements . Based odays 1-4 , 1 mg/m2regimen 1 days, anIn vitro, our study showed that homoharringtonine, cladribine, or aclarubicin treatment alone resulted in inhibition of the cell growth of HL60 and THP1 cells in a dose-dependent manner. These findings support the notion that homoharringtonine, cladribine and aclarubicin can inhibit the growth of AML cells –53. To eApoptosis is a programmed cell death process and an important mechanism by which chemotherapeutic drugs regulate tumor development , 55. CliNecroptosis, a form of cell death defined in recent years, can be inhibited by necrostatin-1. We found that the combined administration of necrostatin-1 or Z-VAD-FMK could reverse HCA-mediated cell death. However, the specific molecular biomarkers evaluated including phosphorylated RIP1, RIP3, and MLKL, which were used to detect the activation of RIP1, RIP3, and MLKL in necroptosis, were not increased after adding Z-VAD-FMK or necrostatin-1 alone to the HCA regimen . This inIn summary, our study suggests that HCA is a very good combination of traditional drugs. HCA synergistically inhibits AML cells and triggers apoptosis. This is the first evaluation of reinduction therapy with the HCA regimen in pediatric primary refractory AML, and the results show that the HCA regimen can be considered a reinduction salvage strategy in children with refractory AML before HSCT or consolidation therapy.


Here, we performed comprehensive high-throughput analyses, including RNA sequencing of CR (complete remission) and relapsed AML patients, and reverse-phase protein arrays of chemoresistant cells to identify METTL3 as a key player regulating AML chemoresistance. Then, METTL3-mediated m6A modification was proved to induce the chemoresistance in vitro and in vivo. Furthermore, AML homing/engraftment was discovered being enhanced by upregulated-METTL3 in chemoresistant cells. And the homing/engraftment and drug-resistance associated phenotypes of chemoresistant cells could be reversed by a METTL3 inhibitor. Mechanistically, METTL3 extended the half-life of ITGA4 mRNA by m6A methylation, and then, increased expression of ITGA4 protein to enhance homing/engraftment of AML cells. The results provide insights into the function of m6A modification on the interaction between AML cells and BM niches and clarify the relationship between METTL3 and AML homing/engraftment, suggesting a therapeutic strategy for the treatment of refractory/relapsed AML with METTL3 inhibitors.Chemoresistant leukemia relapse is one of the most common causes of death for acute myeloid leukemia (AML) patients and the homing/engraftment in bone marrow (BM) are crucial steps for AML cells to acquire chemoresistance by interacting with stromal cell components. No crosstalk between m Acute myeloid leukemia (AML) is a hematologic malignancy characterized by dysregulated proliferation and accumulation of leukemic cells in the hematopoietic system . Inducti6A modification, which is the most prevalent internal chemical modification found in mRNAs [6A modification plays a key role in many physiological and pathological processes, such as viral infections, immune response, tissue regeneration, development of cancer cells and specification of hematopoietic or cancer stem cells [6A modification has been shown to be involved in the pathogenesis and development of AML by regulating the different fates of various mRNAs [6A methylation had been proved associating with cell adhesion [6A methylation and the homing/engraftment of AML cells in BM has been found.In mammalian cells, approximately 0.4% of all adenosines in mRNA are modified by min mRNAs . m6A modem cells –11. Notaus mRNAs –16. Althadhesion , 18, no 6A modification system [6A-dependent manner. Then, a METTL3 inhibitor, STM2457, was proven to significantly reverse the homing/engraftment and drug-resistance of chemoresistant cells. Mechanistically, METTL3 promoted the development of AML chemoresistance via the METTL3-m6A-ITGA4-homing/engraftment axis. Together, the data demonstrate an important role of METTL3 in AML chemoresistance, showing potential application prospects for METTL3-mediated m6A modification in the treatment of refractory/relapsed AML.METTL3 is the core methyltransferase of the mn system . It has n system , 20 and n system . Many efn system , but the6A dot blot, LC-MS/MS for determination of the m6A/A ratio, RNA-seq and m6A-seq, mRNA stability, Western blot and Dual-luciferase reporter assays are described in the Supplementary Materials and Methods section.Cell lines, lentivirus infection, RPPA, Mouse studies and animal housing, EdU and CFA assays, cell migration and adhesion experiments, RT-PCR, mBMMNCs were isolated from primary and relapsed AML patients. All the related procedures for collection of the samples of patients with AML were performed with the approval from the Research and Ethics Committee of Qilu Hospital of Shandong University. All patients who provided clinical specimens signed the written informed consent form. All the procedures were performed in accordance with the 1964 Declaration of Helsinki principles and its later amendments or comparable ethical standards. CD34+ cells were isolated by CD34 MicroBeads according to the manufacturer’s instructions .6A antibody  or rabbit IgG with Protein A/G Magnetic Beads at 4 °C for 1 h. After washing with IP buffer, the m6A IP portion was eluted with 200 μL of elution buffer with Proteinase K at 55 °C for 45 min and purified. qPCR was performed along with the MeRIP-ed RNAs.The MeRIP Kit  was used following the manufacturer’s instructions. Briefly, 200 mg of total RNA was sheared to approximately 300 nt in length by metal ion-induced fragmentation and purified with 450 µL of IP buffer with RNase inhibitors. One-ninth of the fragmented RNA was saved as an input control and then incubated with an mTo identified targets related to chemotherapy resistance with potential for clinical application, we firstly analyzed RNA sequencing (RNA-seq) data from the GSE165430 dataset   mice. Forty-two days later, IDA was injected intraperitoneally for 7 days to treat the disease  in THP-1 cells. Our m6A-seq data showed that 3516 genes had significantly increased levels of m6A modification after METTL3 overexpression. Integrative analysis with the RNA-seq data identified 564 transcriptome-wide potential targets  and the 3’ untranslated region (3’ UTR) of mRNA transcripts in AML cells (Supplementary Fig. 6A modification in the 3’ UTR of ITGA4 mRNA was significantly increased when METTL3 was overexpressed (Fig. 6A sequencing (Supplementary Fig. 6A antibody was significantly increased when METTL3 was overexpressed (Fig. 6A modification, then promoted the expression of ITGA4 on the cell surface and helped the cells access the BM niche.Subsequently, the mechanisms by which METTL3 regulates the expression of ITGA4 were studied. The msed Fig. . Then, bsed Fig. , indicatsed Fig. . Moreovesed Fig. . All the6A modification has been proven to be involved in the carcinogenesis of AML [6A methylation of ITGA4 mRNA, which might guide the clinical application of METTL3 inhibitors in the treatment of AML.Primary resistance to initial treatment and disease relapse are major limitations that have not been addressed for the treatment of AML . Many efs of AML , 20, ands of AML . Notably6A modification was an effective treatment target for AML chemoresistance. METTL3 has been proven to be involved in drug resistance in many cancers, but opposite roles have also been found in various cancer treatment models [6A demethylation on the targeted drug resistance of TKIs [6A methylation [et al. proved that promoter-bound METTL3 introduced m6A methylation within the coding region of SP1 and SP2 transcripts to promote AML maintenance [First, we found that METTL3-mediated mt models –41. Spec of TKIs , but no  of TKIs –47, eluchylation . In the ntenance . These rntenance . The dis6A modification system and AML homing/engraftment has been found. A recent report on STM2457 found that, compared with the initial STM2457-treated mice, the mice after retransplantation of the AML cells that had been treated with STM2457 had a significant increase in lifespan and a marked decrease in the presence of AML cells in peripheral blood [6A methylated target sites were mutated. In addition, METTL3 significantly affected the rate of ITGA4 mRNA degradation. All the results confirmed that METTL3 can increase the stability of ITGA4 mRNA transcript by m6A modification, then promoted the expression of ITGA4 on the cell surface and helped the cells access the BM niche. The study linked the m6A modification system with the BM microenvironment and showed that METTL3-mediated m6A modification affected the communication between cells and other cells or the microenvironment by regulating ITGA4. Why ITGA4 is selected as a key “signal detector” for METTL3-mediated homing of AML cells should be further studied.Second, we showed for the first time that METTL3 enhanced AML chemoresistance by regulating the homing and engraftment of AML cells, which provided a new strategy for targeting AML chemoresistance. AML cell homing to and engraftment in the BM are important for chemoresistance , 49. Manal blood . These r6A modification of ITGA4 mRNA. These findings provide insight into the mechanisms underlying AML chemoresistance and develop a novel chemoresistance regulatory system based on METTL3-mediated m6A modification. These findings will help further elucidate the precise regulatory mechanisms of AML chemoresistance and provide a new perspective for METTL3 as a novel treatment target of AML.Overall, our studies have identified a novel AML chemotherapy drug-resistant protein, METTL3, which can mediate AML cell homing and engraftment by mSupplementary InformationSupplementary Table 1Supplementary Table 2Supplementary Table 3


Calcium mishandling and mitochondrial dysfunction have been increasingly recognized as significant factors involved in the progression procedure of cardiomyopathy. Ca2+ mishandling could cause calcium-triggered arrhythmias, which could enhance force development and ATP consumption. Mitochondrial disorganization and dysfunction in cardiomyopathy could disturb the balance of energy catabolic and anabolic procedure. Close spatial localization and arrangement of structural among T-tubule, sarcoplasmic reticulum, mitochondria are important for Ca2+ handling. So that, we illustrate the regulating network between calcium handling and mitochondrial homeostasis, as well as its intracellular mechanisms in this review, which would be worthy to develop novel therapeutic strategy and restore the function of injured cardiomyocytes. Taffazzin gene mutation  provided by mitochondria . Close aThe microarchitecture of cardiac dyads in cardiomyocytes, which was considered as the structural basis of calcium transits in cardiomyocytes. The release of calcium from the SR and efficient ATP production by mitochondria are essential for normal excitation-contraction (EC) coupling. The increase in cytosolic calcium is augmented by calcium release at numerous dyads. T-tubule depolarization is mediated by coupling L-type Ca2+ channels (LTCCs) next to ryanodine receptors (RyRs), which activates RyR2 channels by a small amount of Ca2+ entry through LTCCs and leads to the release of a large amount of calcium mediated by RyR2 . Dyads oLeft cardiac hypertrophy induced by transverse aortic constriction leads to the proliferation of AMs and ATs and increases the phosphorylation of RyR2, thereby maintaining a higher signal of cytosolic Ca2+ activity . A remodBridging integrator 1  facilitates LTCC localization to T-tubule membranes and plays a critical role in recruiting RyRs to the SR membrane . As a reDysfunction in T-tubules can also impair surrounding ion channels. Changes in the T-system can serve as efficient biomarkers for evaluating heart function and related prognoses. Normal EC coupling relies on close cooperation between the SR network and T-tubule membranes. Most RyR2 clusters are located on (or close to) T-tubules, which suggests that they contribute to the generation of Ca2+ wave development during sarcomere shortening. When RyRs disordered at diastolic phase in pathological conditions, Ca2+ can leak out from the SR network and produce a Ca2+ spark. A major adverse effect of Ca2+ leak is that it reduces the amount of calcium in the SR and attenuates the amplitude of Ca2+ transient. The Ca2+ released from the SR should be taken up by SR calcium ATPase (SERCA) in every cardiac cycle to sustain a steady state with a stable Ca2+ level . Thus, aRyR2 activity is strongly associated with the Ca2+ level in the SR, which is also regulated by the phosphorylation of RyR2 by protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein phosphatase type 1 and type 2A . CalciumThe phosphorylation of RyR2 is the dominant mechanism regulating its function, and several phosphorylation sites have been confirmed to be involved. RyR2 phosphorylation sites at S2808 and S2814, which are phosphorylated by PKA and CaMKII, respectively , mediateActivation of CaMKII-mediated phosphorylation of RYR2 at S2814 can activate the latent arrhythmic potential of catecholaminergic polymorphic ventricular tachycardia (CPVT) caused by RYR2 mutations . CaMKII CaMKII can also phosphorylate phospholamban (PLN) to further activate SERCA, which is important in the uptake of Ca2+ from the cytoplasm to the SR . DecreasThe mechanism underlying Ca2+ handling is intricate. Ca2+ overload in the cytosol increases the affinity of troponin binding Ca2+, contributing to arrhythmogenicity and heart failure. Elucidating the precise mechanisms underlying calcium handling intracellularly and downstream of mitochondrial function is important for understanding the causes and progression of cardiomyopathy and developing novel and precise strategies for treating cardiomyopathy.via the mitochondrial calcium uniporter (mtCU) reduces the mitochondrial membrane potential, potentially inducing ROS generation via perturbation of the pH gradient at the inner mitochondrial membrane (IMM), affecting the rate of mitochondrial energy production, mitochondrial motility, and morphology  consists of a large proportion of the outer mitochondrial membrane (OMM) proteome. TOM70 puncta are frequently associated with MAMs, which regulate Ca2+ shuttling from the SR to mitochondria . Filadi Ru360, a specific inhibitor of the mtCU, can decrease mitochondrial calcium dramatically and prevent mPTP opening after cardiac ischemia . Pan et In sum, the crosstalk between mitochondria and SR Ca2+ handling in cardiomyocytes is increasingly being recognized. The mechanism underlying mitochondrial Ca2+ handling and suitable amounts of mitochondrial Ca2+ on a beat-by-beat basis in normal and damaged cardiomyocytes are unclear. It is thought that the structure of dyads plays an important role in cardiomyocytes, especially in the efficiency of calcium release during EC coupling.Therapeutic strategies have been designed to increase the contractile force of the heart to directly improve the survival of patients with severe heart failure. Although a series of genetic analyses and mechanistic studies have revealed the crosstalk between calcium handling and mitochondrial homeostasis as the central mediator of ventricular arrhythmia and heart failure, the FDA has yet to approve reagents targeting calcium handling and mitochondrial homeostasis for use by cardiovascular patients. via cell-based modification to delivery oligonucleotide .Over the last decades, a large amount researches expanded our knowledge of the Ca2+ handling among the interplays of T-tubule, ER, and mitochondrial in cardiomyocytes. Yet, there are also some unsolved queries and conflicting experimental results. It is urgent to explore the factors involved in coordinating formation of the T-tubular network, along with the potential molecular mechanisms in maintaining homeostasis of intricate architecture between T-tubule and the ER. Moreover, it is also critical to illustrate the transcriptional regulation mechanisms of various proteins reside within the MAMs and the precise mechanisms of calcium handling between ER and mitochondria. Besides, the balance between sufficient ATP consumption and acceptable ROS generation is still required further study. The further understanding of the molecular mechanisms involved in the crosstalk between Ca2+ handling and mitochondria in cardiomyocytes may provide newly concepts on the treatment of cardiovascular disease. And isogeneic therapeutic strategy would be another alternation to handle deadly arrhythmia and improve their prognosis.


This study evaluated the effects of this material on the morphology and physicochemical properties of an established in-vitro biofilm of Enterococcus faecalis. A biofilm of E. faecalis was grown in multichannel plates. The chemicals including Ca2+, OH−, and saturated Ca(OH)2 (ie 21.6 mM) were prepared in order to evaluate which component eradicated or amplified biofilm structure. Various biochemical and microscopic methods were used to investigate the properties of the biofilm. Biofilms treated with Ca(OH)2 absorbed more Ca2+ because of the alkaline pH of the environment and the ions affected the physicochemical properties of the E. faecalis biofilm. A denser biofilm with more cavities and a granular surface was observed in the presence of Ca2+ ions. This resulted in a decrease in the surface-to-biofilm ratio with increases in its biomass, thickness, colony size, and volume. Calcium hydroxide did not destroy E. faecalis biofilms but rather contributed to the biofilm structure. This in-vitro study sheds light on a missing link in the formation of E. faecalis biofilm in which the Ca2+ in Ca(OH)2Calcium hydroxide Ca(OH) Prospective biofilm complications for periapical lesions and tissue breakdown can lead to an inflammation that critically necessitates endodontic treatment2. The anatomical complexity of the root canal system and organization of microorganisms into biofilms are major factors contributing to the difficulty of elimination of root canal infections4.Apical periodontitis is a biofilm-induced inflammatory response around the apex of a tooth, which is caused by microbial infections of the root canal system5. EPS is primarily composed of water (approximately 90%), polymeric substances , proteins, eDNA, and lipids6. The structural properties of biofilm makes it more resistant to antimicrobial agents and environmental stresses compared to planktonic cells7.A biofilm is a surface-attached microbial community encased in a self-produced slimy matrix or extracellular polymeric substance (EPS), that comprises 95–98% EPS and 1–5% microorganisms8. The negative charge of EPS is important during its interaction with polyvalent cations. Calcium can induce production of extracellular material such as alginate in mucoid bacterial strains9. It has also been suggested that calcium, which binds to cell walls, may play a key role in cell-to-cell adhesion in biofilms as well as stabilization of bacterial cell walls10.Divalent cations such as calcium and magnesium have been shown to cross-link with the polymer strands to provide more stable attachment of the biofilm2 is widely used as a temporary root canal medicament to hinder the regrowth of bacteria in the root canal between treatment sessions11. One of the most important reasons to use it in endodontics is its antimicrobial property. The antimicrobial activity of Ca(OH)2 relies on the release of hydroxyl ions (OH-) in the presence of water, which results in cytoplasmic membrane distortion, protein denaturation, and DNA damage12. However, some studies have reported that calcium hydroxide has limited effectiveness in eliminating bacteria from biofilm13.Calcium hydroxide Ca(OH)14. They observed that exposure to calcium resulted in distinct changes in the biofilm. They suggested that chelators like Ethylenediaminetetraacetic acid (EDTA) can absorb these calcium ions in the EPS and facilitating the disruption of biofilm. With this as a basis, we investigated the effect of calcium hydroxide ingredients (Ca2+ and OH-) on the morphology and physicochemical properties of E. faecalis biofilms. This bacterium has been commonly used in endodontic biofilm models because it has been coupled with endodontic treatment failures15.In 2012, van der Waal and van der Sluis hypothesized that calcium plays a key role in the formation of the scaffold and extracellular matrix of a biofilmThe antimicrobial properties of this molecule against planktonic microbial cells have been described but its action against biofilm results to be unclear and controversial. The null hypothesis of this study was that calcium hydroxide does not show antibiofilm activity but rather contributes to the biofilm matrix architecture. E. faecalis biofilms, grown on different artificial surfaces. Evaluation included microscopy  and determination of calcium and polysaccharide content of the biofilm as well as quantity of viable cells. It was found that the different chemical formulations affected the biofilm morphology and physicochemical properties.This laboratory study investigated the effect of calcium hydroxide, calcium and hydroxyl ions on 21-day-old 2 group an increase in cell size was observed  images revealed that in the Ca(OH)ved Fig. b. Furthe2 and Ca2+ groups, biofilm surface was granular due to the huge amount of EPS while in the control and OH- groups, it was smooth oth Fig. . The grages Fig.  showed t2+ ions induced structural changes and a denser biofilm formation  data, the structural properties of biofilm was described in terms of thickness, colony volume, biomass, colony size and surface-biovolume ratio ses Fig.  & S2.Fig2 group had significantly highest average biofilm thickness  followed by the Ca2+, OH-, and control group  from other groups . The difference between the other groups was not significant 2, and Ca2+ groups one Fig.  & S2. Thups Fig.  & S2.2, Ca2+, OH-, and control groups, respectively. Statistical analysis using the Kruskal–Wallis test showed no significant difference between Ca(OH)2 and Ca2+groups (P = 0.727). Meanwhile, the morphological difference pertinent to Ca(OH)2 and OH- groups revealed the cooperation of calcium in biofilm progression where the environment was alkaline.The atomic force microscopy (AFM) microscopy images revealed the variable morphology and roughness of the bacterial biofilms Fig. . The rou2+ ions in the Ca(OH)2 2 Fig. b and Ca2 2+ Fig. c groups.oup Fig. d was veroup Fig. a.Figure 2 group was significantly different from the other groups.The mean concentration of free polysaccharides was measured for in all groups Fig. . A remar2+ ions content of the biofilm was measured by ICP technique. The results showed that the mean Ca2+ concentration in the control, Ca(OH)2, Ca2+, and OH- groups were 30.8, 18,555, 10,948 and 2211 ppm, respectively 2 group, which was in compliance with previous results and confirmed that Ca2+ ions played major role in biofilm progression in alkaline environment.The Caely Fig. . The Ca2- group was lowest followed by the Ca+2, Ca(OH)2, and control groups 2 components, ie Ca2+, and alkaline pH, through a cooperative manner strengthen biofilm. Therefore, Ca(OH)2 did not eradicate E. faecalis biofilm, but promoting growth of biofilm.This study investigated the effect of calcium hydroxide on the morphology and physiochemical properties of 2 and Ca2+ groups formed a granular surface; while, in the control and OH– groups, the surface was smooth. The granular surface was produced by the Ca2+ ion bonds, which led to morphological differences when compared to the smooth surfaces in the absence of Ca2+ ions. These findings are in consistent with those of Safari et al.,who showed that the addition of Ca2+ to the biofilm produced a granular surface16. Moreover, Mangwani et al. observed that the addition of Ca2+ caused cavities in the biofilm. An increased in cavities in the biofilm indicates the accumulation of large amounts of EPS17.The EPS of the biofilm in the Ca(OH)2 and Ca2+ groups were live cells, while in the OH- group the number of viable cell dramatically reduced. In the Ca(OH)2 group, because of alkaline pH of the environment, the uptake of Ca2+ increased in the biofilm such that number of living bacteria was more than other groups18. This finding is consistent with the report pertinent to elevated biomass by adding Ca2+ to the biofilm19.In regard to the qPCR and CLSM results, it can be concluded that most of the green dye displayed in the Ca(OH)2 group. This can be explained by the mechanisms of the Smith–Gilkerson reaction, in which the OH part of the sixth carbon of the saccharide ring in the polysaccharide structure, used to produce chromogenic substance, was associated with large amounts of Ca2+ ions and was unable to interact with the Smith–Gilkerson reagent. We concluded that most of the polysaccharides in the biofilm were not free and had not been measured by the Smith–Gilkerson method.The results of quantitative analysis of free polysaccharides in the EPS revealed that the lowest concentration of free polysaccharides was for the Ca(OH)2+ ions caused a denser biofilm with more cavities and indicates an increase in EPS. The presence of this ion also created a granular surface in the biofilm. Expansion of the biomass and increases in the thickness, colony size, and volume of the biofilm as well as declining the surface-to-biofilm ratio were the results for the Ca2+ ions in the biofilm. The alkaline pH of the environment enhanced absorption of Ca2+ when a biofilm was treated with Ca(OH)2; thus, this ion was able to affect the morphology, structure, and chemical properties of the E. faecalis biofilm as well.In conclusion, the presence of Ca2+, as a part of calcium hydroxide, serves a missing link in the biofilm progression of E. faecalis. Calcium hydroxide does not destroy the biofilm of E. faecalis, but actually participates in strengthening the biofilm of this bacterium.This study has shown that CaE. faecalis strain  was used in all experiments. Brain heart infusion (BHI) agar  and BHI broth culture medium  were prepared according to manufacturer instructions. The isolated colonies were cultured on BHI agar plates and incubated under aerobic conditions at 37 °C for 24 h. Thereafter, a bacterial suspension in normal saline was prepared adjusted to the 0.5 McFarland standard and diluted to equal 106 CFU/mL. Chamber slides, cover glass, mica, and falcon tubes were used in which form the biofilm for microscopic observations. To produce mature biofilms, the BHI broth was replaced every 48 h for 21 days. The samples were incubated at 37 °C in ambient air21.In this study, the standard 2 solution, 1.65 g of powder was dissolved in 1000 mL of deionized water (i.e. 21.6 mM) and stirred for 3 h. After overnight incubation at room temperature, the solution was centrifuged at 8000 RPM for 10 min. The supernatant was used as an 100% Ca(OH)2 solution. To prepare pure Ca2+ ions, a part of aforementioned solution was neutralized using glacial acetic acid at pH 7. To produce the OH– ions, 100% Ca(OH)2 solution was chelated using ethylenediaminetetraacetic acid (EDTA). Thereafter, the Ca2+ content was quantified using Eriochrome Black T indicator solution  to ensure all of the Ca2+ had chelated (https://chem.libretexts.org/@go/page/75718). For convenience, the groups were denoted as Ca(OH)2, Ca2+, and OH-.To prepare the Ca(OH)After 21 days of incubation and formation of a mature biofilm, it was exposed to one of three solutions for 7 days to simulate clinical conditions. All experiments were carried out in triplicate.E. faecalis biofilm under the different solutions, they were examined with scanning electron microscopy (SEM). For this purpose, a 1 cm × 1 cm cover glass was embedded in 6-well plates , and 5 mL of freshly inoculated E. faecalis BHI media was poured into each well.To assess the surface morphology and composition of the 6 CFU/mL. The biofilm culturing and treatments were the same as for the biofilm assay. Thereafter, the cover glass was placed into 4% glutaraldehyde  to fixate the biofilms. After 1 h, the samples were washed with double-distilled water (DDW) and air dried. They then were mounted on a gold-coated SEM stub and photographed using a MIRA3 scanning electron microscope 22. The accumulation of bacteria and biofilm formation under different conditions were investigated.The cumulative number of inoculated bacteria was adjusted to 10CLSM was used to examine the reconstruction of three-dimensional structures in the biofilm. The biofilm was grown on a chamber slides  and exposed to the solutions as described in the biofilm assay. The slides were stained with fluorescein diacetate (FDA) green fluorescent dye  for live bacteria and with propidium iodide (PI) red fluorescent dye  for dead bacteria. The PI was not able to cross the membrane of living cells but can stain the DNA of dead bacteria or eDNA in EPS.www.comstat.dk)23.After staining, the samples were incubated at room temperature for 20 min in a dark place. Then they were gently rinsed with phosphate buffer saline (PBS) to remove non-adherent bacterial cells. For imaging, an inverted microscope  was used at × 10 magnification. The excitation wavelength used for the FDA and PI were 488 nm and 532 nm, respectively, and the emission wavelengths were 500–550 and 580–700 nm, respectively. Four to six regions were randomly selected for each biofilm in each group. The biofilm parameters of biomass, mean thickness, maximum thickness, average colony size, average colony volume, and surface-to-volume ratio were evaluated. The three-dimensional images were analysed using Comstat software  that was combined with an inverted optical microscope  and a Hamamatsu CCD camera, in tapping mode (Mikromasch NSC15 tips) with a scanning speed of 1 line/s. Finally, the images were analysed with JPK IP 16.The roughness value of 6.To ensure that changes in the parameters measured by Comstat in the CLSM experiment were not solely due to the changes in the number of bacteria, the biofilms was stained with Alcian blue . The matrix and acidic extracellular polysaccharides of the biofilm became visible. The biofilm was examined with a light microscope  at a × 400 magnification24. Biofilm was grown in a 50 mL falcon tube and brought into contact with the solutions, as described previously. The samples were dried in an incubator , their weight was measured, and the falcon walls were then carefully scraped using a spatula to remove the biofilm. Next, 2.5 mL of DDW was added to each sample. The samples were then sonicated eight times in 30-s cycles at 60% power with the a sonicator  to release the polymers from the bacterial cells.The amount of free polysaccharides in the biofilms was quantified using the Smith–Gilkerson methodThese samples then were centrifuged at 5000 g for 30 min at 12 °C to separate the supernatants from the bacterial cells. A volume of 1.5 mL of the clear supernatant was removed from the suspension and centrifuged again for 10 min at 12,000 g. Finally, 1 mL of the final clear supernatant was used to determine the polysaccharides concentration. The polysaccharide compounds were hydrolyzed using 0.5 M of hydrochloric acid . Then, 3-methyl-2-benzothiazolinone hydrazine (MBTH) , a chromophore, formed a colour complex and the optical density was measured at 650 nm.E. faecalis was grown to form biofilms and then was put into contact with the solutions as described previously. The samples were dried in a desiccator and the falcons were weighted. Then, 1 mL of nitric acid  and 1 mL of hydrogen peroxide  were added to each sample. Finally, the volume of each sample was increased to 10 mL with DDW and Ca2+ ion concentration was measured by inductively coupled plasma-optical emission spectroscopy  at a wavelength of 397/847 nm25.Inductively coupled plasma was used to measure the amount of calcium in the biofilm matrices. In four falcon tubes, The biofilm was prepared in 96-well flat-bottom plates for each group and for positive (non-treated) and negative (vancomycin-treated) controls. After 21 days of biofilm formation, the plates were gently rinsed twice with PBS to remove the unattached bacteria. To enumerate the viable cells, the RNA of each well was harvested using TRI reagent  following manufacturer protocols with a few modifications. Briefly, 100 µL of lysozyme (20 mg/mL) was added to each well for 10 min to reduce the EPS. Then, RNase free DNase (ThermoFisher Scientific) was poured into each well to digest the eDNA. Finally, 100 µL of TRI reagent was used in each well followed by RNA extraction. The upper phase containing RNA was transferred to a RNeasy column extraction kit  to prepare the pure RNA. DNase treatment was performed to eliminate probable DNA contamination.26 on a C1000 Touch™ Thermal Cycler . The RNA content was translated into the bacterial content in accordance with a standard curve and was reported as colony forming units (CFU) per mL. The experiment carried out in triplicate.The purity and quantity of RNA were detected using NanoDrop 1000 spectrophotometer . The cDNA was constructed using a commercial kit . Quantitative real-time polymerase chain (qPCR) reaction was done to determine the absolute number of viable bacteria in the biofilm using SYBR reagent  and specific primersP < 0.05 indicated statistical significance. The SEM results were descriptively assessed and reported.The normality of the continuous variables was assessed using the Shapiro–Wilk test. In order to check the parameters of CLSM images and polysaccharide quantity, one-way ANOVA and Bonferroni post-hoc tests were used, respectively. The roughness values in the AFM and the calcium content in the biofilms were evaluated by applying the Kruskal–Wallis and Mann–Whitney tests. All data analyses were performed using Stata version 12.0 , and The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information.


Mentoring initiatives for undergraduate and graduate women of color (WOC) have provided peer-to-peer relationships and counterspaces to disrupt the inequitable treatment of students in STEM higher education (HE). This literature synthesis explores intersectionality in STEM HE mentoring through pursuing the following research questions: (1) What impact do the social contexts of WOC have on their mentoring experiences in STEM HE? (2) What role does intersectionality play in the structural organization of WOC mentoring models in STEM HE? (3) How has intersectionality shaped the life experiences of WOC mentors and mentees? and (4) How can mentoring models utilize intersectionality to incorporate the experiences of WOC in STEM HE? Thematic findings from literature related to STEM HE mentoring suggest a reinforcement of deficit mentoring models (Fix the URM), a symbolic application of intersectionality (branding gender–race), and a lack of paradigmatic shifts . Our specific recommendations presented in this article challenge the epistemic oppression and epistemic violence that current STEM HE mentoring models operationalize.The online version contains supplementary material available at 10.1186/s40594-022-00367-7. Our article responds to the call of Ong et al.  out of all bachelor’s degree earned over time for first-time full-time students at a public institution . Given this complexity, we introduce each construct to describe what it signifies; how it is generally used; and how we apply it to this work.What: An intersectional frame considers the social context, which is where, when, and with whom knowledge production occurs. Thus, a social context is more than the physical locale in which individuals find themselves. It concerns how an individual interprets the context and how the context influences an individual’s intersecting identity markers . How: Attention paid to the social context can highlight the perspectives, histories, politics, and communities that influence individual and group identities. Through having a heightened sensitivity to such constructs, we see that there are many historical, interconnected layers contributing to the present-day situation of marginalized groups , and structural domains . How: By building knowledge and socially just actions, social inequality can be both interrogated and transformed. An intersectional framework spotlights that inequality is not “natural” or “inevitable”, despite its being normalized . This move toward critically analyzing intra- and intergroup differences is one step toward socially just action.What: Complexity refers to social inequality, power, relationality, and social contexts as intertwined, fluid, and malleable. While these constructs operate on many levels simultaneously, one cannot be understood without the other. How: When used together, these constructs What: Social justice is action oriented. Through an intersectional lens, social justice is a tool that can be used to effectively confront and transform inequity. How: When intersectionality is used to help students learn how to deconstruct inequitable HE systems, the needs and experiences of WOC can be appropriately centered. The intersectional frame positions social justice as a means through which systemic transformation can occur. Transformation requires more than adding new programs or efforts to a flawed system. Instead, as an intersectional core construct, social justice requires careful exploration of how the system remains unjust; who benefits and remains disenfranchised by the injustice—such as WOC; and how and where subordinated groups have transformed and could transform the system towards more equitable ends. Our application: Although some mentoring models provide a mix of traditional and culturally based strategies for addressing underrepresentation . Additionally, keyword searches included variations of “African American or Black”, “Native American and Indigenous”, “Hispanic and Latina/x”, “Asian American and Asian”, “girls or women”, “girls of color”, “women of color”, and “underrepresented”. This was purposeful because of our understanding from intersectionality that relationality encourages both/and approaches. Specifically, mentors, mentees, and mentoring are all affected by the situated meanings of race, ethnicity, and gender. Admittedly, there are intersections among groups. Exploring African American women who are differently able-bodied or Latina women who self-identify as members of the LGBTQIA + communities, for example, better reflects the heterogeneity beyond race, ethnicity, and gender. We acknowledge that WOC possess and navigate multiple identities in HE and beyond. However, we focus on their race and gender only as entry points to the process of using an intersectional framework.Our review of the literature on STEM mentoring for WOC in HE was done in the following three phases: (1) searching for peer-reviewed empirical articles, (2) abstract screening, and (3) full article screening. We examined the state of the literature from three databases: Academic Search Premier, ERIC, and PsycInfo. We situated the literature search in the geographical area of the United States. Although the U.S.-based articles were from geographically diverse regions, most came from Southwestern states. The selection of keywords was intentional. We selected STEM, mentor, and intersectionality as keywords to explore the strategies used to address the ongoing underrepresentation by gender and race in STEM education. Typical models follow standardized approaches  based on dominant norms to mentor individuals from multicultural and linguistic communities. Such shortsighted techniques inform the research questions to examine for the influence of variables  on STEM mentorship for WOC. For instance, the intersectional construct of context is important for revealing specific saturated sites of violence. Consequently, we used the keywords “higher education” in order to explore this space. The keyword “STEM” in this study is limited in scope and reflects the mainstream reference to the four categories of science, technology, engineering, and mathematics to increase the likelihood of finding related articles. We also used the word “mentor*” (with the asterisk) to allow for verb forms of “mentor” to be included in our search  than White women, they tend to be overrepresented in STEM disciplines  to differentiate article totals and calculate percentages. This grouping revealed 22,306 articles tagged with some combination of “STEM” + additional keywords; 13,086 tagged with some combination of “mentor*” + additional keywords; 107 articles tagged with some combination of “intersectionality” + additional keywords. From the 35,499 total articles, we narrowed our selection to articles with “STEM”, “underrepresented”, “higher education”, and “mentor*” tagged or mentioned in the abstracts because we predicted that combination of keywords would give us a selection of articles most relevant for this study. That search resulted in 99 total articles among the three databases, 16 of which overlapped and 40 of which were eliminated because they were deemed not relevant through a close reading of their abstracts and paper materials.If the abstract provided information based on four criteria, we moved them to full article review: (1) empirical studies whereas conceptual articles without results were excluded; (2) mentoring is centered as a significant HE strategy, meaning details the application and role of mentoring or the mentoring framework; (3) clear articulation of WOC or underrepresented women as mentees disaggregated based on race and/or ethnicity; and (4) the focus of the mentoring program was for undergraduate or graduate STEM students; mentoring faculty to be mentors was not our intent. Also, we mined the references of selected articles to identify additional sources for the literature review. If articles upheld the same standards of the target population  and were U.S.-based studies, they were included. For instance, pilot studies from a multiple-year grant  that focused on the influence of mentoring underrepresented groups and reported outcomes were included.The timeline for our literature review was between 1990 and 2019; we conducted electronic searches of peer-reviewed articles in relevant journals. We began our exploration in 1990 since this was when educational efforts emphasized improving engagement for students of color. Work about culturally relevant teaching rightly assumed centerstage in the 1990s , near-peer mentor , and peer mentoring .The second iteration of coding included examples reflective of the motivation for mentoring. We identified the following five reasons for inclusion by action-oriented agendas: (1) intervention—the creation of STEM programs for minoritized students to improve their success rate; 2) broaden participation—providing the necessary resources to overcome hindrances to student engagement in STEM education; (3) bridge—the formation of an inclusive learning environment in STEM education; (4) cultural responsiveness—a student-centered approach to learning that acknowledged other ways of knowing and being in STEM education; and (5) equity-seeking—dismantling perceived barriers to underrepresented minorities’ educational and career opportunities in STEM education  in STEM HE and how the research contextualizes the needs of that population. We also examined the strategies applied to implementing mentoring specific to WOC in STEM HE and the outcomes of those efforts. Where mentoring in STEM HE did not disaggregate by race or gender but included the underrepresented as a criterion, we looked for evidence of diverse methods to accommodate the demographic diversity.Our exploration of how mentoring models for undergraduate and graduate WOC in STEM applying core constructs of intersectionality revealed surprises and confirmed our suspicions. Altogether, the referenced authors in this next section present empirical data on the results of mentoring WOC. The majority of reviewed work describes mentoring models to cope with social disparities  that define the culture of STEM HE  to interpret barriers, such as the lack of STEM identity development for WOC, encourages decontextualized strategies. Our research noted STEM mentoring models with no identified empirical research on specific STEM disciplines. Instead, research suggests that one mentoring model can be applied to all STEM ranks . Since Asian American women tend to be overrepresented in certain sciences  did not appear in the study; using this critical framework might have led to a different outcome. This example of colorblind and gender-blind mentoring problematized the role of faculty mentoring’s impact on encouraging the representation of racial and gender diversity in STEM HE. Because faculty mentors in STEM higher education influence URM actions, research elevates mentoring’s status as an effective strategy. In some cases, mentors advance potential outcomes to seek resolutions that resist inequities  while excluding all others  opposes relationality as a significant pillar of intersectionality. Additionally, the distinctive experiences of WOC in relation to their social identities are misinterpreted and mistreated  reveal that despite efforts to “upskill” WOC in STEM, the HE STEM mentoring models consist of saturated sites of violence—that is, contexts in which the knowledge of HE remains uncontested and perpetuates dominant narratives of WOC. Without interrogating why HE mentoring models include the above deficit strategies, the “acceptable knowledge [of STEM HE mentoring models] reflects and protects the dominant group’s status of power and privilege, otherwise known as Importantly, we note how intersectionality has been positioned on behalf of WOC in STEM to problematize the normative HE cultures. Seminal work has explored the marginalization of WOC and the barriers to their persistence and participation in STEM  is the establishing of mentor models that serve the needs of White, Western, upper/middle-class, able-bodied, male students and not students of color, women, differently able-bodied individuals, etc. Essentially, we found that WOC’s social contexts did not influence the design of mentor models toward more inclusive practices. In answering our second and third research questions  our literature synthesis revealed that intersectionality is conspicuously absent from mentoring models focused on WOC in STEM HE. Social context, relationality, and power are not taken into account despite the opportunity to include and validate the experiences of WOC. Consequently, this following section responds to our fourth research question .Ultimately, we argue that mentorship is incomplete if not considered through an intersectional lens. In keeping with this central argument and the findings discussed above, we offer a set of guiding principles for transformational mentoring practices. We understand that our proposed strategies may be perceived as conceptually abstract. In part, the challenge lies in delivering nuanced, intersectionality-informed one-on-one mentoring that has never been done. Such an approach is not generalizable or simple. Intersectionality is about recognizing complexity. As was referenced above, in understanding intragroup difference, which was inevitably shaped by Collins’  core conMuch like the recommendations from Boyce  that werTo engage in truly transformational mentoring practices, an authentic and genuine dialogue about the needs and concerns of WOC in STEM HE is crucial. As a first step, we suggest a needs-assessment activity  between the mentor and mentee be performed prior to an in-person meeting. Potential questions to the mentee embedded within the assessment activities are the following: What does a safe space look like in your department and how do you suggest your institution or department create one? What kinds of policies and/or procedures do you believe will assist you in your professional achievements? The same interrogatives should be posed to the mentor. Departments and institutions can sustain these needs-assessment activities by making it a requirement for both mentors and mentees.Data from these assessments can be used in mentoring to identify and highlight the barriers to the participation, retention, and success of WOC in STEM and to brainstorm potential solutions. Key to this process is the notion of relationality, in which the interconnections between social categories, subject formations, and power structures are recognized as mutually productive and reinforcing. While the idea of establishing a dialogue about the needs and concerns of WOC may seem overly simplistic, a thorough reading of the existing research on mentoring proves this step is too often overlooked or considered an afterthought; it is an evaluation of the mentoring performance after the fact. Making dialogue the first guiding principle, or the first step toward transformational mentoring practices that is maintained throughout the mentoring relationship, ensures its inclusion as a key facet of mentoring design and amplifies the voices of WOC in a direct way.One of the most important factors related to the persistence and retention of WOC in STEM HE spaces is the development of a sense of belonging or self-efficacy  of the four guiding principles is centering transformation, or the practice of mentoring for social transformation. Socially transformative mentoring works through centering social justice concerns and equitable patterns of relating in every part of the mentoring process. No part of STEM learning occurs in a vacuum, and pretending that cultural norms and social values do not influence STEM learning and mentoring is akin to “sticking one’s head in the sand”. To solely focus on the academic or professional development elements necessary to advance students through STEM HE or to emphasize the socialization or assimilation of WOC students into dominant STEM norms is to leave aspects of the lived experiences WOC unrecognized and unrealized. It is a failure to engage mentoring with an intersectional frame that implicitly devalues the WOC who participate in mentoring relationships. Rather, conversations between mentors and mentees should include how mentors understand the power hierarchy in their specific context; ways they have navigated it along various lines of identity ; and potential strategies to transform the system using an equity lens. As mentors bring attention to unequal power dynamics, reflect on their sustainability, and co-construct strategies to upend policies and procedures, they will create the “paradigm shift” that Collins  encouragWhile the principles outlined above have started the process of centering social justice concerns and establishing equitable relations, we also offer a list of questions for mentors and mentees to consider in moving toward transformational mentoring practices  need a platform to address hierarchies. Space needs to be provided in which WOC are empowered to challenge and transform interconnected power structures. Is this a one-size-fits-all approach? To some extent, it is; but this approach is also one that is open and non-constraining to individual needs.If the tenor of our current times has taught us anything, it is that established practices and ways of being in the world are untenable. Inequities that make up the foundation of the U.S. education system have been exposed by the COVID-19 pandemic, from disparate access to reliable internet and distance learning technologies to financial insecurity caused by the lack of access to on-campus jobs and housing Patel, . The onlNotable study limitations within our search criteria highlight a lack of specific disciplinary categories  definition of STEM along with medicine), including disciplinary categories of science, technology, engineering, math, astronomy, chemistry, computer science, geoscience, life science, physics, psychology, social science, STEM education, and medicine. In future studies, we recommend adding that level of specificity by disciplines rather than the general reference to STEM as a keyword.How does the resistance knowledge project specifically relate to WOC and why?Until we view WOC as distinct from the socially constructed, and often stereotypical, version that influences decisions on what works for that group, WOC will continue to lag in enrollment numbers and graduation rates in STEM HE. The mistakes of past efforts to attain social equity for WOC follow the same template of: (1) choosing to minimize their unique experiences with prescribed protocols for research that operate from dominant social norms and (2) discussions that present mentoring as a neutral exercise that is transparent and liberating to the mentee. WOC’s narratives as underrepresented in STEM HE contributes to the impression that they, as a group, have the same needs or will positively respond to mentoring as an intervention. Multiple examples in the literature confirm the role of intersectionality, if considered, to serve to justify the application of nontraditional mentoring strategies, therefore making an intersectionality lens less analytical and more descriptive. Moreover, where intersectionality as an analytical lens could expose the flaws in mentor models designed for women and people of color, the omission of intersectionality in the mentoring framework furthers a reliance on dominant norms. Intersectionality has a place in mentoring, especially for contextualizing the significance of diversity when examining the impact on WOC with different lived experiences. Because some studies categorize mentoring for WOC as a resistance knowledge project, this means “the guiding question is less whether to resist prevailing power arrangements and more what forms such resistance might take” Collins, , p. 116.Additional file 1. NCES/IPEDS—graduation/completion rates over time.


A remodeling in calcium homeostasis and the protein kinase AKT signaling pathway often promotes tumorigenic traits in cancer cells. Changes in calcium signaling can be mediated through altered expression or activity of calcium channels and pumps, which constitute a class of targetable therapeutic targets. Currently, the interplay between the two signaling pathways in breast cancer cells is unclear. A better understanding of the association between calcium and AKT signaling, and the molecular players involved may identify novel therapeutic strategies for breast cancers with abnormal AKT signaling. Using fluorescence calcium imaging and gene silencing/knockout techniques, we showed that increased AKT activation results in increased calcium entry, and that this is mediated through ORAI1 calcium channels. Future studies exploring therapeutic strategies to target PTEN-deficient or hyperactivated AKT cancers should consider this novel correlation between AKT activation and ORAI1-mediated calcium influx.Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling. Breast cancer is the most common cancer in women and second leading cause of cancer-related deaths . Breast 2+) homeostasis is also often dysregulated in cancer cells. Changes in cytosolic Ca2+ control various cellular processes including apoptosis, proliferation, gene transcription and cellular energy production  CYT) changes, Ca2+ imaging was performed 96 h post-transfection. Briefly, media was removed, and cells were washed twice with physiological salt solution (PSS) containing nominal Ca2+ (no added CaCl2) before a 15 min incubation with HEPES-buffered PSS nominal at room temperature. Reagents containing BAPTA (100 µM) (Invitrogen) and the Ca2+-mobilizing agonists ATP (Merck-Sigma) or trypsin (Merck-Sigma) were then added. For assessment of unstimulated Ca2+ influx, 1.8 mM CaCl2 was added. To assess the effect of AKT activation on Ca2+ influx, cells were pre-incubated with SC79 (Merck-Sigma) for 1 h prior to imaging. To assess SOCE, cells were first pre-incubated in PSS nominal for 15 min, followed by consequent additions of BAPTA (100 µM) and then cyclopiazonic acid  (Merck-Sigma) to deplete Ca2+ stores. DMSO (0.1%) was included as a control for CPA. CaCl2 (1.8 mM) was then added to the cells to allow SOCE. Changes in fluorescence (F) were measured at 470–495 nm excitation and 515–575 nm emission wavelengths. Fluorescence normalized to baseline fluorescence (F/F0) values were exported from the ScreenWorks (Molecular Devices) software into Microsoft Excel. Relative [Ca2+]CYT changes were represented as normalized fluorescence change over time calculated using the formula: F/F0–1 (ΔF/F0).Cytosolic Ca2+ imaging experiments, each n value represents the mean of three technical replicates, i.e., three individual wells. p values of less than 0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism 9.0 . Statistical tests used for individual experiments were described in the corresponding figure legends. Unless otherwise specified, all data shown in figures are derived from three biological replicates (n = 3). For all Ca2+ signaling in basal breast cancer cells, we first characterized the effect of silencing PTEN, the upstream AKT regulator, on Ca2+ signaling in GCaMP6m-MDA-MB-231 cells. We confirmed that PTEN mRNA levels were significantly reduced with PTEN siRNA (siPTEN)  addition compared to non-target siRNA control (siNT). PTEN silencing also reduced Ca2+ release as a result of trypsin (100 nM) addition   A. To asslar Ca2+ . As showaddition C. These f siPTEN . Indeed, of IP3R ,26,29,3068 cells .2+ signaling changes observed with PTEN silencing are indeed associated with increased AKT activation. We first confirmed that PTEN silencing promotes AKT activation in GCaMP6m-MDA-MB-231 cells. PTEN silencing resulted in a time-dependent decrease in PTEN protein levels, and this was associated with a significant increase in AKT phosphorylation . PTEchannels ,34, we u+ influx . As showeoblasts ,36. Howemembrane ,38. Furt2+ signaling, we assessed if specifically silencing the AKT1 or AKT2 isoforms had any effect on SC79-mediated Ca2+ influx. We first confirmed that AKT1 and AKT2 silencing reduced the protein expression of AKT1 and AKT2, respectively  cells (2+ increases as a result of CPA treatment (Peak 1) compared to WT cells. This reflects lower levels of stored Ca2+ in the endoplasmic reticulum, which is expected since the store-refilling mechanism is impaired in these cells [2+ responses by treating these cells with different concentrations of ATP. Both WT  were observed at 10 µM ATP and above  cells A previouO) cells B. As expse cells . We furt2+ influx in ORAI1KO cells. As shown in 2+ influx in WT-GCaMP6m-MDA-MB-231 cells. However, in ORAI1KO cells, Ca2+ influx induced by SC79 was completely abolished  both share similar structures and function and are both substrates of the upstream kinase PDK1. Increased SGK1 expression enhances ORAI1-dependent Ca2+ influx via preventing NEDD,3,4-mediated degradation [2+ signaling.Our findings that PTEN silencing and AKT activation promoted Ca release ,18. It wrylation . This re complex . Increas complex , where A complex . Alternaradation  or increradation . It is pradation . Further2+ influx was done using a high throughput assay with the genetically encoded GCaMP6m sensor. Future studies could further characterize this initial modest Ca2+ phenotype as a result of increased AKT activation via single cell imaging to identify changes in calcium oscillations. These studies could incorporate the use of newer, genetically encoded ratiometric Ca2+ sensors such as mScarlet [2+. Secondly, the link between increased AKT activity and ORAI1-mediated Ca2+ influx should be investigated in more cell lines. Previous studies in our laboratory have already shown that MDA-MB-468 cells, which are PTEN-deficient and have increased AKT activity also exhibit increased basal Ca2+ influx [2+ influx should optimize for AKT2 isoform-specific knockout such as the use of CRISPR/Cas9 gene knockout.This study has some limitations. Firstly, our initial investigation into the link between increased AKT activity and ORAI1-mediated CamScarlet  or ratio+ influx . Future + influx . Finally2+ influx in MDA-MB-231 breast cancer cells. This Ca2+ influx is regulated partially by AKT2 and is mediated through ORAI1 calcium channels. Future studies assessing the mechanism by which increased AKT activation enhances ORAI1-mediated Ca2+ influx will further define the role of ORAI1 as a therapeutic target in cancers with AKT hyperactivation.Our study showed that both PTEN knockdown and AKT pharmacological activation increased Ca