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During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the α6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax α6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family. [ABSTRACT FROM AUTHOR]

Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca 2+ , across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca 2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca 2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca 2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

In an investigation of pharmaceutical contamination in the Lac du Flambeau Chain of Lakes (hereafter referred to as "the Chain"), few contaminants were detected; only eight pharmaceuticals and one pesticide were identified among the 110 pharmaceuticals and other organic contaminants monitored in surface water samples. This study, conducted in cooperation with the Lac du Flambeau Tribe's Water Resource Program, investigated these organic contaminants and potential biological effects in channels connecting lakes throughout the Chain, including the Moss Lake Outlet site, adjacent to the wastewater treatment plant lagoon. Of the 6 sites monitored and 24 samples analyzed, sample concentrations and contaminant detection frequencies were greatest at the Moss Lake Outlet site; however, the concentrations and detection frequencies of this study were comparable to other pharmaceutical investigations in basins with similar characteristics. Because established water-quality benchmarks do not exist for the pharmaceuticals detected in this study, alternative screening-level water-quality benchmarks, developed using two U.S. Environmental Protection Agency toxicological resources (ToxCast database and ECOTOX knowledgebase), were used to estimate potential biological effects associated with the observed contaminant concentrations. Two contaminants (caffeine and thiabendazole) exceeded the prioritization threshold according to ToxCast alternative benchmarks, and four contaminants (acetaminophen, atrazine, caffeine, and carbamazepine) exceeded the prioritization threshold according to ECOTOX alternative benchmarks. Atrazine, an herbicide, was the most frequently detected contaminant (79% of samples), and it exhibited the strongest potential for biological effects due to its high estimated potency. Insufficient toxicological information within ToxCast and ECOTOX for gabapentin and methocarbamol (which had the two greatest concentrations in this study) precluded alternative benchmark development. This data gap presents unknown potential environmental impacts. Future research examining the biological effects elicited by these two contaminants as well as the others detected in this study would further elucidate the ecological relevance of the water chemistry results generated though this investigation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

SARS-CoV-2 infection, and resulting disease, COVID-19, has a high mortality amongst patients with haematological malignancies. Global vaccine rollouts have reduced hospitalisations and deaths, but vaccine efficacy in patients with haematological malignancies is known to be reduced. The UK-strategy offered a third, mRNA-based, vaccine as an extension to the primary course in these patients. The MARCH database is a retrospective observational study of serological responses in patients with blood disorders. Here we present data on 381 patients with haematological malignancies. By comparison with healthy controls, we report suboptimal responses following two primary vaccines, with significantly enhanced responses following the third primary dose. These responses however are heterogeneous and determined by haematological malignancy sub-type and therapy. We identify a group of patients with continued suboptimal vaccine responses who may benefit from additional doses, prophylactic extended half-life neutralising monoclonal therapies (nMAB) or prompt nMAB treatment in the event of SARS-CoV-2 infection., (© 2022. The Author(s).)

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1ɑ droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin's biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates., (© 2022. The Author(s).)

Study Objectives: The main cause of death in patients with obesity hypoventilation syndrome (OHS) is cardiac rather than respiratory failure. Here, we investigated autonomic-respiratory coupling and serum cardiac biomarkers in patients with OHS and obstructive sleep apnea (OSA) with comparable body mass index and apnea-hypopnea index., Methods: Cardiopulmonary coupling (CPC) and cyclic variation of heart rate analysis was performed on the electrocardiogram signal from the overnight polysomnogram. Cardiac serum biomarkers were obtained in patients with OHS and OSA with a body mass index > 40 kg/m 2 . Samples were obtained at baseline and after 3 months of positive airway pressure (PAP) therapy in both groups., Results: Patients with OHS (n = 15) and OSA (n = 36) were recruited. No group differences in CPC, cyclic variation of heart rate, and serum biomarkers were observed at baseline and after 3 months of PAP therapy. An improvement in several CPC metrics, including the sleep apnea index, unstable sleep (low-frequency coupling and elevated low-frequency coupling narrow band), and cyclic variation of heart rate were observed in both groups with PAP use. However, distinct differences in response characteristics were noted. Elevated low-frequency coupling narrow band coupling correlated with highly sensitive troponin-T ( P  < .05) in the combined cohort. Baseline highly sensitive troponin-T inversely correlated with awake oxygen saturation in the OHS group ( P  < .05)., Conclusions: PAP therapy can significantly improve CPC stability in patients with obesity with OSA or OHS, with key differences. Elevated low-frequency coupling narrow band may function as a surrogate biomarker for early subclinical cardiac disease. Low awake oxygen saturation could also increase this biomarker in OHS., Clinical Trial Registration: Registry: Australian New Zealand Clinical Trials Registry; Name: Obesity Hypoventilation Syndrome and Neurocognitive Dysfunction; URL: https://anzctr.org.au/Trial/Registration/TrialReview.aspx?id=367492; Identifier: ACTRN12615000122550., Citation: Sivam S, Wang D, Wong KKH, et al. Cardiopulmonary coupling and serum cardiac biomarkers in obesity hypoventilation syndrome and obstructive sleep apnea with morbid obesity. J Clin Sleep Med . 2022;18(4):1063-1071., (© 2022 American Academy of Sleep Medicine.)

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling., (© 2021. The Author(s).)

Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies., Competing Interests: Conflict of interest—The authors declare no conflict of interest., (© 2020 Lautenschläger et al.)

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1,2] and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGCs). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications., (© 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chromosomal translocations represent frequent events in leukemia. In t(8;21)+ acute myeloid leukemia, RUNX1 is fused to nearly the entire ETO protein, which contains four conserved nervy homology regions, NHR1-4. Furthermore RUNX1/ETO interacts with ETO-homologous proteins via NHR2, thereby multiplying NHR domain contacts. As shown recently, RUNX1/ETO retains oncogenic activity upon either deletion of the NHR3 + 4 N-CoR/SMRT interaction domain or substitution of the NHR2 tetramer domain. Thus, we aimed to clarify the specificities of the NHR domains. A C-terminally NHR3 + 4 truncated RUNX1/ETO containing a heterologous, structurally highly related non-NHR2 tetramer interface translocated into the nucleus and bound to RUNX1 consensus motifs. However, it failed to interact with ETO-homologues, repress RUNX1 targets, and transform progenitors. Surprisingly, transforming capacity was fully restored by C-terminal fusion with ETO's NHR4 zinc-finger or the repressor domain 3 of N-CoR, while other repression domains failed. With an inducible protein assembly system, we further demonstrated that NHR4 domain activity is critically required early in the establishment of progenitor cultures expressing the NHR2 exchanged truncated RUNX1/ETO. Together, we can show that NHR2 and NHR4 domains can be replaced by heterologous protein domains conferring tetramerization and repressor functions, thus showing that the NHR2 and NHR4 domain structures do not have irreplaceable functions concerning RUNX1/ETO activity for the establishment of human CD34+ cell expansion. We could resemble the function of RUNX1/ETO through modular recomposition with protein domains from RUNX1, ETO, BCR and N-CoR without any NHR2 and NHR4 sequences. As most transcriptional repressor proteins do not comprise tetramerization domains, our results provide a possible explanation as to the reason that RUNX1 is recurrently found translocated to ETO family members, which all contain tetramer together with transcriptional repressor moieties.

Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.

The prosurvival Bcl-2 family proteins Mcl-1 and Bcl-x L inhibit apoptosis by sequestering BH3-only proteins such as Bid and Bim (MODE 1) or the effector proteins Bak and Bax (MODE 2). To better understand the contributions of MODE 1 and MODE 2 in blocking cell death, and thus how to bypass resistance to cell death, we examined prescribed mixtures of Bcl-2 family proteins. In a Bim and Bak mixture, Bcl-x L and Mcl-1 each sequestered not only Bim but also Bak as it became activated by Bim. In contrast, in a Bid and Bak mixture, Bcl-x L preferentially sequestered Bid while Mcl-1 preferentially sequestered Bak. Notably, Bcl-x L could sequester Bak in response to the BH3 mimetic ABT-737, despite this molecule targeting Bcl-x L . These findings highlight the importance of Bak sequestration in resistance to anti-cancer treatments, including BH3 mimetics.

Understanding the factors that affect water quality and the ecological services provided by freshwater ecosystems is an urgent global environmental issue. Predicting how water quality will respond to global changes not only requires water quality data, but also information about the ecological context of individual water bodies across broad spatial extents. Because lake water quality is usually sampled in limited geographic regions, often for limited time periods, assessing the environmental controls of water quality requires compilation of many data sets across broad regions and across time into an integrated database. LAGOS-NE accomplishes this goal for lakes in the northeastern-most 17 US states.LAGOS-NE contains data for 51 101 lakes and reservoirs larger than 4 ha in 17 lake-rich US states. The database includes 3 data modules for: lake location and physical characteristics for all lakes; ecological context (i.e., the land use, geologic, climatic, and hydrologic setting of lakes) for all lakes; and in situ measurements of lake water quality for a subset of the lakes from the past 3 decades for approximately 2600-12 000 lakes depending on the variable. The database contains approximately 150 000 measures of total phosphorus, 200 000 measures of chlorophyll, and 900 000 measures of Secchi depth. The water quality data were compiled from 87 lake water quality data sets from federal, state, tribal, and non-profit agencies, university researchers, and citizen scientists. This database is one of the largest and most comprehensive databases of its type because it includes both in situ measurements and ecological context data. Because ecological context can be used to study a variety of other questions about lakes, streams, and wetlands, this database can also be used as the foundation for other studies of freshwaters at broad spatial and ecological scales., (© The Author 2017. Published by Oxford University Press.)

BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain., Competing Interests: The authors declare no conflict of interest.

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak., (© 2016 Lee et al.; Published by Cold Spring Harbor Laboratory Press.)

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)