Blockage of BCL-XL overcomes venetoclax resistance across BCL2-positive lymphoid malignancies irrespective of BIM status.

Venetoclax, a BCL2 inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large B-cell lymphomas (LBCL), but remissions were generally short, which calls for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples we demonstrated strong synergy between venetoclax and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments, and studies on clones with knockout of expression, or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired venetoclax resistance. Of note, the venetoclax and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the pro-apoptotic BCL2L11/BIM, and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in venetoclax resistance. Immunoprecipitation experiments further suggested that the pro-apoptotic effector BAX belongs to principal mediators of the venetoclax and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new pro-apoptotic combination was confirmed in vivo on a panel of 9 PDX models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and DLBCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days ON / 3 days OFF treatment regimen, which retained the desired anti-tumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2-positive hematologic malignancies irrespective of the BCL1L11/BIM status.

Cyclin dependent kinase 4/6 inhibitor palbociclib synergizes with BCL2 inhibitor venetoclax in experimental models of mantle cell lymphoma without RB1 deletion.

BACKGROUND: Mantle cell lymphoma (MCL) is a chronically relapsing malignancy with deregulated cell cycle progression. We analyzed efficacy, mode of action, and predictive markers of susceptibility to palbociclib, an approved CDK 4/6 inhibitor, and its combination with venetoclax, a BCL2 inhibitor. METHODS: A panel of nine MCL cell lines were used for in vitro experiments. Four patient derived xenografts (PDX) obtained from patients with chemotherapy and ibrutinib-refractory MCL were used for in vivo proof-of-concept studies. Changes of the mitochondrial membrane potential, energy-metabolic pathways, AKT activity, and pro-apoptotic priming of MCL cells were evaluated by JC-1 staining, Seahorse XF analyser, genetically encoded fluorescent AKT reporter, and BH3 profiling, respectively. MCL clones with gene knockout or transgenic (over)expression of CDKN2A, MYC, CDK4, and RB1 were used to estimate impact of these aberrations on sensitivity to palbociclib, and venetoclax. RESULTS: Co-targeting MCL cells with palbociclib and venetoclax induced cytotoxic synergy in vitro and in vivo. Molecular mechanisms responsible for the observed synthetic lethality comprised palbociclib-mediated downregulation of anti-apoptotic MCL1, increased levels of proapoptotic BIM bound on both BCL2, and BCL-XL and increased pro-apoptotic priming of MCL cells mediated by BCL2-independent mechanisms, predominantly palbociclib-triggered metabolic and mitochondrial stress. Loss of RB1 resulted in palbociclib resistance, while deletion of CDKN2A or overexpression of CDK4, and MYC genes did not change sensitivity to palbociclib. CONCLUSIONS: Our data strongly support investigation of the chemotherapy-free palbociclib and venetoclax combination as an innovative treatment strategy for post-ibrutinib MCL patients without RB1 deletion.

Comprehensive profiling of endogenous phytohormones and expression analysis of 1-aminocyclopropane-1-carboxylic acid synthase gene family during fruit development and ripening in octoploid strawberry (Fragaria× ananassa).

The non-climacteric octoploid strawberry (Fragaria × ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N6-(Δ2-isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and progression of strawberry fruit ripening are associated with N6-(Δ2-isopentenyl)adenosine-5'-monophosphate (iPRMP) → N6-(Δ2-isopentenyl)adenosine (iPR) → iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohormones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening.

Complex genetic and histopathological study of 15 patient-derived xenografts of aggressive lymphomas.

Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDXs) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDXs keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We carried out a comparative genetic and histopathological study of 15 PDX models derived from patients with various types of NHL including diffuse large B-cell lymphoma (DLBCL; n = 7), Burkitt lymphoma (BL; n = 1), mantle cell lymphoma (MCL; n = 2), and peripheral T-cell lymphomas (PTCL; n = 5). Whole exome sequencing (WES) of the PDXs and primary lymphoma cells was implemented in 13 out of 15 cases with available DNA samples. Standard immunohistochemistry (IHC) was used to analyze the composition of PDX TME. WES data confirmed that PDXs maintained the genetic heterogeneity with the original primary lymphoma cells. In contrast, IHC analysis revealed the following recurrently observed alterations in the composition of PDX tumors: more blastoid lymphoma cell morphology, increased proliferation rate, lack of non-malignant cellular components including T cells and (human or murine) macrophages, and significantly lower intratumoral microvessel density and microvessel area composed of murine vessels. In addition, PDX tumors derived from T-NHL displayed additional differences compared to the primary lymphoma samples including markedly lower desmoplasia (i.e., the extent of both reticular and collagen fibrosis), loss of expression of cytotoxic granules (i.e., perforin, TIA, granzyme B), or loss of expression of T-cell specific antigens (i.e., CD3, CD4, CD8). Our data suggest that despite keeping the same genetic profiles, PDX models of aggressive NHL do not recapitulate the microenvironmental heterogeneity of the original lymphomas. These findings have implications on the relevance of PDX models in the context of preclinical research.

Apolipoprotein L1 variability is associated with increased risk of renal failure in the Czech population.

BACKGROUND: With stage 5 chronic kidney disease (CKD5) more prevalent in the Czech Republic than in most European countries, genetic susceptibility is potentially implicated. METHODS: In a group of 1489 CKD5 kidney transplantation patients (93% with complete clinical characteristics; mean age 52.0 years, 37% females) and 2559 healthy controls (mean age 49.0 years, 51% females), we examined the prevalence of six APOL1 SNPs (rs73885319, rs71785313, rs13056427, rs136147, rs10854688 and rs9610473) and one newly detected 55-nucleotide insertion/deletion polymorphism. RESULTS: The rs73885319 and rs71785313 variants were monomorphic in the Czech Caucasian population. Genotype frequencies of the three SNPs examined (rs13056427, rs136147 and rs9610473) were almost identical in patients and controls (all P values were between 0.39 and 0.91). Minor homozygotes of rs10854688 were more common between the patients (13.2%) than in controls (10.7%) (OR [95% CI]; 1.32 [1.08-1.64]; P < 0.01). Prevalence of the newly detected 55-bp APOL1 deletion was significantly higher in CKD5 patients (3.0% vs. 1.7%; OR [95% CI]; 1.80 [1.16-2.80]; P < 0.01) compared to controls. Frequencies of some individual APOL1 haplotypes were borderline different between patients and controls. CONCLUSION: We found an association between rs10854688 SNP within the APOL1 gene and end-stage renal disease in the Czech Caucasian population. Further independent studies are required before a conclusive association between the newly detected APOL1 insertion/deletion polymorphism and CKD5 can be confirmed.

Anti-apoptotic MCL1 Protein Represents Critical Survival Molecule for Most Burkitt Lymphomas and BCL2-negative Diffuse Large B-cell Lymphomas.

The pro-survival MCL1 protein is overexpressed in many cancers, including B-cell non-Hodgkin lymphomas (B-NHL). S63845 is a highly specific inhibitor of MCL1. We analyzed mechanisms of sensitivity/resistance to S63845 in preclinical models of diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. Annexin V-based cytotoxic assays, Western blot analysis, protein co-immunoprecipitation, and cell clones with manipulated expression of BCL2 family proteins were used to analyze mechanisms of sensitivity to S63845. Experimental in vivo therapy with S63845 and/or venetoclax was performed using patient-derived xenografts (PDX) of treatment-refractory B-NHL. A subset of DLBCL and majority of Burkitt lymphoma cell lines were sensitive to S63845. The level of BCL2 protein expression was the major determinant of resistance to S63845: BCL2 serves as a buffer for pro-apoptotic proteins released from MCL1 upon exposure to S63845. While BCL2-negative lymphomas were effectively eliminated by single-agent S63845, its combination with venetoclax was synthetically lethal in BCL2-positive PDX models. Concerning MCL1, both, the level of MCL1 protein expression, and its occupational status represent key factors mediating sensitivity to S63845. In contrast to MCL1-BIM/BAK1 complexes that prime lymphoma cells for S63845-mediated apoptosis, MCL1-NOXA complexes are associated with S63845 resistance. In conclusion, MCL1 represents a critical survival molecule for most Burkitt lymphomas and a subset of BCL2-negative DLBCLs. The level of BCL2 and MCL1 expression and occupational status of MCL1 belong to the key modulators of sensitivity/resistance to S63845. Co-treatment with venetoclax can overcome BCL2-mediated resistance to S63845, and enhance efficacy of MCL1 inhibitors in BCL2-positive aggressive B-NHL.

Hormonome Dynamics During Microgametogenesis in Different Nicotiana Species.

Plant microgametogenesis involves stages leading to the progressive development of unicellular microspores into mature pollen. Despite the active and continuing interest in the study of male reproductive development, little is still known about the hormonomics at each ontogenetic stage. In this work, we characterized the profiles and dynamics of phytohormones during the process of microgametogenesis in four Nicotiana species (Nicotiana tabacum, Nicotiana alata, Nicotiana langsdorffii, and Nicotiana mutabilis). Taking advantage of advanced HPLC-ESI-MS/MS, twenty to thirty endogenous hormone derivatives were identified throughout pollen ontogenesis, including cytokinins, auxins, ABA and its derivatives, jasmonates, and phenolic compounds. The spectra of endogenous phytohormones changed dynamically during tobacco pollen ontogeny, indicating their important role in pollen growth and development. The different dynamics in the accumulation of endogenous phytohormones during pollen ontogenesis between N. tabacum (section Nicotiana) and the other three species (section Alatae) reflects their different phylogenetic positions and origin within the genus Nicotiana. We demonstrated the involvement of certain phytohormone forms, such as cis-zeatin- and methylthiol-type CKs, some derivatives of abscisic acid, phenylacetic and benzoic acids, in pollen development for the first time here. Our results suggest that unequal levels of endogenous hormones and the presence of specific derivatives may be characteristic for pollen development in different phylogenetic plant groups. These results represent the currently most comprehensive study of plant hormones during the process of pollen development.

Rejection-associated Phenotype of De Novo Thrombotic Microangiopathy Represents a Risk for Premature Graft Loss.

BACKGROUND: Thrombotic microangiopathy (TMA) significantly affects kidney graft survival, but its pathophysiology remains poorly understood. METHODS: In this multicenter, retrospective, case-control paired study designed to control for donor-associated risks, we assessed the recipients' risk factors for de novo TMA development and its effects on graft survival. The study group consists of patients with TMA found in case biopsies from 2000 to 2019 (n = 93), and the control group consists of recipients of paired kidney grafts (n = 93). Graft follow-up was initiated at the time of TMA diagnosis and at the same time in the corresponding paired kidney graft. RESULTS: The TMA group displayed higher peak panel-reactive antibodies, more frequent retransplantation status, and longer cold ischemia time in univariable analysis. In the multivariable regression model, longer cold ischemia times (odds ratio, 1.18; 95% confidence interval [CI], 1.01-1.39; P = 0.043) and higher peak pretransplant panel-reactive antibodies (odds ratio, 1.03; 95% CI, 1.01-1.06; P = 0.005) were found to be associated with increased risk of de novo TMA. The risk of graft failure was higher in the TMA group at 5 y (hazard ratio [HR], 3.99; 95% CI, 2.04-7.84; P < 0.0001). Concomitant rejection significantly affected graft prognosis at 5 y (HR, 6.36; 95% CI, 2.92-13.87; P < 0.001). De novo TMA associated with the active antibody-mediated rejection was associated with higher risk of graft failure at 5 y (HR, 3.43; 95% CI, 1.69-6.98; P < 0.001) compared with other TMA. CONCLUSIONS: Longer cold ischemia and allosensitization play a role in de novo TMA development, whereas TMA as a part of active antibody-mediated rejection was associated with the highest risk for premature graft loss.

Quantitative In Vivo Monitoring of Hypoxia and Vascularization of Patient-Derived Murine Xenografts of Mantle Cell Lymphoma Using Photoacoustic and Ultrasound Imaging.

Tumor oxygenation and vascularization are important parameters that determine the aggressiveness of the tumor and its resistance to cancer therapies. We introduce dual-modality ultrasound and photoacoustic imaging (US-PAI) for the direct, non-invasive real-time in vivo evaluation of oxygenation and vascularization of patient-derived xenografts (PDXs) of B-cell mantle cell lymphomas. The different optical properties of oxyhemoglobin and deoxyhemoglobin make it possible to determine oxygen saturation (sO2) in tissues using PAI. High-frequency color Doppler imaging enables the visualization of blood flow with high resolution. Tumor oxygenation and vascularization were studied in vivo during the growth of three different subcutaneously implanted patient-derived xenograft (PDX) lymphomas (VFN-M1, VFN-M2 and VFN-M5 R1). Similar values of sO2 (sO2 Vital), determined from US-PAI volumetric analysis, were obtained in small and large VFN-M1 tumors ranging from 37.9 ± 2.2 to 40.5 ± 6.0 sO2 Vital (%) and 37.5 ± 4.0 to 35.7 ± 4.6 sO2 Vital (%) for small and large VFN-M2 PDXs. In contrast, the higher sO2 Vital values ranging from 57.1 ± 4.8 to 40.8 ± 5.7 sO2 Vital (%) (small to large) of VFN-M5 R1 tumors corresponds with the higher aggressiveness of that PDX model. The different tumor percentage vascularization (assessed as micro-vessel areas) of VFN-M1, VFN-M2 and VFN-M5 R1 obtained by color Doppler (2.8 ± 0.1%, 3.8 ± 0.8% and 10.3 ± 2.7%) in large-stage tumors clearly corresponds with their diverse growth and aggressiveness. The data obtained by color Doppler were validated by histology. In conclusion, US-PAI rapidly and accurately provided relevant and reproducible information on tissue oxygenation in PDX tumors in real time without the need for a contrast agent.

Cytarabine nanotherapeutics with increased stability and enhanced lymphoma uptake for tailored highly effective therapy of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare subtype of B-cell non-Hodgkin lymphoma (B-NHL) with chronically relapsing clinical course. Implementation of cytarabine (araC) into induction and salvage regimen became standard of care for majority of MCL patients. In this study, tailored N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer nanotherapeutics containing covalently bound araC (araC co-polymers) were designed, synthesized and evaluated for their anti-lymphoma efficacy in vivo using a panel of six patient-derived lymphoma xenografts (PDX) derived from newly diagnosed and relapsed / refractory (R/R) MCL. While free araC led to temporary inhibition of growth of MCL tumors, araC co-polymers induced long-term disappearance of the engrafted lymphomas with no observed toxicity even in the case of PDX models derived from patients, who relapsed after high-dose araC-based treatments. The results provide sound preclinical rationale for the use of HPMA-based araC co-polymers in induction, salvage or palliative therapy of MCL patients.

Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants.

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

Physiological and Transcriptomic Response of Grey Poplar (Populus ×canescens Aiton Sm.) to Cadmium Stress.

(1) Background: Populus ×canescens (Aiton) Sm. is a fast-growing woody plant belonging to the family Salicaceae. Two poplar genotypes characterized by unique phenotypic traits (TP11 and TP20) were chosen to be characterized and tested for a physiological and transcriptomic response to Cd stress. (2) Methods: A comparative analysis of the effects of exposure to high cadmium (Cd) concentrations (10 µM and 100 µM) of TP11 and TP20 was performed. (3) Results: Neither of the tested Cd concentration negatively affected plant growth; however, the chlorophyll content significantly decreased. The potassium (K) content was higher in the shoots than in the roots. The magnesium concentrations were only slightly affected by Cd treatment. The zinc content in the shoots of TP20 was lower than that in the shoots of TP11. Cd accumulation was higher in the roots than in the shoots. After 10 days of exposure, 10 µM Cd resulted in comparable amounts of Cd in the roots and shoots of TP20. The most significant change in transcript amount was observed in endochitinase 2, 12-oxophytodienoate reductase 1 and phi classglutathione S-transferase. (4) Conclusions: Our study provided new insights for effective assessing the ability of different poplar genotypes to tolerate Cd stress and underlying Cd tolerance.

Overcoming resistance to rituximab in relapsed non-Hodgkin lymphomas by antibody-polymer drug conjugates actively targeted by anti-CD38 daratumumab.

B-cell non-Hodgkin lymphomas (B-NHL) represent the most common type of hematologic malignancies in the Western hemisphere. The therapy of all B-NHL is based on the combination of different genotoxic cytostatics and anti-CD20 monoclonal antibody (mAb) rituximab. Unfortunately, many patients relapse after the mentioned front-line treatment approaches. The therapy of patients with relapsed/refractory (R/R) B-NHL represents an unmet medical need. We designed, developed and tested novel actively targeted hybrid mAb-polymer-drug conjugate (APDC) containing anti-CD20, anti-CD38 or anti-CD19 mAbs. Biocompatible copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) with cytostatic agent doxorubicin attached via stimuli-sensitive hydrazone bond were employed for the mAb grafting. Anti-lymphoma efficacy of the APDC nanotherapeutics was evaluated in vivo on a panel of three patient-derived lymphoma xenografts derived from two patients with R/R B-NHL and one patient with so far untreated B-NHL. In both PDX models derived from patients with R/R B-NHL, the targeting with anti-CD38 antibody daratumumab demonstrated highly improved anti-lymphoma efficacy compared to the targeting with anti-CD20 rituximab, two experimental anti-CD19 antibodies and non-targeted controls. The results represent a proof-of-concept of a new algorithm of personalized anti-tumor therapy based on highly innovative APDC biomaterials.

High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle.

Impaired myocardial bioenergetics is a hallmark of many cardiac diseases. There is a need of a simple and reproducible method of assessment of mitochondrial function from small human myocardial tissue samples. In this study we adopted high-resolution respirometry to homogenates of fresh human cardiac muscle and compare it with isolated mitochondria. We used atria resected during cardiac surgery (n = 18) and atria and left ventricles from brain-dead organ donors (n = 12). The protocol we developed consisting of two-step homogenization and exposure of 2.5% homogenate in a respirometer to sequential addition of 2.5 mM malate, 15 mM glutamate, 2.5 mM ADP, 10 µM cytochrome c, 10 mM succinate, 2.5 µM oligomycin, 1.5 µM FCCP, 3.5 µM rotenone, 4 µM antimycin and 1 mM KCN or 100 mM Sodium Azide. We found a linear dependency of oxygen consumption on oxygen concentration. This technique requires < 20 mg of myocardium and the preparation of the sample takes <20 min. Mitochondria in the homogenate, as compared to subsarcolemmal and interfibrillar isolated mitochondria, have comparable or better preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c is up to 11.7±1.8% vs. 15.7±3.1%, p˂0.05 and 11.7±3.5%, p = 0.99, resp.) and better efficiency of oxidative phosphorylation (Respiratory Control Ratio = 3.65±0.5 vs. 3.04±0.27, p˂0.01 and 2.65±0.17, p˂0.0001, resp.). Results are reproducible with coefficient of variation between two duplicate measurements ≤8% for all indices. We found that whereas atrial myocardium contains less mitochondria than the ventricle, atrial bioenergetic profiles are comparable to left ventricle. In conclusion, high resolution respirometry has been adapted to homogenates of human cardiac muscle and shown to be reliable and reproducible.

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles.

INTRODUCTION: Propofol causes a profound inhibition of fatty acid oxidation and reduces spare electron transfer chain capacity in a range of human and rodent cells and tissues-a feature that might be related to the pathogenesis of Propofol Infusion Syndrome. We aimed to explore the mechanism of propofol-induced alteration of bioenergetic pathways by describing its kinetic characteristics. METHODS: We obtained samples of skeletal and cardiac muscle from Wistar rat (n = 3) and human subjects: vastus lateralis from hip surgery patients (n = 11) and myocardium from brain-dead organ donors (n = 10). We assessed mitochondrial functional indices using standard SUIT protocol and high resolution respirometry in fresh tissue homogenates with or without short-term exposure to a range of propofol concentration (2.5-100 µg/ml). After finding concentrations of propofol causing partial inhibition of a particular pathways, we used that concentration to construct kinetic curves by plotting oxygen flux against substrate concentration during its stepwise titration in the presence or absence of propofol. By spectrophotometry we also measured the influence of the same propofol concentrations on the activity of isolated respiratory complexes. RESULTS: We found that human muscle and cardiac tissues are more sensitive to propofol-mediated inhibition of bioenergetic pathways than rat's tissue. In human homogenates, palmitoyl carnitine-driven respiration was inhibited at much lower concentrations of propofol than that required for a reduction of electron transfer chain capacity, suggesting FAO inhibition mechanism different from downstream limitation or carnitine-palmitoyl transferase-1 inhibition. Inhibition of Complex I was characterised by more marked reduction of Vmax, in keeping with non-competitive nature of the inhibition and the pattern was similar to the inhibition of Complex II or electron transfer chain capacity. There was neither inhibition of Complex IV nor increased leak through inner mitochondrial membrane with up to 100 µg/ml of propofol. If measured in isolation by spectrophotometry, propofol 10 µg/ml did not affect the activity of any respiratory complexes. CONCLUSION: In human skeletal and heart muscle homogenates, propofol in concentrations that are achieved in propofol-anaesthetized patients, causes a direct inhibition of fatty acid oxidation, in addition to inhibiting flux of electrons through inner mitochondrial membrane. The inhibition is more marked in human as compared to rodent tissues.

Immunoglobulin abnormalities in 1677 solid organ transplant recipients. Implications for posttransplantation follow-up.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a severe complication of solid organ transplantation (SOT). However, there is no consensus on PTLD screening methods. Gammopathies (GP), which occur in 10-25% of SOT recipients, have been linked to subsequent development of PTLD. Therefore, GP detection methods, such as serum protein electrophoresis (SPE), serum protein immunofixation (SIFE), urine protein immunofixation (UIFE) and the quantitative measurement of serum free light chains (SFLC) are candidate methods for PTLD screening. OBJECTIVE: We aimed to assess the frequency of PTLD and GP, association of GP with subsequent PTLD, allograft loss or death and the diagnostic performance of SPE/SIFE in PTLD screening. The main objective was to explore, whether GP detection methods can be used to enhance the efficiency of PTLD screening and to formulate a concise algorithm for posttransplantation (post-Tx) follow-up. METHODS: We performed a cohort study on 1677 SOT recipients with SPE/SIFE data who underwent kidney, liver, heart, pancreas, Langerhans islets or multiple organ transplantation at the Institute of Clinical and Experimental Medicine between 1966 and 2015. The median (IQR) of follow-up time was 8.0 (4.0-12.0) years. RESULTS: The frequencies of PTLD and GP in SOT recipients were 2.8% and 6.4%, respectively. The frequencies of transient GP, GP of undetermined significance and malignant GP were 33%, 63% and 4% respectively. The median time between SOT and GP detection was 2.0 (interquartile range 1.0-7.0) years. GP was associated with a significantly higher risk of PTLD, allograft loss and death, with hazard ratios (95% confidence intervals) of a 6.06 (2.51-14.64), 2.61 (1.49-4.6) and 1.99 (1.2-3.3), respectively. Additionally, GP was associated with 2.98-fold increased risk of allograft loss in kidney transplant patients. SPE diagnostic sensitivity and specificity for PTLD were 14.8% and 93.9%, respectively. PTLD was diagnosed more often and earlier if SPE/SIFE was included in the post-Tx follow-up. CONCLUSIONS: GP after SOT is associated with a high risk of PTLD, allograft loss and poor survival. The combination of SPE, SIFE, SFLC and UIFE is optimal for GP detection. These methods aid in identifying patients who are at risk for PTLD or allograft damage and should be included in regular post-Tx follow-up.

Cotargeting of BCL2 with Venetoclax and MCL1 with S63845 Is Synthetically Lethal In Vivo in Relapsed Mantle Cell Lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it. EXPERIMENTAL DESIGN: We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples. RESULTS: We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. CONCLUSIONS: Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Impact of jasmonic acid on lignification in the hemp hypocotyl.

Phytohormones are crucial molecules regulating plant development and responses to environmental challenges, including abiotic stresses, microbial and insect attacks. Most notably, phytohormones play important roles in the biosynthesis of lignocellulosics. Jasmonates are involved in secondary growth and secondary metabolism, such as phenylpropanoids and lignin biosyntheses. At the physiological and molecular levels, the actions of phytohormones depend on subtle concentration changes, as well as antagonistic equilibria between two or more of these molecules. In this article, we investigate the consequences of jasmonic acid (JA) spraying on young hemp hypocotyls. First, we show that JA application results in changes in the monomeric composition of lignin. Second, we highlight that, five days after application, JA leads to an increase in salicylic acid (SA) content in hemp hypocotyls. These results are discussed in the light of the known antagonism between JA and SA at both the physiological and molecular levels.

Effective doxorubicin-based nano-therapeutics for simultaneous malignant lymphoma treatment and lymphoma growth imaging.

In this study, we report the in vivo anti-lymphoma efficacy and diagnostic potential of newly designed near-infrared fluorescent dye containing polymer-doxorubicin conjugates using murine models of malignant lymphomas including one cell line-derived xenograft (RAJI) and two patient-derived lymphoma xenografts (VFN-D1 and VFN-M2). Two types of passively targeted conjugates differing in architecture of the polymer backbone were synthesized. One of the conjugates was designed using a single linear polymer chain, and the second was more sophisticated with a star-shaped high-molecular-weight (HMW) polymer employing a dendrimer core. The linear HPMA copolymers were linked to the dendrimer core via a one-point attachment, thus forming a hydrophilic polymer shell. Both polymer-doxorubicin conjugates were long-circulating with reduced side effects. Both polymer prodrugs were designed as stimuli-sensitive systems in which the anti-cancer drug doxorubicin was attached to the hydrophilic copolymers via a pH-labile hydrazone linkage. Such polymer prodrugs were fairly stable in aqueous solutions at pH 7.4, and the drug was readily released in mildly acid environments at pH 5-6.5 by hydrolysis of the hydrazone bonds. In addition, polymers were labelled with near-infrared fluorescent dye enabling long term in vivo visualization. Malignant lymphomas represent the most common type of haematological malignancies. Therapy for the majority of malignant lymphomas consists of multi-agent chemotherapy based on an anthracycline doxorubicin, the most prominent side effect of which is cardiotoxicity. We have demonstrated significant anti-lymphoma efficacy of the polymer-doxorubicin conjugates when compared to equally toxic doses of conventional (unbound) doxorubicin in all tested models. Favourable pharmacokinetics for carried drug and labelled polymer carrier was observed, showing predominant uptake of the drug and polymer itself in the tumour mass. In addition, we have observed a promising diagnostic potential of fluorescently labelled polymer prodrugs. Dynamically analyzed fluorescence intensity over subcutaneously xenografted lymphomas closely corresponded to changes in the lymphoma tumour volumes, thereby enabling a non-invasive assessment of treatment efficacy.