Decoding Cell Death: From a Veritable Library of Babel to Vade Mecum?

Programmed cell death (PCD) is a requisite feature of development and homeostasis but can also be indicative of infections, injuries, and pathologies. In concordance with these heterogeneous contexts, an array of disparate effector responses occur downstream of cell death and its clearance-spanning tissue morphogenesis, homeostatic turnover, host defense, active dampening of inflammation, and tissue repair. This raises a fundamental question of how a single contextually appropriate response ensues after an event of PCD. To explore how complex inputs may together tailor the specificity of the resulting effector response, here we consider (a) the varying contexts during which different cell death modalities are observed, (b) the nature of the information that can be passed on by cell corpses, and (c) the ways by which efferocyte populations synthesize signals from dying cells with those from the surrounding microenvironment.

When Visual Circuits Collide: Motion Processing in the Brain.

How is sensory information transformed by each station of a synaptic circuit as it flows progressively deeper into the brain? In this issue of Cell, Mauss et al. describe a set of connections in the fly brain that combines opposing directional signals, and they hypothesize that this motif limits global motion noise as the fly moves through space.

Cenabis Bene: Treg Cells Invite Macrophages to Dine.

Resolution of the immune response requires a coordinated effort to dampen inflammatory mediators and remove dying cells and debris. In this issue of Immunity, Proto et al. (2018) describe a circuit by which regulatory T cells enhance macrophage consumption of apoptotic cells during resolution.

Induction of the Mdm2 gene and protein by kinase signaling pathways is repressed by the pVHL tumor suppressor.

The tumor suppressor von Hippel-Lindau, pVHL, is a multifaceted protein. One function is to dock to the hypoxia-inducible transcription factor (HIF) and recruit a larger protein complex that destabilizes HIF via ubiquitination, preventing angiogenesis and tumor development. pVHL also binds to the tumor suppressor p53 to activate specific p53 target genes. The oncogene Mdm2 impairs the formation of the p53-pVHL complex and activation of downstream genes by conjugating nedd8 to pVHL. While Mdm2 can impact p53 and pVHL, how pVHL may impact Mdm2 is unclear. Like p53 somatic mutations, point mutations are evident in pVHL that are common in renal clear cell carcinomas (RCC). In patients with RCC, Mdm2 levels are elevated, and we examined whether there was a relationship between Mdm2 and pVHL. TCGA and DepMap analysis revealed that mdm2 gene expression was elevated in RCC with vhl point mutations or copy number loss. In pVHL reconstituted or deleted isogenetically match RCC or MEF cell lines, Mdm2 was decreased in the presence of pVHL. Furthermore, through analysis using genetic and pharmacological approaches, we show that pVHL represses Mdm2 gene expression by blocking the MAPK-Ets signaling pathway and blocks Akt-mediated phosphorylation and stabilization of Mdm2. Mdm2 inhibition results in an increase in the p53-p21 pathway to impede cell growth. This finding shows how pVHL can indirectly impact the function of Mdm2 by regulating signaling pathways to restrict cell growth.

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin.

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

Activation of the Snail transcription factor induces Mdm2 gene expression.

Epithelial-like tumor cells can become metastatic by undergoing molecular and phenotypic reprogramming in a process referred to as epithelial-to-mesenchymal transition (EMT). In response to EMT genes that promote migration and condition the tumor microenvironment to permit intravasation into the bloodstream, dissemination, and extravasation into new organs are induced. While the mutant p53 has been implicated in extravasation, one negative regulator of p53, the oncogene Mdm2, is required in the early stages of metastasis and the driver of EMT. This activity is independent of Mdm2's role in the p53-Mdm2 autoregulatory feedback loop. Herein, we examine the EMT transcription factor Snail as a downstream effector of kinase signaling pathways. We show that the activation of upstream receptors and KRas signaling increase Snail levels. Snail binds to Ebox DNA motifs, and Mdm2 has two Ebox DNA binding domains in the second promoter. Snail binds to the second Ebox and induces Mdm2 gene expression. Knockdown of endogenous Snail by shRNA shows a decrease in Mdm2 and is associated with reduced migration. The reintroduction of Mdm2 in shSnail cells restores cellular migration. These data integrate upstream pathways that induce Snail-Mdm2 to promote the metastasis of tumor cells.

High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid.

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

A midline thalamic circuit determines reactions to visual threat.

How our internal state is merged with our visual perception of an impending threat to drive an adaptive behavioural response is not known. Mice respond to visual threats by either freezing or seeking shelter. Here we show that nuclei of the ventral midline thalamus (vMT), the xiphoid nucleus (Xi) and nucleus reuniens (Re), represent crucial hubs in the network controlling behavioural responses to visual threats. The Xi projects to the basolateral amygdala to promote saliency-reducing responses to threats, such as freezing, whereas the Re projects to the medial prefrontal cortex (Re→mPFC) to promote saliency-enhancing, even confrontational responses to threats, such as tail rattling. Activation of the Re→mPFC pathway also increases autonomic arousal in a manner that is rewarding. The vMT is therefore important for biasing how internal states are translated into opposing categories of behavioural responses to perceived threats. These findings may have implications for understanding disorders of arousal and adaptive decision-making, such as phobias, post-traumatic stress and addictions.

Neuronal ROS-induced glial lipid droplet formation is altered by loss of Alzheimer's disease-associated genes.

A growing list of Alzheimer's disease (AD) genetic risk factors is being identified, but the contribution of each variant to disease mechanism remains largely unknown. We have previously shown that elevated levels of reactive oxygen species (ROS) induces lipid synthesis in neurons leading to the sequestration of peroxidated lipids in glial lipid droplets (LD), delaying neurotoxicity. This neuron-to-glia lipid transport is APOD/E-dependent. To identify proteins that modulate these neuroprotective effects, we tested the role of AD risk genes in ROS-induced LD formation and demonstrate that several genes impact neuroprotective LD formation, including homologs of human ABCA1, ABCA7, VLDLR, VPS26, VPS35, AP2A, PICALM, and CD2AP Our data also show that ROS enhances Aß42 phenotypes in flies and mice. Finally, a peptide agonist of ABCA1 restores glial LD formation in a humanized APOE4 fly model, highlighting a potentially therapeutic avenue to prevent ROS-induced neurotoxicity. This study places many AD genetic risk factors in a ROS-induced neuron-to-glia lipid transfer pathway with a critical role in protecting against neurotoxicity.

The Origin of the Problem: Characterization of Paraguayan Septoria steviae, Causal Agent of Septoria Leaf Spot of Stevia, Based on Multilocus Sequence Analysis.

Septoria leaf spot is a significant disease affecting cultivated stevia, potentially reducing yields by > 50%. The disease is caused by Septoria steviae, first identified in 1978 in Japan as a new pathogen of stevia. Understanding the origin of S. steviae could clarify how it spread to new production areas. To investigate this, 12 isolates of Septoria sp. were obtained from stevia's native range in the Amambay forests and field plantings in Paraguay from 2018 to 2020. These isolates underwent colony morphology and molecular characterization of Actin, ß-Tubulin, Calmodulin, ITS, LSU, RPB2, and TEF1α loci. GenBank sequences from S. steviae isolates collected in France, Japan, and the United States were included. Multilocus sequence phylogenetic analysis generated a maximum likelihood (ML) tree. The morphological characteristics of Paraguayan isolates were similar to those of previously reported S. steviae type cultures from Japan. The ML analysis showed that Paraguayan isolates formed a monophyletic group with S. steviae isolates from France, Japan, and the United States. During blotter tests, pycnidia and cirri of S. steviae were observed on multiple stevia seed surfaces from different sources. Further characterization confirmed viable pathogenic conidia of S. steviae. This observation suggests that S. steviae could be associated with stevia seed, possibly spreading from the center of origin to other countries. This research is the first to genetically characterize S. steviae from Paraguay and propose its potential spread mechanism from the center of origin to the rest of the world.

Occurrence and Distribution of Meloidogyne spp. in Fields Rotated with Sweetpotato and Host Range of a North Carolina Population of Meloidogyne enterolobii.

Root-knot nematodes (RKNs, Meloidogyne spp.) are some of the most economically important and common plant parasitic nematodes in North Carolina (NC) cropping systems. Soil samples collected from fields planted with crops rotated with sweetpotato (Ipomoea batatas [L.] Lam.) in 39 NC counties in 2015 to 2018 were processed at the NC Nematode Assay Laboratory. The occurrence of second-stage juvenile (J2) RKN populations was examined based on collection year, month, county, and previous planted crop. The highest number of RKN-positive samples originated from Cumberland (53%), Sampson (48%), and Johnston (48%) counties. The highest average RKN population density was detected in Sampson (147 J2/500 cm3 of soil) and Nash (135 J2/500 cm3 of soil) counties, while Wayne (7 J2/500 cm3 of soil) and Greene (11 J2/500 cm3 of soil) counties had the lowest average RKN population density. Meloidogyne enterolobii is a new invasive species that is impacting sweetpotato growers of NC. The host status of an NC population of M. enterolobii, the guava RKN, was determined by examining eggs per gram of fresh root (ER) and the final nematode egg population divided by the initial population egg count (reproductive factor, RF) in greenhouse experiments. This included 18 vegetable, field, and cover crops and weed species. The tomato 'Rutgers' was used as a susceptible control. Cabbage 'Stonehead', pepper 'Red Bull', and watermelon 'Charleston Gray' and 'Fascination' were hosts and had similar mean ER values to the positive control, ranging from 64 to 18,717. Among field crops, cotton, soybean 'P5018RX', and tobacco were hosts with ER values that ranged from 185 to 706. Members of the Poaceae family such as sweet corn (Zea mays) and sudangrass (Sorghum × drummondii) were nonhosts to M. enterolobii, and the mean ER values ranged from 1.85 to 7. The peanut 'Tifguard' and winter wheat (Triticum aestivum) also had lower ER values than the vegetable hosts. Growers should consider planting less susceptible hosts or nonhosts such as peanut, sudangrass, sweet corn, and winter wheat in 2- to 3-year crop rotations to lower populations of this invasive nematode.

New Roles for Canonical Transcription Factors in Repeat Expansion Diseases.

The presence of microsatellite repeat expansions within genes is associated with >30 neurological diseases. Of interest, (GGGGCC)>30-repeats within C9orf72 are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). These expansions can be 100s to 1000s of units long. Thus, it is perplexing how RNA-polymerase II (RNAPII) can successfully transcribe them. Recent investigations focusing on GGGGCC-transcription have identified specific, canonical complexes that may promote RNAPII-transcription at these GC-rich microsatellites: the DSIF complex and PAF1C. These complexes may be important for resolving the unique secondary structures formed by GGGGCC-DNA during transcription. Importantly, this process can produce potentially toxic repeat-containing RNA that can encode potentially toxic peptides, impacting neuron function and health. Understanding how transcription of these repeats occurs has implications for therapeutics in multiple diseases.

Validation of Standard Area Diagrams to Estimate the Severity of Septoria Leaf Spot on Stevia in Paraguay, Mexico, and the United States.

Septoria leaf spot (SLS) affects stevia leaves, reducing their quality. Estimates of SLS severity on different genotypes are made to identify resistance and as a basis to compare management approaches. The use of standard area diagrams (SADs) can improve the accuracy and reliability of severity estimates. In this study, we developed new SADs with six illustrations (0.5, 1, 10, 25, 40, and 75% severity). The SADs were validated by raters with and without experience in estimating SLS. Raters evaluated 40 leaf photos with SLS severities ranging from 0 to 100% without and with the SADs. Agreement (ρc), bias (Cb), precision (r), and intracluster correlation (ρ) coefficients were significantly closer to "true" severity values when the SADs was used by inexperienced (ρc = 0.89; Cb = 0.97; r = 0.90, ρ = 0.81) and experienced (ρc = 0.94; Cb = 0.99; r = 0.95, ρ = 0.91) raters. The SADs were tested under field conditions in Paraguay, Mexico, and the United States, with inexperienced raters assigned to two groups, one SADs trained and the other not trained, that estimated SLS severity three times: first, all raters without SADs and no time limit for the estimates; second, only the SADs-trained group used SADs and no time limit; and third, only the SADs-trained group used SADs, with a time limit of 10 s imposed per specimen assessment. Agreement and reliability of SLS severity estimates significantly improved when raters used the SADs without a time limit. The use of the new SADs improved the accuracy, precision, and reliability of SLS severity estimates, enhancing the uniformity in assessment across different stevia programs.

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

Effects of Formulation on the Palatability and Efficacy of In-Feed Praziquantel Medications for Marine Finfish Aquaculture.

Praziquantel (PZQ) provides an effective treatment against monogenean parasitic infestations in finfish. However, its use as an in-feed treatment is challenging due to palatability issues. In this study, five formulations of PZQ beads (1−4 mm) were developed using marine-based polymers, with allicin added as a flavouring agent. All formulations attained PZQ loading rates ≥74% w/w, and the beads were successfully incorporated into fish feed pellets at an active dietary inclusion level of 10 g/kg. When tested for palatability and digestibility in small yellowtail kingfish, the PZQ-loaded beads produced with alginate-chitosan, alginate-Cremophor® RH40, and agar as carriers resulted in high consumption rates of 99−100% with no digesta or evidence of beads in the gastrointestinal tract (GIT) of fish fed with diets containing either formulation. Two formulations produced using chitosan-based carriers resulted in lower consumption rates of 68−75%, with undigested and partly digested beads found in the fish GIT 3 h post feeding. The PZQ-loaded alginate-chitosan and agar beads also showed good palatability in large (≥2 kg) yellowtail kingfish infected with gill parasites and were efficacious in removing the parasites from the fish, achieving >90% reduction in mean abundance relative to control fish (p < 0.001). The two effective formulations were stable upon storage at ambient temperature for up to 18 months, showing residual drug content >90% compared with baseline levels. Overall, the palatability, efficacy and stability data collected from this study suggest that these two PZQ particulate formulations have potential applications as in-feed anti-parasitic medications for the yellowtail kingfish farming industry.

Meta-Analysis of the Field Efficacy of Seed- and Soil-Applied Nematicides on Meloidogyne incognita and Rotylenchulus reniformis Across the U.S. Cotton Belt.

Meta-analysis was used to compare yield protection and nematode suppression provided by two seed-applied and two soil-applied nematicides against Meloidogyne incognita and Rotylenchulus reniformis on cotton across 3 years and several trial locations in the U.S. Cotton Belt. Nematicides consisted of thiodicarb- and fluopyram-treated seed, aldicarb and fluopyram applied in furrow, and combinations of the seed treatments and soil-applied fluopyram. The nematicides had no effect on nematode reproduction or root infection but had a significant impact on seed cotton yield response ([Formula: see text]), with an average increase of 176 and 197 kg/ha relative to the nontreated control in M. incognita and R. reniformis infested fields, respectively. However, because of significant variation in yield protection and nematode suppression by nematicides, five or six moderator variables (cultivar resistance [M. incognita only], nematode infestation level, nematicide treatment, application method, trial location, and growing season) were used depending on nematode species. In M. incognita-infested fields, greater yield protection was observed with nematicides applied in furrow and with seed-applied + in-furrow than with solo seed-applied nematicide applications. Most notable of these in-furrow nematicides were aldicarb and fluopyram (>131 g/ha) with or without a seed-applied nematicide compared with thiodicarb. In R. reniformis-infested fields, moderator variables provided no further explanation of the variation in yield response produced by nematicides. Furthermore, moderator variables provided little explanation of the variation in nematode suppression by nematicides in M. incognita- and R. reniformis-infested fields. The limited explanation by the moderator variables on the field efficacy of nematicides in M. incognita- and R. reniformis-infested fields demonstrates the difficulty of managing these pathogens with nonfumigant nematicides across the U.S. Cotton Belt.

Do adolescents consider mind-body skills groups an acceptable treatment for depression: results from a pilot study.

BACKGROUND: Mind-Body Skills Groups (MBSGs) have shown promise in reducing adolescent depression symptoms; however, little is known about adolescents' perspectives on this treatment. The objective of this study was to understand the acceptability of a new treatment for depressed adolescents in primary care settings. METHODS: Adolescents participating in a 10-week MBSG treatment were interviewed to understand their perspectives on the acceptability and effectiveness of the treatment. Interviews were collected at post-intervention and at a 3-month follow-up visit. RESULTS: A total of 39 adolescents completed both the post-intervention and 3-month follow-up interview. At post-intervention and follow-up, 84% of adolescents stated the MBSGs helped them. When asked how the MBSGs helped them, 3 areas were identified: learning new MBSG activities and skills, social connection with others within the group, and outcomes related to the group. Many adolescents reported no concerns with the MBSGs (49% at post- intervention; 62% at follow-up). Those with concerns identified certain activities as not being useful, wanting the group to be longer, and the time of group (after school) being inconvenient. Most adolescents reported that their life had changed because of the group (72% at post-intervention; 61% at follow-up), and when asked how, common responses included feeling less isolated and more hopeful. CONCLUSIONS: Adolescents found the MBSGs to be helpful and acceptable as a treatment option for depression in primary care. Given the strong emphasis on treatment preference autonomy and the social activities within the group, MBSGs appear well-suited for this age group. TRIAL REGISTRATION: NCT03363750 ; December 6th, 2017.

How can healthcare professionals provide guidance and support to parents of adolescents? Results from a primary care-based study.

BACKGROUND: This study explored the rewards and difficulties of raising an adolescent and investigated parents' level of interest in receiving guidance from healthcare providers on parenting and adolescent health topics. Additionally, this study investigated whether parents were interested in parenting programs in primary care and explored methods in which parents want to receive guidance. METHODS: Parents of adolescents (ages 12-18) who attended an outpatient pediatric clinic with their adolescent were contacted by telephone and completed a short telephone survey. Parents were asked open-ended questions regarding the rewards and difficulties of parenting and rated how important it was to receive guidance from a healthcare provider on certain parenting and health topics. Additionally, parents reported their level of interest in a parenting program in primary care and rated how they would like to receive guidance. RESULTS: Our final sample included 104 parents, 87% of whom were interested in a parenting program within primary care. A variety of parenting rewards and difficulties were associated with raising an adolescent. From the list of parenting topics, communication was rated very important to receive guidance on (65%), followed by conflict management (50%). Of health topics, parents were primarily interested in receiving guidance on sex (77%), mental health (75%), and alcohol and drugs (74%). Parents in the study wanted to receive guidance from a pediatrician or through written literature. CONCLUSIONS: The current study finds that parents identify several rewarding and difficult aspects associated with raising an adolescent and are open to receiving guidance on a range of parenting topics in a variety of formats through primary care settings. Incorporating such education into healthcare visits could improve parents' knowledge. Healthcare providers are encouraged to consider how best to provide parenting support during this important developmental time period.

Death begets a new beginning.

Cell death is a perpetual feature of tissue microenvironments; each day under homeostatic conditions, billions of cells die and must be swiftly cleared by phagocytes. However, cell death is not limited to this natural turnover-apoptotic cell death can be induced by infection, inflammation, or severe tissue injury. Phagocytosis of apoptotic cells is thus coupled to specific functions, from the induction of growth factors that can stimulate the replacement of dead cells to the promotion of tissue repair or tissue remodeling in the affected site. In this review, we outline the mechanisms by which phagocytes sense apoptotic cell death and discuss how phagocytosis is integrated with environmental cues to drive appropriate responses.