Purpose: Spinal cord injury (SCI) has a damaging impact on patients, amid being a worldwide problem with no effective treatment. Herein, we reported a method for functional therapy of SCI in rats, wherein we combined thermo-sensitive hydrogel with Sonic Hedgehog (SHH) expressed in rat bone-marrow derived mesenchymal stem cells (RMSCs).Methods: Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from Sprague-Dawley (SD) female rats. The SHH was optimized and transferred into RMSCs via cationic liposomes, while thermo-sensitive hydrogel was reformed with hyaluronate (HA) and Pluronic F127. Then, a rat model with SCI was established accordingly by male SD rats and randomized into sham, model, RMSCs with hydrogel and SHH-RMSCs with hydrogel. The evaluation of SCI repair based on Basso, Beattie Bresnahanlocomotor rating scale  and inclined plate score. Immunofluorescence, immunohistochemistry and hematoxylin-eosin were utilized to explore the expression of protein  and histopathology.Results: It was demonstrated that transfection of SHH with cationic liposomes exhibited more effect in RMSCs than lipofectamine 2000. As shown in SEM, 3.5% HA-F127 demonstrated porous structure. In the MTT and dead/live assay, 3.5% HA-F127 showed good biocompatibility for RMSCs. Both RMSCs and SHH-RMSCs groups could significantly promote BBB and inclined plate scores (p < 0.01) compared with the model. Furthermore, the SHH-RMSC group was significantly improved than RMSC with the expression of related proteins, where NF200, MBP, and GAP43 were principally enhanced with the GFAP expression being virtually down-regulated.Conclusion: All in all, the results suggested that transplantation of RMSCs with SHH could improve the function of SCI and promote nerve regeneration. The pathological process of traumatic injuries such as spinal cord injury (SCI) is complicated coupled with high disability and mortality. SCI often leads to sensory and motor deficits, as well as acutely causing bradyarrhythmia, vasodilation and autonomic dysreflexia, ectopic beats, hypotension, and neurogenic shock . AdditioCurrent treatment options for SCI include the use of high-dose sodium succinate methylprednisolone and early surgery, which aim to prevent further injury. Because the poor neurologic recovery affecting the patients daily lives, it is very worthwhile to find a more effective treatment method for spinal cord injury . TherefoAs mesenchymal stem cells, marrow stromal cells (MSCs) have multiple abilities to differentiate into triploids, such as bone , cartilaIn recent years, genetic engineering technology has become more and more prosperous and has been applied in the treatment of clinical diseases. At present, vectors of gene therapy are mainly allocated into two classes, namely non-viral and viral vectors. In particular, viral vectors have the advantages of high transfection and transformation rates, but clinical applications have been limited owing to their small capacity, difficult preparation, high immunogenicity, and other potential safety issues. Comparably, the non-viral vectors have the advantages of natural degradation, simple preparation, low toxicity and repeatable transfection, especially low immunogenicity . Common Hydrogels can form polymer chains that cross physically or chemically the forming 3D-porous structure (3D)-porous structure . In viewSonic hedgehog (SHH) is an essential signal in the formation of the neuronal pattern, regulation of cell fate, axonal guidance, proliferation, survival and differentiation during the development of the central nervous system (CNS). Gli is the downstream signal molecule of SHH, which has been shown to promote the protective effect of SCI recovery, amid the potential to maintain biological activity at the injury site for the short term. Presently, available literature suggests that signal of SHH/Gli1 in activated astrocytes plays a crucial role in blood-SC barrier permeability and recovery of locomotor function after SCI . FurtherIn this study, cationic liposomes were formed by the thin film dispersion method, while RMSCs were transfected with SHH gene that was delivered by cationic liposomes. The expression of SHH gene in RMSCs was optimized and combined with thermo-sensitive hydrogel scaffold, which may provide suitable scaffold materials for repairing SCI in rats, wherein the repair effect was investigated.Merck KGaA  supplied 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), while SinoPharm Chemical Reagent Co., Ltd. Provide chloroform and ethanol. Chromatographically grade methanol was supplied by Hanbon Sci. And Tech. . Production of double distilled (DD) water was accomplished with a Millipore Water Purifying System . Other analytically grade chemicals were obtained commercially. We bought MSCgo™ Rapid Osteogenic Differentiation Medium, fetal bovine serum (FBS), DMEM medium and D-hanks solution from Biological Industries (Israel). Anti-NF200 , anti-GAP43 , anti-GFAP , anti-MBP , anti-CD44-APC-conjugate (#80813), mAb IgG2b Isotype Control (APC Conjugate) (#34828) and alexa Fluor 488 goat anti-rabbit IgG  were purchased from Cell Signaling Technology Co., Ltd. Anti-CD29-APC-conjugate (17-0291-82), anti-vimentin-APC-conjugate (MA5-28601), TGF-beta1, oil red O, HA and F127 were obtained from Sigma Co., Ltd. All the antibodies were obtained from Cell Signaling Technology, Inc. The DsDNA fluorimetry test kits were obtained by Jiaxing Yakangbo Medical Examination Co., Ltd.The Center for Laboratory Animal Research at Jiangsu University  supplied the male SD rats  for establishment of SCI model and the female SD rats  for isolating RMSCs. We maintained the rats for 3 days at room temperature, 40%–70% relative humidity and 12/12 cycle of ligh/dark with free water and food access. The animal study was reviewed and approved by the board of institutional review at the Institute of Jiangsu University.2, and 37°C. After 24 h, the non-adherent cells were discarded after medium refreshing. Then, the culture medium was refreshed every 3 days. After RMSCs had achieved 80% confluence, we digested and passaged the cells at a density of 2.0 × 105 cells per ml for subculturing. The cells morphology and growth were observed under a microscope.RMSCs were harvested from adult male SD rats (6–8 weeks). In brief, SD rats were applied for intraperitoneal anaesthesia . And femurs and tibias were cut off aseptically, removed the skin and muscles. A hole was then created in the knee joint end of each bone with a 26-gauge needle, and marrow was flushed with D-hanks solution containing 2% FBS. The solution was centrifuged for 5 min at 800 rpm. The supernatant was aspirated gently, and pallet was resuspended in DMEM comprising streptomycin (100 μg/mL) penicillin (100 U/mL), and 10% FBS. Cells were seeded in tissue culture flask and cultured in incubator with 5% CO4 cells per mL) in a 6-well plate with MSCgo™ Rapid Osteogenic Differentiation Medium was accomplished before incubation under 5% CO2 at 37°C, while medium renewal was carried out once in 3 days. Later, we fixed the cells after 10 days with ethanol (95%) for 10 min, before addition of Alizarin Red staining solution and 30 min of incubation at 37°C. After twice washing, the stained cells were detected under optical microscope.Seeding of RMSCs (4.5 × 104 cells per mL) in a 6-well plate was carried out for 10 days with DMEM, which contained 3-isobutyl-1-methyl-xanthine , insulin (5 μg/mL), and dexamethasone (1 µM) for inducing adipogenic differentiation, while the medium was renewed once in 3 days. The cells were then stained with oil red O for 15 min, rinsed with 85% propylene glycol for 3 min, before washing in DD water for 3 times and recording with optical microscope.Seeding of RMSCs (4.5 × 104 cells per mL) in a 6-well plate was carried out for 2 weeks in DMEM medium that contained insulin (6.25 mmol/L), transforming growth factor β1 , and 10% FBS (v/v), as well as subsequent cell inducement and assays with Type II collagen-FITC. Meanwhile.Seeding of RMSCs (4.5 × 10Detection of cell surface markers (CD29 and CD44) and intracellular markers (vimentin) was performed by flow cytometry antibody staining as following procedure. RMSCs were incubated with APC-conjugated mAb, including anti-CD29, anti-CD44, anti-vimentin. Data were acquired and analyzed on a BD Accuri TM C6 Plus with FlowJo V10 software.4 cells per mL) were inoculated in a 24-well plate. Meanwhile, the cells were counted every day for a week in triplicate, before we plotted growth curve with abscissa representing culture time and ordinate denoting the average number of cells.RMSCs were digested into single-cell suspension and 5 min of centrifugation at 1000 rpm. The precipitated cells was resuspended and counted, while the adjusted cells (1 × 10The preparation of cationic liposomes was carried out using the thin film dispersion method  (28). Thvia laser light scattering Zetasizer  at temperature of 25°C and angle of 90° by diluting with DD water  as previously described . Morin vitro. The experiment was randomized into three groups, viz., experimental (cationic liposome transfected SHH), control (cationic liposome) and positive control groups . In experimental group, different concentrations of SHH plasmid were mixed with cationic liposome solution (without FBS) at room temperature (RT) and incubated for 20 min. This mixture was added to RMSCs and cultured in the incubator. After 4 h, we replaced the culture medium with fresh DMEM. Afterwards, cultivation of the cells was carried out for 8 days prior to testing.Plasmids carrying the SHH gene were constructed by Sangon Biotech, shanghai, China. The third generation RMSCs was transfected with SHH plasmids 4, 2 × 104, 4 × 104 and 8 × 104 cells per well) were explored. The procedure was in accordance with the above method (Section 2.6.1). The SHH was detected by ELISA and Western blot (WB) to optimize the transfection condition, namely, the concentration of plasmids and the number of cells.Different concentrations of SHH plasmids  were mixed with cationic liposomes, while different RMSCs numbers  of the growth curve.RMSCs  were seeded in a 24 well plate (500 µl per well) at 2 × 10Amounts of Pluronic F127 (0.6 g) and sodium hyaluronate  were weighed and mixed at 4°C. Next, the mixed HA-F127 solution was placed under a 37°C water bath to gelatinize, while the transaction time was recorded, respectively.via a Philips XL-30E scanning electron microscope.The HA-F127 hydrogel was lyophilized by the freeze dryer. The lyophilized material was then placed on conductive tape and metal sprayed for 60 s to observe the morphology For competition swelling, we placed the HA-F127 hydrogel in PBS (pH 7.4) for 24 h at room temperature. Using a universal material testing machine, we subjected the gel to a mechanical compression test at room temperature, wherein the compression speed was 2 mm/min. Each experimental group was repeated in triplicate and the average value was recorded.The HA-F127 hydrogel was immersed in PBS (pH7.4) and placed in a 37°C thermostat air bath to completely expand. After 24 h, the samples were taken out, before we used filter paper to absorb water on the surface. Later, we accurately weighed the swelled hydrogels as W1, while the samples were lyophilized totally and weighed as W2. The calculation of the swelling ratio (SR) of the HA-F127 hydrogel was performed using formula , while ta. The hydrogel was placed in a 0.02% (w/v) sodium azide solution in PBS before shaking at 37°C, while the hydrogel was weighed and recorded as Wb at different times. The remaining mass (RM %) of the hydrogel degradation over time was calculated by the following formula (The HA-F127 hydrogel was added to PBS (pH7.4) before swelling in a constant temperature shaking box at 37°C for 24 h. Afterwards, the hydrogel was taken out, wiped with filter paper to remove residual surface moisture, before weighing the hydrogel mass as W formula . All samThe HA-F127 hydrogel containing SHH plasmid was collected in a tube comprising PBS  before placement in a shaker operating at constant temperature of 37°C. Aliquot (300 μl) of release solution was withdrawn at 1, 2, 3, 4, 5, 6, 7, and 8 days, prior to supplementation with equal amount of fresh PBS (pH = 7.4) in the tube. Ds-DNA kit was used to determine the concentration of SHH gene, while the cumulative release rate was calculated.The preparation of a solution of 3-  -2,5-diphenyl-tetrazolium bromide (MTT) was carried out as follows: 250 mg of MTT were dissolved in 50 mL of PBS in dark conditions and filtered with a 0.22 μm filter before storage at 4°C. Next, HA-F127 solution was prepared at 3.5% (w/v) with DMEM as stock solution. All the RMSCs were divided into 6 groups and cultivated with different concentrations of HA-F127 solution, including culture and diluted stock solution of HA-F127 , while each group was examined in quintuple. Then, the proliferation of different groups was calculated with MTT method. Briefly, 50 µl MTT solution were added into each well prior to 4 h incubation. Next, we discarded the supernatant, before addition of DMSO (150 µl). The samples were mixed, prior to measuring the absorbance value of each group at 490 nm with the enzyme-linked immunosorbent monitor.viz., 24, 72, and 120 h according to the protocol. We washed the cells with PBS (pH 7.4) prior to observation and recording under florescent microscope. The number of living and dead cells was recorded, and the ratio of dead to live cells was calculated with ImageJ.RMSCs were cultured in 3.5% HA-F127 and detected with Live/dead kit  at different times, via intraperitoneal injection of 10% (w/v) chloral hydrate , and tying to an animal stereotaxic instrument, before hair trimming and disinfection of the skin. Later, laminectomy at the level of the T9−T11 vertebral column was performed to expose the spinal cord. Complete spinal cord transection at the T10 level was performed using a microscopic scissor after lifting the spinal cord with a self-made hook. The hook was passed through the gap to ensure that there were no residual fibers on the bottom and lateral sides of the canal. After the hemostasis, cells were added dropwise in situ. Finally, the skin and other tissues were sutured, while the gentamicin was injected for 4 days.Rat SCI models were constructed as an existing study  (30). Brn = 10), model (n = 10), RMSC treated (n = 10) and SHH-RMSC treated groups (n = 40).Male SD rats (200–250 g) were randomized into four groups, namely sham operation , growth-associated protein 43 (GAP43), myelin basic protein (MBP), and neurofilament 200 (NF200) expressions at protein level in the experimental group was accomplished by immunohistochemical staining. Paraffin-embedded tissue sections were firstly deparaffinized with 100% xylene, followed by rehydration using gradient ethanol and soaked in the boiled citric acid repair solution. Then, the sections were blocked with 5% BSA. The samples were stained with primary antibodies at 4°C overnight followed by incubation with secondary antibodies and SABC reagents. After that, staining of the slices were done with DAB, lightly counterstained with hematoxylin for 0.5–2 min before recording image with microscope.After dewaxing the paraffin sections, the slices from all the groups were stained with hematoxylin eosin for 5 min before 10 min of washing with DD water. The slices were then treated with a 1% hydrochloric acid-ethanol solution for 30 before 30 s of washing with DD water. Subsequently, the slices were stained with 0.5% eosin for 1–3 min and washed with DD water for 30 s, before subsequent washing with 80 and 95% ethanol, absolute ethanol, and xylene for 1–3 min. After sealing with neutral gum, the slices were observed and imaged under microscope.Paraffin sections were deparaffinized before we soaked (lasted 5–10 min) all the slices in 3% hydrogen peroxide, and thricely washed with PBS. Next, the slices were boiled in the citric acid repair solution and washed twice with PBS. And the sections were incubated in blocking solution of 5% BSA. The samples were stained with primary antibodies at 4°C overnight followed by incubation with fluorescently labeled secondary antibodies. Washing of the slices with PBS was performed before staining with DAPI for 15 min. Finally, antifluorescence quencher was dropwise added in the dark, prior to observation under a fluorescence microscope.p < 0.05. SPSS software  was used for calculations, while. Plotting of graphs was done with Origin Software® (Origin-Lab Corporation).Presentation of data derived from the experiments was accomplished with mean and standard deviation (SD). Statistically, we evaluated differences within varied groups through ANOVA and least significant difference (LSD) test. Significance level was statistically accepted at The fresh separated RMSCs were deposited on the beneath of culturing flask, which was a round shape and bright cytoplasm with good refraction. After 24 h, the RMSCs began to attach to the bottom, while the cytoplasm extended outward. The adherent cells appeared to be spindle, triangular, fan-shaped and round with fibroblasts like morphology after 48 h  were examined with cationic liposomes to optimize the transfection condition. Through detection of SHH expression at protein level with ELISA and WB technique, we observed (p < 0.05) and 80,000 cells per well (p < 0.01). The growth curve  and varied cell numbers , while the viability in 72 h was higher than 24 h. Live/dead results indicated that the RMSCs survived and proliferated well in the 3.5% HA-F127.The RMSCs embedded in 3.5% HA-F127 were cultivated and stained for live/death assays at 24, 72, and 120 h, accordingly. As indicated in p < 0.01).The BBB scores  of all rp < 0.01).As shown in inclined plate test , angle oAs shown in Levels of GAP43, NF200, MBP, and GFAP in the four groups were detected by immunofluorescence and immunohistochemistry . GAP43, p < 0.01), and a down-regulated expression of GFAP (p < 0.01).GAP43, NF200, MBP, and GFAP protein levels in the four groups were detected by Western blotting . In the In recent years, the study of RMSCs has been of great interest in the fields of regenerative medicine and animal biotechnology alike. In our study, we combined the thermo-sensitive hydrogel (3.5% HA-F127) with SHH expressed RMSCs as via cationic liposomes and expect RMSCs to improve neurology recovery in our study. The SHH plays a crucial role in nervous development by stimulating production of neuron and oligodendrocyte from ventral cord progenitor cells, enhancement of synaptic plasticity, inhibition of astrocyte formation and guidance of axon guidance growth  gel for the treatment of SCI. Assays showed that the SHH treated RMSC group had a better effect on SCI repair, including improvement of BBB and angle scores, higher expression of GAP43, NF200, and MBP and reduced expression of GFAP. These findings demonstrated that cationic liposomes could provide a safe and efficient platform for gene delivery, while RMSC transfected with SHH could promote SCI repair and enhance nerve regeneration in SCI.


Inhibitors of poly(ADP‐ribose) polymerase (PARP) proteins potentiate antitumor activity of platinum chemotherapy. This study sought to determine the safety and tolerability of PARP inhibitor talazoparib with carboplatin and paclitaxel.2 days 1, 8, 15 of 21‐day cycles in patients with advanced solid tumors. Patients enrolled using a 3 + 3 design in two cohorts with talazoparib for 7 (schedule A) or 3 days (schedule B). After induction with 4–6 cycles of triplet therapy, patients received one of three maintenance options: (a) continuation of triplet (b) carboplatin/talazoparib, or (c) talazoparib monotherapy.We conducted a phase I study of talazoparib with carboplatin AUC5‐6 and paclitaxel 80 mg/mForty‐three patients were treated. The MTD for both schedules was talazoparib 250mcg daily. The main toxicity was myelosuppression including grade 3/4 hematologic treatment‐related adverse events (TRAEs). Dose modification occurred in 87% and 100% of patients for schedules A and B, respectively. Discontinuation due to TRAEs was 13% in schedule A and 10% in B. Ten out of 22 evaluable patients in schedule A and 5/16 patients in schedule B had a complete or partial response. Twelve out of 43 patients received ≥6 cycles of talazoparib after induction, with a 13‐month median duration of maintenance.2 on days 1, 8, 15 of 21‐day cycles. This regimen is associated with significant myelosuppression, and in addition to maximizing supportive care, modification of the chemotherapy component would be a consideration for further development of this combination with the schedules investigated in this study.We have established the recommended phase II dose of Talazoparib at 250mcg on a 3‐ or 7‐day schedule with carboplatin AUC6 and paclitaxel 80 mg/m Carboplatin, paclitaxel and talazoparib followed by talazoparib maintenance is feasible with appropriate supportive care, and warrants further investigation across solid tumor types, particularly those with DNA repair pathway alterations, and supports further development of PARP inhibitor maintenance strategies in the advanced disease setting. Here, we report the safety profile, maximum tolerated dose (MTD), and efficacy of carboplatin and paclitaxel combined with talazoparib on a 3‐ or 7‐day schedule.22.1BRCA1/2 mutation or (3) deleterious somatic BRCA1/2 mutation  reasonable expectation of response to carboplatin and paclitaxel per treating physician (2) deleterious germline 2.2This was a multicenter, open‐label phase 1 study. Patients were enrolled at three study sites under an IRB‐approved protocol (NCT02317874). Written informed consent was obtained from all participants prior to enrollment in the trial. The study was conducted in compliance with the Declaration of Helsinki. Patients enrolled in two independent cohorts with separate dosing schedules  talazoparib on a 7‐day schedule in combination with carboplatin and paclitaxel, and (2) talazoparib on a 3‐day schedule in combination with carboplatin and paclitaxel, in patients with advanced solid malignancies.A standard 3 + 3 designDescriptive summaries of demographics, incidence, and severity of toxicities and disease response are presented. Dose intensity during induction for each agent was defined as percent of full dose for the number of induction cycles received. Talazoparib plasma concentrations were summarized using descriptive statistics.2.4The MTD was defined as the highest dose level where <33% of patients experienced a DLT prior to cycle 2. Growth factor support was not permitted in cycle 1, but was permitted in subsequent cycles per the treating physician's discretion. DLTs were defined as: absolute neutrophil count (ANC) < 1000/mcL lasting >7 days, grade 3/4 febrile neutropenia, grade 4 thrombocytopenia lasting >7 days or requiring platelet transfusion, or grade 3/4 thrombocytopenia associated with clinically significant bleeding, grade ≥3 nausea, vomiting, diarrhea, or electrolyte abnormalities lasting >48 h despite maximal medical therapy, grade ≥3 fatigue lasting >7 days despite maximal medical therapy, any other grade ≥3 non‐hematologic toxicity, or delay in starting cycle 2 by ≥2 weeks due to toxicity. Patients must have completed at least day 1 through day 8 of planned cycle 1 treatment to be considered evaluable, unless a DLT occurred in this timeframe, in which case they would be considered evaluable and classified as a DLT regardless of whether day 8 treatment was completed. Patients who did not complete cycle 1 for reasons other than safety  OR experienced a ≥ 7‐day treatment delay within Cycle 1 for reasons other than safety, were also deemed inevaluable. Dose modification was required for grade 3/4 ANC lasting >7 days or with fever, grade 4 thrombocytopenia >7 days or requiring platelet transfusion. Dose modifications of paclitaxel were required for intolerable grade 2 or ≥ grade 3 peripheral neuropathy. Up to two dose reductions were allowed.2.5For pharmacokinetic (PK) analyses, plasma samples were collected before and approximately 4 h after talazoparib administration on C1D1, C1D7 (schedule A), or C1D3 (schedule B), C2D1, and D1 of cycles 3–5 for cycles coinciding with radiographic assessment. On each day when pharmacodynamic (PD) or plasma samples were collected, talazoparib was dosed in clinic (prior to paclitaxel and carboplatin administration). Plasma concentrations of talazoparib were evaluated by a validated LC/MS assay. For PD analyses, whole blood samples were obtained before and approximately 4 h after talazoparib administration on C1D1, C1D3 (schedule B), or C1D7 (schedule A), and C2D1, for assessment of PAR inhibition and DNA damage response pathway activation  in peripheral blood mononuclear cells (PBMCs) see Data .2.6ARID1A, ATM, ATRX, BARD1, BLM, BRCA1, BRCA2, BRIP1 (FANCJ), CDK12, CHEK1, CHEK2, FANCA, FANCD2, FANCE, FANCF, FANCL, FANCM, MRE11, MSH2, MSH6, NBN, PALB2, PPP2R2A, RAD50, RAD51B, RAD51C, RAD51D, RAD54L, WRN, and XRCC3. When feasible, the NGS results were sent to the patient's treating physician for referral to genetic counseling if indicated.Standard of care germline genetic testing was available for 30/43 patients and archival tumor tissue (available for 18/43 patients) was submitted for somatic next‐generation sequencing (NGS) with the Foundation One NGS panel. The following genes were included in the analysis: 33.1BRCA1/2 mutations, and 11.6% had received prior PARP inhibitor. Patients in both schedules received a median of four cycles of induction therapy with carboplatin, paclitaxel, and talazoparib (range 1–9), and 37.2% went on to receive one of the protocol‐approved maintenance therapy regimens  and 12/38 partial (PR) responses. All three patients with CRs were enrolled in schedule A. An additional 17 patients across both cohorts had stable disease (SD) Table . Among pTwelve patients received at least six cycles of talazoparib maintenance therapy (with or without carboplatin) after completing at least four cycles of initial triplet therapy. In this group, the median duration of maintenance therapy was 12.9 months, with a range from 5.5 to 55.5+ months Table ; two pat3.4The mean plasma concentrations of talazoparib 4 h after the first dose of cycle 1 were 0.612 ng/ml (44.1 CV%) and 0.58 ng/ml (44.9 CV%) for schedule A and B, respectively. For Schedule A, the mean talazoparib plasma concentration on C1D7 4 h post‐dose was 2.95 ng/ml (44.8 CV%). For Schedule B, the mean talazoparib plasma concentration was 2.19 (55.7 CV%). Plasma concentrations 4 h following the first dose of cycles 2 and 3 were similar for both schedules.Pharmacodynamic analyses in schedule A demonstrated no significant change in markers of DNA damage response (Rad51 foci and γH2AX intensity) or PARP inhibition (PAR intensity) in peripheral blood mononuclear cells (PBMCs) from baseline to C1D7 or C2D1 Figure  and as s3.5gBRCA1 and 10 patients had gBRCA2 mutations. Two patients had germline mutations in other DNA damage response (DDR) pathway genes (ATM and ARID1A), and five patients had deleterious mutations in DDR pathway genes  identified on Foundation One NGS. Two out of 3 complete responses and 8/10 partial responses occurred in patients with DDR pathway alterations  pathway mutation status was conducted for patients with standard of care germline testing (30/43 patients) and/or sufficient archival tumor tissue for Foundation One NGS panel sequencing (18/43 patients), which does not distinguish between germline and somatic mutations Figure . Seven ps Figure . Six out42, and talazoparib 250 mcg for both the 3‐day and 7‐day schedule. However, this regimen was associated with significant myelosuppression during the induction phase, with a third of patients receiving growth factor after cycle 1, and a third requiring transfusion support. Dose modifications were also frequently required, most commonly omitting one weekly paclitaxel dose. Unexpectedly, there was no evidence of improved tolerability for the 3‐day versus 7‐day schedule. However, the rate of febrile neutropenia was low, and supportive care allowed patients to complete induction chemotherapy with relatively preserved chemotherapy intensity and few discontinuations due to toxicity, similar to what has been reported in other studies evaluating PARPi with chemotherapy.This phase I study identified a recommended phase 2 dose of carboplatin AUC 6, paclitaxel 80 mg/mBRCA1/2 mutations are well‐established predictive biomarkers of PARPi sensitivity,PALB2 mutations and somatic BRCA1/2 mutations in metastatic breast cancerBRCA1/2 and ATM alterations in metastatic castrate resistant prostate cancerGermline The majority of patients on this study with objective responses did have DDR pathway genomic alterations Figure . HoweverThe phase III BROCADE3 trial found that the addition of intermittent veliparib to carboplatin plus paclitaxel in advanced gBRCA‐mutated breast cancer with 0–2 prior lines of therapy improved PFS and duration of response,Overall, the induction‐maintenance design in our study did facilitate a shorter duration of cytotoxic chemotherapy in the advanced disease setting, at the expense of significant toxicity during the induction phase in both schedules. The exploratory analysis of somatic and germline DDR mutation status, available for the majority of enrolled patients, did demonstrate disease responses in patients with DDR alterations in tumor types such as head and neck squamous cell cancer in which the impact on DDR mutation status on PARPi/platinum sensitivity is less well characterized. Limitations include the finding that the MTD for talazoparib was significantly lower for both the 3‐ and 7‐day schedules compared to monotherapy PARPi studies. However, while maximal PARP inhibition is seen with doses ≥600mcg in the monotherapy setting, pharmacodynamic studies demonstrated significant inhibition at doses as low as 200 mcg, suggesting that the MTD here would be expected to result in significant target engagement.In conclusion, the combination of talazoparib with carboplatin and paclitaxel is feasible with an intermittent, lower dose schedule of talazoparib and appropriate supportive care. However, modification of the cytotoxic chemotherapy component of the regimen would be a consideration in further development, given the challenging toxicity profile. Future studies of PARPi combined with single agent platinum or other DNA damaging chemotherapy may maximize the effect of DNA damage with a more favorable toxicity profile, potentially expanding use of PARPi to other solid tumors. Patient selection for PARPi based on genomic alterations affecting DNA repair should continue to be explored in future trials.Ticiana A. Leal: Consulting ‐ Boehringer‐Ingelheim, Jazz Pharmaceuticals, Genentech, Lilly; Advisory Boards ‐ AstraZeneca, EMD Serono, Merck, Boehringer‐Ingelheim, Blueprint, Bayer, Janssen, Mirati. Lisa M. Barroilhet: Advisory boards ‐ Clovis, Astrazeneca, Kiayatec. Kari B. Wisinski: Research support ‐ Pfizer, Context, Novartis; Scientific Advisory boards ‐ Eisai, AstraZeneca, Pfizer, Sanofi, Daiichi Sankyo, Novartis. The other authors declare no potential conflict of interest.Ticiana A. Leal: Conceptualization, resources, funding acquisition, investigation, methodology, supervision, writing‐original draft, writing‐review, and editing. Marina N. Sharifi: Data curation, formal analysis, investigation, visualization, methodology, writing‐original draft, writing‐review, and editing. Nancy Chan: Conceptualization, resources, methodology, writing‐review, and editing. Robert Wesolowski: Conceptualization, resources, methodology, writing‐review, and editing. Anita A. Turk: Data curation, formal analysis, visualization, methodology, writing‐review, and editing. Justine Y. Bruce: Resources, writing‐review, and editing. Ruth M. O'Regan: Resources, writing‐review, and editing. Jens Eickhoff: Resources, methodology, writing‐review, and editing. Lisa M. Barroilhet: Resources, writing‐review, and editing. Jyoti Malhotra: Conceptualization, resources, methodology, writing‐review, and editing. Janice Mehnert: Resources, writing‐review, and editing. Eugenia Girda: Resources, writing‐review, and editing. Elizabeth Wiley: Resources and investigation. Natalie Schmitz: Resources, formal analysis, investigation, visualization, methodology, writing‐review, and editing. Shannon Andrews: Resources, formal analysis, investigation, and methodology. Glenn Liu: Conceptualization, resources, methodology, writing‐review, and editing. Kari B. Wisinski: Conceptualization, resources, supervision, funding acquisition, methodology, writing‐original draft, writing‐review, and editing.This study was conducted under an IRB‐approved protocol (NCT02317874). Written informed consent was obtained from all participants prior to enrollment in the trial. The study was conducted in compliance with the Declaration of Helsinki.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


Bone fracture healing is an overly complex process in which inflammation, osteogenesis, and angiogenesis are tightly coupled, and delayed fracture repair is a very common health risk. One of the major causes of delayed healing is the formation of insufficient vasculature. Precise regulation of blood vessels in bone and their interplay with especially osteogenic processes has become an emerging topic within the last years; nevertheless, regulation of angiogenesis in (early) diseased fracture repair is still widely unknown. Here, we aim to develop an in vitro model for the analysis of early fracture healing which also enables the analysis of angiogenesis as a main influencing factor. As smoking is one of the main risk factors for bone fractures and developing a delay in healing, we model smoking and non-smoking conditions in vitro to analyze diverging reactions. Human in vitro fracture hematomas mimicking smokers’ and non-smokers’ hematomas were produced and analyzed regarding cell viability, inflammation, osteogenic and chondrogenic differentiation, and angiogenic potential. We could show that smokers’ blood hematomas were viable and comparable to non-smokers. Smokers’ hematomas showed an increase in inflammation and a decrease in osteogenic and chondrogenic differentiation potential. When analyzing angiogenesis, we could show that the smokers’ hematomas secrete factors that drastically reduced HUVEC proliferation and tube formation. With an angiogenesis array and gene expression analysis, we could identify the main influencing factors: Anpgt1/2, Tie2, and VEGFR2/3. In conclusion, our model is suitable to mimic smoking conditions in vitro showing that smoking negatively impacts early vascularization of newly formed tissue. Delayed fracture healing is one of the main causes of prolonged hospital stays. It is a burden for the patient and additionally produces large costs for the health care system . BetweenIntact vasculature is mandatory for physiological tissue homeostasis by transporting oxygen, nutrients, and metabolic waste products . The fraTreatment to avoid a delay in fracture repair usually combines mechanical stabilization and biological stimulation using cellular bone matrices, mainly focusing on promoting osteogenesis. More advanced matrices are also designed to deliver growth factors such as recombinant human BMP-2, PDGF-BB, or VEGF to the site of fracture. Nevertheless, these matrices are still not broadly used for therapy .Within this study, we want to analyze the effect of smoking on early fracture repair in vitro, as it is one of the main risk factors for the development of fracture healing disorders. Cigarette smoke consumption not only interferes with the inflammatory status by, for instance, increasing oxidative stress and altered cytokine secretion, but also influences bone metabolism via toxic components of the smoke, oxygenation, altered hormone status, etc. ,19. The For our analysis, we modified an in vitro fracture hematoma model ,23 to diHuman immortalized bone marrow-derived mesenchymal stem cell (hB-MSCs) line SCP-1 was obtained from Prof. Schieker. SCP-1 cells were immortalized by hTERT lentiviral gene transfer, which prevented senescence by maintaining all essential functions including the differentiation ability . A trans6 cells/mL cell suspension of SCP-1 cells in clotting medium , 10 mM calcium chloride ) was mixed in a 1:1 ratio  with human peripheral blood. Blood was drawn max. At 2 h prior to clot formation and from healthy male volunteers using EDTA monovettes. Both smokers’ and non-smokers’ blood was drawn from healthy  male volunteers. Smokers were on average 33 years old, non-smokers 31. The mixture was allowed to coagulate for 1 h at 37 °C, humidified atmosphere, 5% CO2. Then, formed clots were transferred to fresh 96-well plates, and 100 µL culture medium  were added. In vitro hematomas were cultured within a hypoxia incubator chamber  filled with a hypoxic gas mixture ) in a humidified atmosphere for up to 48 h.Hematomas were produced following Pfeiffenberger et al. 2019 . TherefoFor simulation of smoking conditions in vitro, a combination of smokers’ blood and Cigarette Smoke Extract (CSE) pre-stimulated SCP-1 cells was used. For stimulation of SCP-1 cells, CSE was prepared as described . A 5% CSHematomas were stained with a 0.1% CalceinAM  solution in culture medium for 20 min, 37 °C, dark. The stain was removed, and in vitro hematomas were washed with PBS once. Images were taken using an EVOS FL Cell imaging system. Clot diameters were determined using ImageJ .For histological analyses, in vitro hematomas were fixed in 4% formaldehyde, embedded in paraffin, cut in 4 µm-thick slices, and mounted on glass slides . Sections were stained with hematoxylin–eosin and Movat pentachrome stains  and evalUGT1A6) gene, whereas male DNA content was assessed by detecting y-chromosomal specific sex-determining region Y (SRY) gene. Sample-specific DNA content was determined by respective standard curves. PCRs were run in total volumes of 20 µL containing 10 µL Green Master Mix (2X) High Rox , 1 µL primer forward, 1 µL primer reverse, 7 µL of RNase/DNase-free water, and 1 µL of template DNA. The cycling program included 15 min initial denaturation at 95 °C followed by 40 cycles of denaturation , primer annealing , and elongation , and a final elongation . Melting curves of amplified DNA were recorded to ensure specific amplification only. PCRs were run within a StepOnePlus™ Real-Time PCR System  and analyzed using the StepOne Software v2.3 , where cycle threshold (CT) and baseline were set automatically. Primer details are listed in To determine the ratio of incorporated SCP-1 to blood cells, sex-specific qPCR was performed as previously described . Since tMitochondrial activity was assessed through Resazurin conversion. In brief, 100 µL of 0.001% Resazurin working solution  was added per well or in vitro hematoma. After incubation at 37 °C, formed Resorufin was measured at λEx = 544 nm/λEm = 590–10 nm using the Omega plate reader.® 2.0 Cell Viability Assay  following the manufacturer’s instructions. For analysis of 3D cultures, the incubation time was increased to 30 min. Luminescence was quantified using the Omega plate reader.Adenosine triphosphate (ATP) content was determined by using the CellTiter-GloRelease of lactate dehydrogenase (LDH) in the culture supernatant was determined by CyQUANT™ LDH Cytotoxicity Assay  following the manufacturer’s instructions using 50 µL culture supernatant per technical replicate.Levels of IL-6, TNF-α, and CCL2 in the culture supernatant were determined by enzyme-linked-immunosorbent-assay (ELISA) following the manufacturer’s instructions .2, pH 10.5 ) was added per in vitro hematoma and incubated at 37 °C for 1 h. Clots were removed from the solution and absorbance at λ = 405 nm was measured using the Omega Plate reader. ALP activity was normalized to mitochondrial activity.As a measure for osteogenic differentiation, alkaline phosphatase (ALP) activity, was determined by conversion of 4-Nitrophenyl phosphate (pNpp) to 4-Nitrophenol (pNp) . A 200 µRNA was isolated from a pool of 12 in vitro hematomas per condition using Chloroform/Phenol extraction. Prior to RNA isolation, in vitro hematomas were homogenized in RNA isolation solution using homogenization pestles. cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit  following the manufacturer’s instructions. Reverse transcription-polymerase chain reactions (RT-PCRs) were conducted using a total reaction volume of 15 µL consisting of 7.5 µL Red HS Master Mix , 0.75 µL primer forward and reverse, respectively, 2 µL template cDNA, and 4 µL RNase/DNase free water. PCRs were run within an Arktik™ Thermocycler  with 2 min, 95 °C initial denaturation; between 23 and 40 cycles of 15 s of each denaturation (95 °C), primer annealing (52–64 °C), elongation (72 °C); and a final elongation step . Detailed information on primers used and the cycling conditions are listed in Human angiogenesis array C1000  was performed following the manufacturer’s instructions, using pooled culture supernatants collected after 48 h of culture. Chemiluminescent signals were recorded using an INTAS Chemocam  and analyzed using ImageJ. Obtained data were normalized to the internal controls.Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell Growth Basal Medium 2  supplemented with 2% FCS, 1% Antibiotic/Antimycotic , 0.5 ng/mL human VEGFA165, 10 ng/mL human FGF-b, 5 ng/mL human Epidermal growth factor (EGF), 20 ng/mL hRIGF-R3 , 22.5 µg/mL Heparin , 0.2 µg/mL Hydrocortisone , and 1 µg/mL L-ascorbic acid . HUVECs were cultured on 0.1% Gelatin -coated flasks and subcultured when confluent. The medium was changed every 2 to 3 days. For experiments, cells were used in passages 7 to 10.HUVECs were seeded on with 0.1% Gelatin-pre-coated 96-well plates. Cells were stimulated with a 1:1 mixture of culture supernatant and EBM-2. Proliferation was indirectly assessed after 48 h by means of mitochondrial activity and ATP content as described in 4 HUVECs were seeded per well and the respective stimuli were added 1:1 diluted in plain EBM-2 to a total volume of 200 µL. Tube formation assays were performed using pooled culture supernatants. Cells were incubated in humidified atmosphere, 37 °C, 5% CO2 for 18 h. Before microscopy, tubes were stained with 0.1% CalceinAM for 10 min, 37 °C. Microscopic images were captured using an EVOS FL imaging system  and analyzed using the ImageJ angiogenesis analyzer [48-well plates were coated with 6 µL GeltrexTM  using a thin-layer angiogenesis assay method . A totalanalyzer . Data wep < 0.05, ** p < 0.01, *** p < 0.001. Data are shown as box plots showing all data points if not indicated differently.Statistics were made using Graph Pad Prism 8 . When comparing solely two experimental groups, data were compared using non-parametric Mann–Whitney tests. If more than two experimental groups were compared, non-parametric Kruskal–Wallis tests followed by Dunns’ multiple comparison tests were performed. The number of biological replicates (N) and technical replicates (n) is given in the figure legend for each experiment performed. Levels of significances were defined as * As described in the materials and methods section, experiments were performed using blood from non-smokers and smokers paired with SCP-1 cells either pre-stimulated with CSE or not. As active smokers not only show a diverging blood composition but also impaired mesenchymal stem cells, two main experimental conditions were defined: smokers’ blood and CSE-pre-stimulated SCP-1 cells mimicking the smokers’ hematomas (S) and non-smokers’ blood combined with unstimulated SCP-1 cells mimicking the non-smokers hematomas (Ctrl). The respective control conditions, combining smokers’ blood with unstimulated SCP-1 cells (E1) and non-smokers’ blood with pre-stimulated SCP-1 cells (C2), were also measured. As the main part of the manuscript solely focuses on the two experimental groups, results of the control conditions are displayed in First, smokers’ and non-smokers’ hematomas were compared in terms of viability and stability to allow later comparison of results. As shown in The inflammatory status of hematomas was determined by expression and release of CCL2, TNF-α, and IL-6. The results are shown in RUNX2, ALP, BMP4, and Noggin as well as the chondrogenic differentiation marker SOX9 were all significantly lower in smokers’ hematomas  on the cell surface. VEGFRs are receptor tyrosine kinases and ligand binding triggers the phosphorylation of intracellular tyrosine residues which can activate various signaling pathways including phospholipase Cγ (PLCγ)–ERK1/2 pathway, the Pi3K-AKT-mTOR pathway, and SRC and small GTPases . Mainly In contrast to VEGF, which induces vessel sprouting and early angiogenesis, angiopoietins, especially Angpt1, are described to be important modulators in angiogenic maturation and development . Both Anvice versa. However, osteogenesis, angiogenesis, and inflammation are highly interdependent and it is known that also the endothelium stimulates osteogenesis, for instance via endothelial Notch, which has been shown to promote both angiogenesis and osteogenesis [Additionally, also the reduction in MMPs and the increase in TIMPs in smokers’ early fracture repair point out the reduced ability for matrix remodeling and therefore angiogenesis. MMPs are involved in leveling the path for the ingrowth of new blood vessels but also help in callus remodeling and increase the bioavailability of different growth factors stored in the ECM . This alogenesis . Howeverogenesis . In contogenesis . The exa


Candida glabrata, an opportunistic pathogenic yeast intrinsically tolerant to fluconazole. Analyses of the transcriptomic profile of C. glabrata after the combination of copper and fluconazole showed that the expression of the multidrug transporter gene CDR1 was decreased, suggesting that fluconazole efflux could be affected. In agreement, we observed that copper inhibits the transactivation of Pdr1, the transcription regulator of multidrug transporters and leads to the intracellular accumulation of fluconazole. Copper also decreases the transcriptional induction of ergosterol biosynthesis (ERG) genes by fluconazole, which culminates in the accumulation of toxic sterols. Co-treatment of cells with copper and fluconazole should affect the function of proteins located in the plasma membrane, as several ultrastructural alterations, including irregular cell wall and plasma membrane and loss of cell wall integrity, were observed. Finally, we show that the combination of copper and fluconazole downregulates the expression of the gene encoding the zinc-responsive transcription regulator Zap1, which possibly, together with the membrane transporters malfunction, generates zinc depletion. Supplementation with zinc reverts the toxic effect of combining copper with fluconazole, underscoring the importance of this metal in the observed synergistic effect. Overall, this work, while unveiling the molecular basis that supports the use of copper to enhance the effectiveness of fluconazole, paves the way for the development of new metal-based antifungal strategies.The synergistic combinations of drugs are promising strategies to boost the effectiveness of current antifungals and thus prevent the emergence of resistance. In this work, we show that copper and the antifungal fluconazole act synergistically against  Candida, also named invasive candidiasis, represent the most common fungal disease among hospitalized patients receiving immunosuppressive or intensive antibacterial therapies  are a major health concern, affecting many people worldwide and causing more than 1.2 million deaths per year . IFIs caherapies . Candidaes occur . Candidaifungals .C. glabrata standing out among them  and treated with 625 μM CuSO4, 32 μg/mL fluconazole, or with both compounds for 24 or 48 h. For fungicidal assays, serial dilutions of treated or untreated cultures were plated on YPD agar plates for colony forming units (CFUs) count. Whenever the CFU count was lower than that corresponding to OD600 0.8 , the treatment was considered fungicidal. For zinc supplementation assays 4 mM of ZnSO4 was used. Spot assays were carried out by spotting 5 μL of sequential dilutions (C. glabrata: from 8 × 106 to 80 cells/mL and Saccharomyces cerevisiae: 4 × 106 to 40 cells/mL) of exponentially growing cultures onto SC agar plates containing the indicated treatments. Unless otherwise stated, the plates were incubated for 48 h at 37°C. Spot assays were repeated at least three times. To construct the C. glabrata Δzap1 mutant (Cg Δzap1), a histidine cassette was constructed by amplifying CgHIS3 gene with primers for the flanking region of the CgZAP1 gene (pZAP1_KO_FW: 5′CATAATT GAGTATAAAAAGGACTACAAAAGCTAAGAAACTACAGAC CGGCTTGGGTACAAGAGGATAAC3′/ZAP1_KO_RV: 5′GGA CGAGTTGGAGTTATGTCTATATATTTATTGGACTATGTACA GTATAGTGAATGGGCAATGCTATGGTGTTGTTGAAACCA CCACGAG3′). C. glabrata competent cells and transformation were performed with the Zymo Transformation Kit (Zymo Research), according to manufacturer’s instructions. Mutant cells were confirmed by colony PCR using specific ZAP1 primers (ZAP1_A1: 5′TGATTGTGGTGGGAGATGC3′ and ZAP1_A4: 5′GTTGATCACAGCGAGAAGC3′), followed by fragment digestion with XcmI. To construct the pZAP1 plasmid, the CgZAP1 gene was amplified by PCR with primers for the flanking region of this gene (ZAP1_A1N: 5′AATGCAAGAGGGACAAATCG3′ and ZAP1_A4). The PCR product was inserted into the SmaI-digested pCgACT14 vector using T4 DNA ligase (New England Biolabs Inc.), according to manufacturer’s instructions.The strains used in this study are listed in C. glabrata Pdr1 was investigated using a LexA-based one-hybrid system. The CgPDR1 ORF (CAGL0A00451g) was amplified by PCR using specific oligonucleotides (PDR1_ATG_II_FW: 5′ATGCAAACATTAGA AACTACATCAAAATC3′/PDR1_KpnI_Rv: 5′ccGGTACCTCAC AAGTAAACATCAGAAAATAGGTC3′). The PCR product was digested with KpnI and inserted into the Sma/-KpnI digested Yap8-LexA plasmid (LexA sequence. The plasmid (LexA-PDR1) sequence was confirmed by sequencing. The S. cerevisiae EGY48 strain was then co-transformed with LexA-PDR1 and pSH18-34 (Thermo Fisher), which contains eight LexA operators fused to the LacZ reporter gene. Positive transformants were selected in SD agar plates, after 48 h incubation at 30°C. For the transactivation assay, the co-transformed strain was grown in SD medium until exponential phase and left untreated or treated with 64 μg/mL of fluconazole, 625 μM of CuSO4, or with both compounds, for 2 h. Total proteins were extracted from cell cultures as described in The transactivation activity of  plasmid , in framCandida glabrata cultures were grown under the indicated conditions, harvested by centrifugation and RNA was isolated using the phenol:chloroform method (RPL10 gene (CAGL0K12826g), which encodes an ortholog of the S. cerevisiae ribosomal L10 protein, was used as reference gene. All assays were performed using biological triplicates and technical duplicates. The following primer pairs were used: CDR1: 5′CATGGCCACTTTTGGTCTTT3′/5′CAGCAATGGAGACAC GCTTA3′; PDR1: 5′CAGCAATGGAGACACGCTTA3′/5′AG TGGGCACGTCAGAGACAG3′; ERG11: 5′TATGGTCGCCTTG CCATT3′/5′GACCCATGGGATCCAGTAGA3′; ZAP1: 5′GAAG CCTTACGAGTGCCATA3′/5′TGCACTTCAGTGGTTTCTCA3′; RPL10: 5′GAGATTCTTTCCACTTGAGAGTCAGA3′/5′CTC TCATACCTTGTTGCAATCTATCC3′.m method . RNA samFlow cytometry analysis for ROS detection was performed as described by Cultures grown for 24 h in the presence of the indicated compounds were harvested by centrifugation, washed with PBS and resuspended in PBS containing 50 μg/mL of propidium iodide (PI). After a 15 min incubation period, samples were washed with PBS and filtered. Assays were performed in an S3e cell sorter (Bio-rad) and PI signal was detected using the 488 nm excitation laser and the FL3 detection channel. Unstained cells were used to establish the baseline of cells exhibiting positive PI signal. Five biological replicates of each condition were analyzed and a total of 100,000 cells were counted in each sample.600. All assays were made using biological quadruplicates.Cultures were grown under the indicated conditions for 24 h, harvested and washed with 10 mM EDTA and metal-free water as described in g for 5 min and supernatant was recovered. For the GC-MS analysis, 1 μL of each sample was injected into a DB5 column  in spitless mode using helium as carrier gas at a flow rate of 1 mL/min. The inlet was held at 200°C. The column oven was held at 50°C for 1 min, and then ramped up, at a rate of 50°C per min, to 260°C, followed by a ramp rate of 4°C per min up to the final temperature of 320°C, and then held for 5 min. The total run time was 25.2 min. Analyses were performed at REQUIMTE – LAQV, Universidade Nova de Lisbon, Caparica, Portugal. Sterols were identified according to their retention time (relative to cholestane) and by comparison with GC-MS profiles described in Sterol extraction and gas chromatography–mass spectrometry (GC-MS) analyses were performed according to the protocols described by Cultures were left untreated or treated as indicated, for 24 h. Ergosterol extraction and quantification was done as described in Candida glabrata cultures were grown under the indicated conditions for 24 h, harvested, resuspended in 2 mL MilliQ water and normalized against OD600. Tinidazole (100 μg) was added to samples prior to extraction. Extraction was then carried out using glass beads and methanol. After 30 s of vortex agitation, samples were incubated at 30°C and 200 rpm for 1 h, followed by centrifugation at 4000 × g for 7 min at 4°C. The supernatant was then recovered and centrifuged again at 9500 × g for 10 min, to remove any remaining debris. Analyses were performed using a Waters 2695 Alliance HPLC system  equipped with a Waters 486 Absorbance Detector set at 245 nm. Samples (80 μL) were injected into a reverse phase Symmetry C18 column  using an isocratic eluent mixture of ammonium acetate buffer/acetonitrile/methanol (80:15:5% v/v) at 0.7 mL/min flow rate. Column temperature was held at 30°C and samples were kept at 10°C. The Empower 2 software  was used for data acquisition. The retention time of fluconazole (13.4 min) was compared with standards for identification, and the peak area was used for quantification. Quantification of fluconazole was normalized to the internal control . Standard curves for fluconazole and tinidazole were plotted to measure the amount of compound present in each sample. All assays were performed using biological quadruplicates.600 0.8), treated with 625 μM CuSO4, 32 μg/mL fluconazole, or with both compounds for up to 2.5 h and harvested. Total proteins were extracted from cell cultures as described in The BVGC3 strain was grown until exponential phase  in SC medium and left untreated or treated with 1.25 mM CuSO4, 32 μg/mL fluconazole, or a combination of both for 1 h. Biological triplicates were used for each condition. Cells were harvested and RNA was isolated. RNA samples were treated with DNase and purified using the RNeasy Kit (Qiagen). Library preparation and sequencing was done at the Genomics Facility of Instituto Gulbenkian da Ciência, Portugal, using the SMART-SEQ2 protocol, adapted from C. glabrata genome (GCA_000002545.2 downloaded from NCBI genome database) using Bowtie2 program and obtained more than 90% of aligned reads  and fold change (FC) higher than 2. The functional annotation was performed with FungiFun  index (∑FIC = FICCu + FICFluc). The MIC for fluconazole (MICFluc) and copper (MICCu) were determined to be 64 μg/mL and 625 μM, respectively. For each drug combinations the FIC was calculated as follows: FICCu = MICCu+Fluc/625 and FICFluc = MICFluc+Cu/64. FIC index ≤ 0.5 indicates a synergistic effect.The assays were performed in SC medium using 96-well flat bottom plates, according to the CLSI standard M27-A3 , followiCandida glabrata cultures were grown under the indicated conditions for 24 h and harvested by centrifugation. Samples were fixed with a mixture of 0.4% glutaraldehyde and 4% formaldehyde in sodium cacodylate buffer 0.1 M for 18 h at room temperature, followed by three washing steps with sodium cacodylate buffer 0.1 M. Samples were dehydrated by sequential washing with four ethanol solutions . After the last dehydration step, ethanol was removed and tert-butyl alcohol was added. Samples were incubated at 30°C for 1 h, placed on ice until completely frozen, freeze-dried by lyophilization and kept at room temperature until the scanning electron microscopy (SEM) analyses were performed. Samples were gold sputtered with 8 nm gold in an electron sputter (Cressington 108) and imaged in a Hitachi SU8010 scanning electron microscope, at 1.5 kV. Approximately 120 cells were counted per condition and the proportion of cells exhibiting surface abnormalities was calculated.Candida glabrata cultures were grown under the indicated conditions for 24 h, harvested by centrifugation and fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.3. Following primary fixation for 2 h at 4°C and wash in the cacodylate buffer, cells were, pelleted and embedded in 2% agar for further processing. Samples were further fixed for 3 h in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer pH 7.3. Then, samples were washed in 0.1 M acetate buffer, pH 5.0 and fixed in 1% uranyl acetate in the same buffer for 1 h. Dehydration was carried out with increasing concentrations of ethanol. After passing through propylene oxide, samples were embedded in Epon-Araldite, using SPI-Pon as an Epon 812 substitute. Thin sections were made with diamond knives and stained with 2% aqueous uranyl acetate and Reynold’s lead citrate. The stained sections were analyzed and photographed in a JEOL 1200-EX electron microscope.GSE162741.RNA-sequencing raw data is available at Gene Expression Omnibus (GEO) under the accession number C. glabrata at concentrations where these compounds cause only mild growth defects when used separately  and 16-fold (4 μg/mL) when added at concentrations of 156 and 313 μM, respectively. Fluconazole decreased the MICCu fourfold (156 μM) and eightfold (78 μM) when combined with copper at concentrations of 16 and 32 μg/mL, respectively (4) and 16 μg/mL (fluconazole), as depicted in C. glabrata strain with a lower MIC for fluconazole . Unlike C. albicans , potentially specific to this organism, should underlie such effect.These data indicate that in C. glabrata  were conducted using edgeR  to identify the most relevant affected pathways.In yeast species, the response to copper overload requires the transcriptional activation of copper detoxification genes, such as those encoding metallothioneins , by a coglabrata ]. The adglabrata , which iand CDR2 . Therefong edgeR , and a sAfter exposure to fluconazole, only 34 genes were found differentially expressed, whereas copper and Cu + Fluc altered the expression of 1164 and 1451 genes, respectively. The overlapping and specific alterations of each transcriptomic profile are illustrated in ERG pathway, such as ERG11, ERG1, ERG2, CYB5, and NCP1, which were upregulated  and the transcriptional regulator of copper detoxification, AMT1 . Fe-S clusters are primary targets of copper toxicity ectively . As antiectively . Under ttoxicity  and theitoxicity . Anothertoxicity . These gnditions , suggestle alone .UPC2A and ERG11 that encode the transcriptional regulator of the pathway , found induced in response to both copper and Cu + Fluc (C. glabrata (2FCCu −1.38) and Cu + Fluc (log2FCCu+Fluc −1.74), contrasting with its upregulation following treatment with fluconazole  drug detoxification through Cdr1 and (2) ergosterol biosynthesis. In addition, zinc homeostasis may be deregulated when copper and fluconazole are combined, which might pose a threat because zinc is an essential metal whose intracellular levels need to be delicately balanced.CDR1 were noticeably downregulated in response to copper and Cu + Fluc . The S. cerevisiae EGY48 strain was then co-transformed with LexA-PDR1 and the pSH18-34 plasmid, which contains the LacZ reporter gene, encoding β-galactosidase under the control of 8 LexA operators   . InteresG genes) . Accordicultures . A similcultures . Corrobocultures . Contrarcultures .C. glabrata exposed to each condition, by GC-MS. A scheme summarizing the GC-MS results integrated with the transcriptomic data can be found in To have a comprehensive understanding of the impact of copper and fluconazole on ergosterol metabolism, we analyzed the sterol profile of C. glabrata untreated cells have a well-defined oval shape with a smooth surface  is important for an adequate response to the stress imposed by the combination of copper and fluconazole.In recent years, there was a growing interest in exploiting the antifungal properties of copper. Trojan horse strategies that deceive the fungus copper homeostatic mechanisms  or the cC. glabrata . These sterol metabolites alter the physicochemical properties of the plasma membrane, thus affecting membrane-associated processes like transport, growth, and division . UnexpecC. glabrata, sterol metabolism is controlled by two Zn2Cys6 transcription factors, Upc2a and Upc2b, orthologs of S. cerevisiae Upc2 and Emc22, respectively , but not with the metal alone , ergosterol metabolism (ERG genes), or zinc homeostasis. Also contrasting with our data , ERG (Upc2), and zinc homeostasis (Zap1). Therefore, when copper and fluconazole are combined, cells fail to detoxify the drug and accumulate toxic intermediates of the sterol metabolism. This accumulation, possibly together with copper-induced lipid damages, should affect the activity of membrane metal transporters. Zinc transporters malfunction together with the downregulation of reatment .Overall, our findings strongly support continued investment in bi-functional drugs, combining azole and copper chelating groups. Such a strategy, while ensuring a synchronized uptake of both compounds, would deceive the yeast copper homeostatic mechanisms, and possibly decrease the effective copper concentration needed to achieve the same synergistic effect, which would certainly reduce the toxicity to human cells.https://www.ncbi.nlm.nih.gov/geo/, GSE162741.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: AG-C and CP contributed to the conceptualization and wrote the original draft. AG-C, CA, WA, and AM performed the experiments. AG-C, CA, CP, VP, and AP analyzed the data. PP performed the clinical sample collection. CP performed the project administration and funding acquisition. All authors contributed to writing – review and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.