ABSTRACT
During the development of the nervous system, there is an overproduction of neurons and synapses. Hebbian competition between neighboring nerve endings and synapses performing different activity levels leads to their elimination or strengthening. We have extensively studied the involvement of the brain-derived neurotrophic factor-Tropomyosin-related kinase B receptor neurotrophic retrograde pathway, at the neuromuscular junction, in the axonal development and synapse elimination process versus the synapse consolidation. The purpose of this review is to describe the neurotrophic influence on developmental synapse elimination, in relation to other molecular pathways that we and others have found to regulate this process. In particular, we summarize our published results based on transmitter release analysis and axonal counts to show the different involvement of the presynaptic acetylcholine muscarinic autoreceptors, coupled to downstream serine-threonine protein kinases A and C (PKA and PKC) and voltage-gated calcium channels, at different nerve endings in developmental competition. The dynamic changes that occur simultaneously in several nerve terminals and synapses converge across a postsynaptic site, influence each other, and require careful studies to individualize the mechanisms of specific endings. We describe an activity-dependent balance (related to the extent of transmitter release) between the presynaptic muscarinic subtypes and the neurotrophin-mediated TrkB/p75NTR pathways that can influence the timing and fate of the competitive interactions between the different axon terminals. The downstream displacement of the PKA/PKC activity ratio to lower values, both in competing nerve terminals and at postsynaptic sites, plays a relevant role in controlling the elimination of supernumerary synapses. Finally, calcium entry through L- and P/Q- subtypes of voltage-gated calcium channels (both channels are present, together with the N-type channel in developing nerve terminals) contributes to reduce transmitter release and promote withdrawal of the most unfavorable nerve terminals during elimination (the weakest in acetylcholine release and those that have already become silent). The main findings contribute to a better understanding of punishment-rewarding interactions between nerve endings during development. Identifying the molecular targets and signaling pathways that allow synapse consolidation or withdrawal of synapses in different situations is important for potential therapies in neurodegenerative diseases.
ABSTRACT
Age-related conditions, such as sarcopenia, cause physical disabilities for an increasing section of society. At the neuromuscular junction, the postsynaptic-derived neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT-4) have neuroprotective functions and contribute to the correct regulation of the exocytotic machinery. Similarly, presynaptic muscarinic signalling plays a fundamental modulatory function in this synapse. However, whether or not these signalling pathways are compromised in ageing neuromuscular system has not yet been analysed. The present study analyses, through Western blotting, the differences in expression and activation of the main key proteins of the BDNF/NT-4 and muscarinic pathways related to neurotransmission in young versus ageing Extensor digitorum longus (EDL) rat muscles. The main results show an imbalance in several sections of these pathways: (i) a change in the stoichiometry of BDNF/NT-4, (ii) an imbalance of Tropomyosin-related kinase B receptor (TrkB)-FL/TrkB-T1 and neurotrophic receptor p 75 (p75NTR), (iii) no changes in the cytosol/membrane distribution of phosphorylated downstream protein kinase C (PKC)ßI and PKCε, (iv) a reduction in the M2-subtype muscarinic receptor and P/Q-subtype voltage-gated calcium channel, (v) an imbalance of phosphorylated mammalian uncoordinated-18-1 (Munc18-1) (S313) and synaptosomal-associated protein 25 (SNAP-25) (S187), and (vi) normal levels of molecules related to the management of acetylcholine (Ach). Based on this descriptive analysis, we hypothesise that these pathways can be adjusted to ensure neurotransmission rather than undergoing negative alterations caused by ageing. However, further studies are needed to assess this hypothetical suggestion. Our results contribute to the understanding of some previously described neuromuscular functional age-related impairments. Strategies to promote these signalling pathways could improve the neuromuscular physiology and quality of life of older people.
Subject(s)
Aging , Brain-Derived Neurotrophic Factor , Neuromuscular Junction , Receptor, trkB , Signal Transduction , Brain-Derived Neurotrophic Factor/metabolism , Animals , Neuromuscular Junction/metabolism , Aging/metabolism , Rats , Receptor, trkB/metabolism , Nerve Growth Factors/metabolism , Male , Receptors, Muscarinic/metabolism , Synaptic Transmission , Receptors, Nerve Growth Factor/metabolism , Rats, WistarABSTRACT
BACKGROUND: Protein kinase A (PKA) enhances neurotransmission at the neuromuscular junction (NMJ), which is retrogradely regulated by nerve-induced muscle contraction to promote Acetylcholine (ACh) release through the phosphorylation of molecules involved in synaptic vesicle exocytosis (SNAP-25 and Synapsin-1). However, the molecular mechanism of the retrograde regulation of PKA subunits and its targets by BDNF/TrkB pathway and muscarinic signalling has not been demonstrated until now. At the NMJ, retrograde control is mainly associated with BDNF/TrkB signalling as muscle contraction enhances BDNF levels and controls specific kinases involved in the neurotransmission. Neurotransmission at the NMJ is also highly modulated by muscarinic receptors M1 and M2 (mAChRs), which are related to PKA and TrkB signallings. Here, we investigated the hypothesis that TrkB, in cooperation with mAChRs, regulates the activity-dependent dynamics of PKA subunits to phosphorylate SNAP-25 and Synapsin-1. METHODS: To explore this, we stimulated the rat phrenic nerve at 1Hz (30 minutes), with or without subsequent contraction (abolished by µ-conotoxin GIIIB). Pharmacological treatments were conducted with the anti-TrkB antibody clone 47/TrkB for TrkB inhibition and exogenous h-BDNF; muscarinic inhibition with Pirenzepine-dihydrochloride and Methoctramine-tetrahydrochloride for M1 and M2 mAChRs, respectively. Diaphragm protein levels and phosphorylation' changes were detected by Western blotting. Location of the target proteins was demonstrated using immunohistochemistry. RESULTS: While TrkB does not directly impact the levels of PKA catalytic subunits Cα and Cß, it regulates PKA regulatory subunits RIα and RIIß, facilitating the phosphorylation of critical exocytotic targets such as SNAP-25 and Synapsin-1. Furthermore, the muscarinic receptors pathway maintains a delicate balance in this regulatory process. These findings explain the dynamic interplay of PKA subunits influenced by BDNF/TrkB signalling, M1 and M2 mAChRs pathways, that are differently regulated by pre- and postsynaptic activity, demonstrating the specific roles of the BDNF/TrkB and muscarinic receptors pathway in retrograde regulation. CONCLUSION: This complex molecular interplay has the relevance of interrelating two fundamental pathways in PKA-synaptic modulation: one retrograde (neurotrophic) and the other autocrine (muscarinic). This deepens the fundamental understanding of neuromuscular physiology of neurotransmission that gives plasticity to synapses and holds the potential for identifying therapeutic strategies in conditions characterized by impaired neuromuscular communication.
Subject(s)
Brain-Derived Neurotrophic Factor , Cyclic AMP-Dependent Protein Kinases , Neuromuscular Junction , Receptor, trkB , Signal Transduction , Synapsins , Synaptosomal-Associated Protein 25 , Animals , Male , Rats , Brain-Derived Neurotrophic Factor/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Neuromuscular Junction/metabolism , Phosphorylation , Rats, Wistar , Receptor, trkB/metabolism , Receptors, Muscarinic/metabolism , Synapsins/metabolism , Synaptosomal-Associated Protein 25/metabolismABSTRACT
Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon ß-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of ß-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.
Subject(s)
Molecular Dynamics Simulation , Protein Binding , Receptors, CXCR , Humans , Receptors, CXCR/metabolism , Receptors, CXCR/genetics , Binding Sites , Protein Conformation , beta-Arrestin 1/metabolism , beta-Arrestin 1/genetics , Ligands , HEK293 Cells , Mutagenesis, Site-Directed , Allosteric Regulation , Structure-Activity RelationshipABSTRACT
PURPOSE: Early treatment of high-risk smoldering myeloma has been shown to delay progression to multiple myeloma (MM). We conducted this trial with curative intention using a treatment approach employed for newly diagnosed patients with MM. METHODS: Patients with high-risk smoldering myeloma (>50% progression risk at 2 years) and transplant candidates were included and received induction therapy with carfilzomib, lenalidomide, and dexamethasone (KRd), six cycles, followed by high-dose melphalan (200 mg/m2) autologous stem-cell transplantation (HDM-ASCT), two KRd consolidation cycles, and Rd maintenance for 2 years. The primary end point was undetectable measurable residual disease (uMRD) rate by next-generation flow after ASCT. Sustained uMRD 4 years after ASCT was the secondary end point. RESULTS: Between June 2015 and June 2017, 90 patients were included, and 31% met at least one SixtyLightchain MRI (SLiM)-hypercalcemia, renal impairment, anemia, bone disease (CRAB) criterion. After a median follow-up of 70.1 months, 3 months after ASCT, in the intention-to-treat population, 56 (62%) of 90 patients had uMRD, and 4 years later, it was sustained in 29 patients (31%). Five patients progressed to MM, and the 70-month progression rate was 94% (95% CI, 84 to 89). The presence of any SLiM CRAB criteria predicted progression to MM (four of the five patients; hazard ratio, 0.12; 95% CI, 0.14 to 1.13; P = .03). Thirty-six patients showed biochemical progression, and failure to achieve uMRD at the end of treatment predicted it. The 70-month overall survival was 92% (95% CI, 82 to 89). Neutropenia and infections were the most frequent adverse events during treatment, resulting in one treatment-related death. Three second primary malignancies have been reported. CONCLUSION: Although a longer follow-up is needed, this curative approach is encouraging and more effective than active MM, with 31% of the patients maintaining the uMRD 4 years after HDM-ASCT.
ABSTRACT
Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.
Subject(s)
Chemokine CXCL12 , Receptors, CXCR4 , Humans , Receptors, CXCR4/metabolism , Chemokine CXCL12/metabolism , Chemokine CXCL11/metabolism , Signal Transduction , Ligands , Binding, CompetitiveABSTRACT
The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor.
ABSTRACT
Purpose: CAR-T cell therapy has proven to be a disruptive treatment in the hematology field, however, less than 50% of patients maintain long-term response and early predictors of outcome are still inconsistently defined. Here, we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify phenotypic features as early biomarkers associated with toxicity and outcomes. Experimental design: In this study, monitoring by flow cytometry and digital PCR (dPCR), and immunophenotypic characterization of circulating CAR-T cells from 48 patients treated with Tisa-cel or Axi-cel was performed. Results: Validation of the flow cytometry reagent for the detection of CAR-T cells in blood revealed CD19 protein conjugated with streptavidin as the optimal detection method. Kinetics of CAR-T cell expansion in blood confirmed median day of peak expansion at seven days post-infusion by both flow cytometry and digital PCR. Circulating CAR-T cells showed an activated, proliferative, and exhausted phenotype at the time of peak expansion. Patients with increased expansion showed more severe CRS and ICANs. Immunophenotypic characterization of CAR-T cells at the peak expansion identified the increased expression of co-inhibitory molecules PD1 and LAG3 and reduced levels of the cytotoxicity marker CD107a as predictors of a better long-term disease control. Conclusions: These data show the importance of CAR-T cells in vivo monitoring and identify the expression of PD1LAG3 and CD107a as early biomarkers of long-term disease control after CAR-T cell therapy.
Subject(s)
Lymphoma, Large B-Cell, Diffuse , Receptors, Chimeric Antigen , Humans , Receptors, Chimeric Antigen/genetics , Kinetics , B-Lymphocytes/pathology , T-Lymphocytes/pathology , Lymphoma, Large B-Cell, Diffuse/pathologyABSTRACT
Allogeneic hematopoietic stem cell transplantation (HSCT) represents the best therapeutic option for many patients with acute myeloid leukemia (AML). However, relapse remains the main cause of mortality after transplantation. The detection of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) in AML, before and after HSCT, has been described as a powerful predictor of outcome. Nevertheless, multicenter and standardized studies are lacking. A retrospective analysis was performed, including 295 AML patients undergoing HSCT in 4 centers that worked according to recommendations from the Euroflow consortium. Among patients in complete remission (CR), MRD levels prior to transplantation significantly influenced outcomes, with overall (OS) and leukemia free survival (LFS) at 2 years of 76.7% and 67.6% for MRD-negative patients, 68.5% and 49.7% for MRD-low patients (MRD < 0.1), and 50.5% and 36.6% for MRD-high patients (MRD ≥ 0.1) (p < 0.001), respectively. MRD level did influence the outcome, irrespective of the conditioning regimen. In our patient cohort, positive MRD on day +100 after transplantation was associated with an extremely poor prognosis, with a cumulative incidence of relapse of 93.3%. In conclusion, our multicenter study confirms the prognostic value of MRD performed in accordance with standardized recommendations.
ABSTRACT
BACKGROUND: Bidirectional communication between presynaptic and postsynaptic components contribute to the homeostasis of the synapse. In the neuromuscular synapse, the arrival of the nerve impulse at the presynaptic terminal triggers the molecular mechanisms associated with ACh release, which can be retrogradely regulated by the resulting muscle contraction. This retrograde regulation, however, has been poorly studied. At the neuromuscular junction (NMJ), protein kinase A (PKA) enhances neurotransmitter release, and the phosphorylation of the molecules of the release machinery including synaptosomal associated protein of 25 kDa (SNAP-25) and Synapsin-1 could be involved. METHODS: Accordingly, to study the effect of synaptic retrograde regulation of the PKA subunits and its activity, we stimulated the rat phrenic nerve (1 Hz, 30 min) resulting or not in contraction (abolished by µ-conotoxin GIIIB). Changes in protein levels and phosphorylation were detected by western blotting and cytosol/membrane translocation by subcellular fractionation. Synapsin-1 was localized in the levator auris longus (LAL) muscle by immunohistochemistry. RESULTS: Here we show that synaptic PKA Cß subunit regulated by RIIß or RIIα subunits controls activity-dependent phosphorylation of SNAP-25 and Synapsin-1, respectively. Muscle contraction retrogradely downregulates presynaptic activity-induced pSynapsin-1 S9 while that enhances pSNAP-25 T138. Both actions could coordinately contribute to decreasing the neurotransmitter release at the NMJ. CONCLUSION: This provides a molecular mechanism of the bidirectional communication between nerve terminals and muscle cells to balance the accurate process of ACh release, which could be important to characterize molecules as a therapy for neuromuscular diseases in which neuromuscular crosstalk is impaired.
Subject(s)
Neurotransmitter Agents , Synapsins , Animals , Rats , Phosphorylation , Biological Transport , HomeostasisABSTRACT
In recent years, we have studied by immunohistochemistry, intracellular recording, and western blotting the role of the muscarinic acetylcholine receptors (mAChRs; M1, M2, and M4 subtypes) in the mammalian neuromuscular junction (NMJ) during development and in the adult. Here, we evaluate our published data to emphasize the mAChRs' relevance in developmental synaptic elimination and their crosstalk with other metabotropic receptors, downstream kinases, and voltage-gated calcium channels (VGCCs). The presence of mAChRs in the presynaptic membrane of motor nerve terminals allows an autocrine mechanism in which the secreted acetylcholine influences the cell itself in feedback. mAChR subtypes are coupled to different downstream pathways, so their feedback can move in a broad range between positive and negative. Moreover, mAChRs allow direct activity-dependent interaction through ACh release between the multiple competing axons during development. Additional regulation from pre- and postsynaptic sites (including neurotrophic retrograde control), the agonistic and antagonistic contributions of adenosine receptors (AR; A1 and A2A), and the tropomyosin-related kinase B receptor (TrkB) cooperate with mAChRs in the axonal competitive interactions which lead to supernumerary synapse elimination that achieves the optimized monoinnervation of musculoskeletal cells. The metabotropic receptor-driven balance between downstream PKA and PKC activities, coupled to developmentally regulated VGCC, explains much of how nerve terminals with different activities finally progress to their withdrawal or strengthening.
Subject(s)
Axons , Neuromuscular Junction , Animals , Neuromuscular Junction/metabolism , Axons/metabolism , Receptors, Muscarinic/metabolism , Acetylcholine/metabolism , Calcium Channels/metabolism , Mammals/metabolismABSTRACT
Low-cost sensors can provide inaccurate data as temperature and humidity affect sensor accuracy. Therefore, calibration and data correction are essential to obtain reliable measurements. This article presents a training and testing method used to calibrate a sensor module assembled from SO2 and NO2 electrochemical sensors (Alphasense B4 and B43F) alongside air temperature (T) and humidity (RH) sensors. Field training and testing were conducted in the industrialized coastal area of Quintero Bay, Chile. The raw responses of the electrochemical (mV) and T-RH sensors were subjected to multiple linear regression (MLR) using three data segments, based on either voltage (SO2 sensor) or temperature (NO2). The resulting MLR equations were used to estimate the reference concentration. In the field test, calibration improved the performance of the sensors after adding T and RH in a linear model. The most robust models for NO2 were associated with data collected at T < 10 °C (R2 = 0.85), while SO2 robust models (R2 = 0.97) were associated with data segments containing higher voltages. Overall, this training and testing method reduced the bias due to T and HR in the evaluated sensors and could be replicated in similar environments to correct raw data from low-cost electrochemical sensors. A calibration method based on training and sensor testing after relocation is presented. The results show that the SO2 sensor performed better when modeled for different segments of voltage data, and the NO2 sensor model performed better when calibrated for different temperature data segments.
Subject(s)
Air Pollutants , Nitrogen Dioxide , Air Pollutants/analysis , Calibration , Environmental Monitoring , Humidity , Nitrogen Dioxide/analysisABSTRACT
During the nervous system development, synapses are initially overproduced. In the neuromuscular junction (NMJ) however, competition between several motor nerve terminals and the synapses they made ends with the maturation of only one axon. The competitive signaling between axons is mediated by the differential activity-dependent release of the neurotransmitter ACh, co-transmitters, and neurotrophic factors. A multiple metabotropic receptor-driven downstream balance between PKA and PKC isoforms modulates the phosphorylation of targets involved in transmitter release and nerve terminal stability. Previously, we observed in the weakest endings on the polyinnervated NMJ that M1 mAChR receptors reduce ACh release through the PKC pathway coupled to an excess of Ca2+ inflow through P/Q- N- and L-type voltage-gated calcium channels (VGCC). This signaling would contribute to the elimination of this nerve terminal. Here, we investigate the involvement of the P/Q-, N-, and L-subtype channels in transgenic B6.Cg-Tg (Thy1-YFP)16-Jrs/J mice during synapse elimination. Then, the axon number and postsynaptic receptor cluster morphologic maturation were evaluated. The results show that both L- and P/Q-type VGCC (but not the N-type) are equally involved in synapse elimination. Their normal function favors supernumerary axonal loss by jointly enhancing intracellular calcium [Ca2+]i. The block of these VGCCs or [Ca2+]i i sequestration results in the same delay of axonal loss as the cPKCßI and nPKCε isoform block or PKA activation. The specific block of the muscle cell's contraction with µ-conotoxin GIIIB also delays synapse maturation, and thus, a retrograde influence from the postsynaptic site regulating the presynaptic CaV1.3 may contribute to the synapse elimination.
Subject(s)
Calcium Channels , Neuromuscular Junction , Animals , Axons/metabolism , Calcium/metabolism , Calcium Channels/metabolism , Mice , Neuromuscular Junction/metabolism , Protein Isoforms/metabolism , Signal Transduction , Synapses/metabolismABSTRACT
The use of reduced-intensity conditioning (RIC) regimens has decreased the risk of nonrelapse mortality (NRM) after allogeneic stem cell transplantation (alloSCT). In contrast, disease relapse remains the most frequent cause of treatment failure and death. Owing to both their antimyeloma effect and immunomodulatory properties, novel drugs could improve outcomes after alloSCT. This phase II European Myeloma Network trial was designed to evaluate the combination of alloSCT with novel agents. The study was conducted to evaluate the toxicity and efficacy of RIC intensified with bortezomib (Bz) prior to alloSCT for high-risk (HR) multiple myeloma (MM) patients, as well as the efficacy of post-transplantation maintenance with Bz and lenalidomide (Len). Patients received RIC with Bz on days -9 and -2, fludarabine on days -6 to -4, and melphalan on day -3. Patients who were in complete response (CR) or near CR at day +100 post-transplantation received 6 cycles of Bz every 56 days, and the remaining received Bz, Len, and dexamethasone. Len maintenance was started on day +180 at a dose of 5 mg and continued until relapse or toxicity occurred. Of the 24 patients included, 21 were evaluable on day +100, including 12 in CR, 4 in very good partial response, 3 in partial response, and 2 with relapse or progression. The cumulative incidence (CuI) of relapse was 13.6% (95% confidence interval [CI], 3.2% to 31.3%) at 1 year and 28.5% (95% CI, 11.1% to 48.9%) at 2 years. The CuI of NRM was 21.1% (95% CI, 7.4% to 39.4%) at 2 years. With a median follow-up of 39 months (range, 1 to 67 months), the median event-free survival (EFS) was 29 months, and median overall survival (OS) was not reached. EFS and OS at 3 years were 42.5% (95% CI, 21.9% to 61.7%) and 74.01% (95% CI, 50.9% to 87.5%), respectively. The use of Bz within an RIC regimen allows for a high response rate after alloSCT. Maintenance with Bz and Len is feasible and provides remarkable results in terms of EFS and OS in HR MM patients.
Subject(s)
Multiple Myeloma , Bortezomib/therapeutic use , Humans , Lenalidomide/therapeutic use , Multiple Myeloma/drug therapy , Neoplasm Recurrence, Local/drug therapy , Transplantation Conditioning/methods , Transplantation, HomologousABSTRACT
At the neuromuscular junction (NMJ), motor neurons and myocytes maintain a bidirectional communication that guarantees adequate functionality. Thus, motor neurons' firing pattern, which is influenced by retrograde muscle-derived neurotrophic factors, modulates myocyte contractibility. Myocytes can be fast-twitch fibers and become easily fatigued or slow-twitch fibers and resistant to fatigue. Extraocular muscles (EOM) show mixed properties that guarantee fast contraction speed and resistance to fatigue and the degeneration caused by Amyotrophic lateral sclerosis (ALS) disease. The TrkB signaling is an activity-dependent pathway implicated in the NMJ well-functioning. Therefore, it could mediate the differences between fast and slow myocytes' resistance to fatigue. The present study elucidates a specific protein expression profile concerning the TrkB signaling that correlates with higher resistance to fatigue and better neuroprotective capacity through time. The results unveil that Extra-ocular muscles (EOM) express lower levels of NT-4 that extend TrkB signaling, differential PKC expression, and a higher abundance of phosphorylated synaptic proteins that correlate with continuous neurotransmission requirements. Furthermore, common molecular features between EOM and slow soleus muscles including higher neurotrophic consumption and classic and novel PKC isoforms balance correlate with better preservation of these two muscles in ALS. Altogether, higher resistance of Soleus and EOM to fatigue and ALS seems to be associated with specific protein levels concerning the TrkB neurotrophic signaling.
ABSTRACT
Renal impairment (RI) is common in patients with multiple myeloma (MM) and new therapies that can improve renal function are needed. The phase III IKEMA study (clinicaltrials gov. Identifier: NCT03275285) investigated isatuximab (Isa) with carfilzomib and dexamethasone (Kd) versus Kd in relapsed MM. This subgroup analysis examined results from patients with RI, defined as estimated glomerular filtration rate <60 mL/min/1.73 m². Addition of Isa prolonged progression-free survival (PFS) in patients with RI (hazard ratio: 0.27; 95% confidence interval [CI]: 0.11-0.66; median PFS not reached for Isa-Kd versus 13.4 months for Kd [20.8-month follow-up]). Complete renal responses occurred more frequently with Isa-Kd (52.0%) versus Kd (30.8%) and were durable in 32.0% versus 7.7% of patients, respectively. Treatment exposure was longer with Isa-Kd, with median number of started cycles and median duration of exposure of 20 versus 9 cycles and 81.0 versus 35.7 weeks for Isa-Kd versus Kd, respectively. Among patients with RI, the incidence of patients with grade ≥3 treatment-emergent adverse events was similar between the two arms (79.1% in Isa-Kd vs. 77.8% in Kd). In summary, the addition of Isa to Kd improved clinical outcomes with a manageable safety profile in patients with RI, consistent with the benefit observed in the overall IKEMA study population.
Subject(s)
Multiple Myeloma , Renal Insufficiency , Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Dexamethasone/adverse effects , Humans , Multiple Myeloma/complications , Multiple Myeloma/drug therapy , Oligopeptides , Renal Insufficiency/complicationsABSTRACT
Due to the continuous monitoring process of critical patients, Intensive Care Units (ICU) generate large amounts of data, which are difficult for healthcare personnel to analyze manually, especially in overloaded situations such as those present during the COVID-19 pandemic. Therefore, the automatic analysis of these data has many practical applications in patient monitoring, including the optimization of alarm systems for alerting healthcare personnel. In this paper, explainable machine learning techniques are used for this purpose, with a methodology based on age-stratification, boosting classifiers, and Shapley Additive Explanations (SHAP) proposed. The methodology is evaluated using MIMIC-III, an ICU patient research database. The results show that the proposed model can predict mortality within the ICU with AUROC values of 0.961, 0.936, 0.898, and 0.883 for age groups 18-45, 45-65, 65-85 and 85+, respectively. By using SHAP, the features with the highest impact in predicting mortality for different age groups and the threshold from which the value of a clinical feature has a negative impact on the patient's health can be identified. This allows ICU alarms to be improved by identifying the most important variables to be sensed and the threshold values at which the health personnel must be warned.
Subject(s)
COVID-19 , Pandemics , Humans , Intensive Care Units , Machine Learning , SARS-CoV-2ABSTRACT
The Exome Aggregation Consortium has collected the protein-encoding DNA sequences of almost 61,000 unrelated humans. Analysis of this dataset for G protein-coupled receptor (GPCR) proteins (available at GPCRdb) revealed a total of 463 naturally occurring genetic missense variations in the histamine receptor family. In this research, we have analyzed the distribution of these missense variations in the four histamine receptor subtypes concerning structural segments and sites important for GPCR function. Four missense variants R1273.52×52H, R13934.57×57H, R4096.29×29H, and E4106.30×30K, were selected for the histamine H1 receptor (H1R) that were hypothesized to affect receptor activity by interfering with the interaction pattern of the highly conserved D(E)RY motif, the so-called ionic lock. The E4106.30×30K missense variant displays higher constitutive activity in G protein signaling as compared to wild-type H1R, whereas the opposite was observed for R1273.52×52H, R13934.57×57H, and R4096.29×29H. The E4106.30×30K missense variant displays a higher affinity for the endogenous agonist histamine than wild-type H1R, whereas antagonist affinity was not affected. These data support the hypothesis that the E4106.30×30K mutation shifts the equilibrium towards active conformations. The study of these selected missense variants gives additional insight into the structural basis of H1R activation and, moreover, highlights that missense variants can result in pharmacologically different behavior as compared to wild-type receptors and should consequently be considered in the drug discovery process.
Subject(s)
Receptors, Histamine H1/genetics , GTP-Binding Proteins/metabolism , HEK293 Cells , Humans , Mutation, Missense , Receptors, Histamine H1/metabolismABSTRACT
Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.