Metastasis-Free Survival Versus Treatment-Free Survival in Biochemically Recurrent Prostate Cancer: The EMBARK Trial.

What is most important to patients with BCR prostate cancer? Metastasis-free versus treatment-free survival.

Double Chromogen-based Immunohistochemical Staining: An Efficient Approach for Utilizing Long-term Formalin-fixed Tissue in Biobanks.

The New South Wales Brain Tissue Resource Centre is a human brain bank that provides top-quality brain tissue for cutting-edge neuroscience research spanning various conditions from alcohol use disorder to neurodegenerative diseases. However, the conventional practice of preserving brain tissue in formalin poses challenges for immunofluorescent staining primarily due to the formalin's tendency, over time, to create cross-links between antigens, which can obscure epitopes of interest. In addition, researchers can encounter issues such as spectral bleeding, limitations in using multiple colors, autofluorescence, and cross-reactivity when working with long-term formalin-fixed brain tissue. The purpose of the study was to test chromogen-based double immunolabeling to negate the issues with immunofluorescent staining. Colocalization of antigens was explored using chromogens 3-amino-9-ethylcarbazole (AEC) and 3,3,-diaminobenzidine in a sequential staining procedure where the AEC signal was eliminated by alcohol treatment. Combinations of 2 or 3 primary antibodies from the same or different species were trialed successfully with this protocol. The colocalization of antigens was also demonstrated with pseudocoloring that mimicked immunofluorescence staining. This staining technique increases the utility of archival formalin-fixed tissue samples.

Phosphatidylethanol in post-mortem brain: correlation with blood alcohol concentration and alcohol use disorder.

Phosphatidylethanol (PEth) is an alcohol derivative that has been employed as a blood-based biomarker for regular alcohol use. This study investigates the utility of phosphatidylethanol (PEth) as a biomarker for assessing alcohol consumption in post-mortem brain tissue. Using samples from the New South Wales Brain Tissue Resource Centre, we analysed PEth(16:0/18:1) levels in the cerebellum and meninges of individuals with varying histories of alcohol use, including those diagnosed with alcohol use disorder (AUD) and controls. Our findings demonstrate a significant correlation between PEth levels and blood alcohol content (BAC) at the time of death, supporting the biomarker's sensitivity to recent alcohol intake. Furthermore, this study explores the potential of PEth levels in differentiating AUD cases from controls, taking into consideration the complexities of diagnosing AUD post-mortem. The study also examined the relationship between PEth levels and liver pathology, identifying a link with the severity of liver damage. These results underscore the value of PEth as a reliable indicator of alcohol consumption and its potential contributions to post-mortem diagnostics and consequently, research into alcohol-related brain damage.

A Survey of Community Perceptions on Brain Donation for Research.

Postmortem brain donation for medical research is a little-known form of organ donation. While most brain research is carried out using animal models, many neurological diseases are uniquely human. Greater availability of human postmortem brain tissue from diseased individuals and controls would therefore improve the development of treatments for neurological and neuropsychiatric diseases. Globally, organ donation for medical research is dwarfed by organ donation for transplantation. In 2021, 36% of Australians were registered organ donors for transplantation, with public "in-principle" support even higher, at 76%. In contrast, there are little data on Australian or international brain donation rates for research. A 30-item online survey was conducted to ascertain knowledge of, and attitudes toward, brain donation in Australia. Of the respondents, 12/237 (5%) were current brain donors and excluded from further analysis. Of the remaining 225, 75% were registered organ donors for transplant. The vast majority (n = 189/225, 84%) of respondents supported or strongly supported the principle of brain donation. However, of those registered for transplantation or whole-body donors, 93/170 (55%) were not aware that brain donation was possible, while 50%, alternatively or also, thought that registering as an organ donor for transplantation rendered them a brain donor by default. Only 9/225 (4%) respondents indicated that they would definitely not donate their brain in the future, while 27 remained unsure. There is prominent public support for brain donation in Australia, with 84% of respondents willing to donate their brain. Yet, the extent of public misconceptions on brain donation for research suggests the need for further education on all types of organ donation, so individuals may make informed decisions.

Central markers of neuroinflammation in alcohol use disorder: A meta-analysis of neuroimaging, cerebral spinal fluid, and postmortem studies.

INTRODUCTION AND AIMS: There is emerging evidence that heavy long-term alcohol consumption may alter the neuroimmune profile. We conducted a meta-analysis of the association between alcohol use disorder (AUD) and the extent of neuroinflammation using cerebrospinal (CSF), PET (Positron Emission Tomography), and postmortem studies. DESIGN AND METHODS: A comprehensive search of electronic databases was conducted using the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) for AUD-related terms in combination with neuroinflammatory markers and cytokine- and chemokine-related terms for CSF, PET, and postmortem studies. Participants had to meet established criteria for AUD and/or heavy alcohol consumption with dependence features and be compared with healthy controls. Papers retrieved were assessed for inclusion criteria and a critical appraisal was completed using the Newcastle-Ottawa Scale. A meta-analysis was conducted on postmortem and PET studies. RESULTS: Eleven papers met the inclusion criteria with CSF, PET, and postmortem studies included in the final analysis. Postmortem studies demonstrate significant heterogeneity (𝑄 (14) = 62.02, 𝑝 < 0.001), with the alcohol group showing higher levels of neuroimmune markers than controls (𝑑 = 1.50 [95% CI 0.56, 2.45]). PET studies demonstrated a lower [11 C] PBR28 total volume of distribution (V T ) for translocator protein in the hippocampus (g = -1.95 [95% CI -2.72, -1.18], p < 0.001) of the alcohol group compared to controls. CONCLUSION: There is emerging evidence across multiple diagnostic modalities that alcohol impacts neuroimmune signaling in the human brain.

Adherence to oral oncolytics filled through an internal health-system specialty pharmacy compared with external specialty pharmacies.

BACKGROUND: Oral oncolytics are becoming increasingly common in the treatment of solid and hematological malignancies. Medication adherence is especially important to ensure adequate drug levels to treat active malignancies, notably in curative-intent therapy. Further data are needed to quantify and confirm the effects of internal health-system specialty pharmacies (HSSPs) on medication adherence. OBJECTIVE: To confirm the effect of an internal HSSP compared with external specialty pharmacies on oncolytic adherence as measured by proportion of days covered (PDC), medication possession ratio (MPR), and time to treatment (TTT). METHODS: This single-center retrospective cohort study included patients receiving oral oncolytics through an internal HSSP or external specialty pharmacies between January 2019 and June 2020. Fill data were extracted from pharmacy claims databases and electronic medical records. The primary adherence outcome was patient-level PDC. Secondary adherence outcomes included patient-level MPR and TTT. For PDC and MPR analyses, patients with at least 3 fills per oncolytic were included. All patients were included for the TTT analysis. Chi-square or Fisher's exact tests were used to analyze categorical differences between pharmacy groups. Differences in continuous variables across pharmacy groups were evaluated using Wilcoxon rank-sum tests. RESULTS: 871 prescriptions met inclusion criteria: 549 patients were included in the PDC/MPR analysis, and 758 patients were included in the TTT analysis (patients might have multiple prescriptions). Patients who filled at an internal HSSP had a higher median PDC compared with those who filled at external specialty pharmacies (0.99 [IQR = 0.89-1.00] vs 0.91 [IQR = 0.76-0.98]; P < 0.01). The adherence rate as measured by MPR was higher for patients who used an internal HSSP compared with those who used external specialty pharmacies (MPR = 1.00 [IQR = 0.90-1.00] vs 0.93 [IQR = 0.76-1.00]; P < 0.01). Median TTT was lower for patients using the internal HSSP vs an external specialty pharmacy (5 days [IQR = 2-13] vs 27 days [IQR = 2-82], respectively; P < 0.01). CONCLUSIONS: Internal HSSP services improved adherence as measured by PDC and MPR. Significantly lower TTT was seen with the internal HSSP compared with external pharmacies. These data confirm and support use of internal HSSPs to dispense oral oncolytics for treatment of solid and hematological malignancies. DISCLOSURES: This study received no financial support. The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

An up-to-date evaluation of abiraterone for the treatment of prostate cancer.

Introduction: Abiraterone acetate, an oral 17-alpha-hydroxylase inhibitor, effectively prevents the synthesis of androgens from steroid precursors. Abiraterone has become a standard of care in patients with metastatic prostate cancer due to its efficacy in both castrate-sensitive and castrate-resistant disease when given in combination with androgen deprivation therapy (ADT). Abiraterone may have a role in additional aspects of prostate cancer treatment in the future.Areas covered: The present article focuses on the development and establishment of abiraterone among the available treatment options for prostate cancer. A literature search was performed in PubMed/Medline for prior studies and reviews of the drug. Current clinical trials were examined in the Clinicaltrials.gov database.Expert opinion: Abiraterone has shown efficacy in castrate-resistant metastatic prostate cancer, providing an additional degree of hormonal sensitivity for tumors resistant to ADT. Impressively, abiraterone in conjunction with ADT as a first-line treatment for castrate-sensitive prostate cancer also confers a significant overall survival benefit compared to ADT alone. With minimal additional toxicity, abiraterone has established itself as a well-tolerated, convenient, and effective treatment option. Ongoing studies are expected to broaden the drug's indications as well as its preference among other prostate cancer therapies.

Coexisting Lewy body disease and clinical parkinsonism in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is associated with a range of clinical phenotypes and shows progressive degeneration of upper and/or lower motor neurons, and phosphorylated 43 kDa TAR DNA-binding protein (pTDP-43) inclusions in motor and non-motor pathways. Parkinsonian features have been reported in up to 30% of ALS patients, and Lewy bodies, normally associated with Lewy body disease (LBD), have been reported in a small number of ALS cases, with unknown clinical relevance. This study investigates the prevalence of clinically relevant LBD in a prospectively studied ALS cohort to determine whether concomitant pathology contributes to the clinical heterogeneity. METHODS: All ALS cases held by the New South Wales Brain Bank (n = 97) were screened for coexisting LBD consistent with clinical disease (Braak ≥ stage IV). Relevant clinical and genetic associations were determined. RESULTS: Six cases had coexisting LBD Braak ≥ stage IV pathology. The age at symptom onset (69 ± 7 years) and disease duration (4 ± 3 years) in ALS cases with coexisting LBD did not differ from ALS cases. Three patients had lower limb onset and two patients had bulbar onset. Two patients developed the clinical features of Parkinson's disease, with one receiving a dual diagnosis. All cases had no known relevant family history or genetic abnormalities. CONCLUSION: The prevalence of clinically relevant LBD pathology in ALS is higher than in the general population, and has implications for clinical and neuropathological diagnoses and the identification of biomarkers.

Novel disease resistance gene paralogs created by CRISPR/Cas9 in soy.

KEY MESSAGE: Novel disease resistance gene paralogues are generated by targeted chromosome cleavage of tandem duplicated NBS-LRR gene complexes and subsequent DNA repair in soybean. This study demonstrates accelerated diversification of innate immunity of plants using CRISPR. Nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) gene families are key components of effector-triggered immunity. They are often arranged in tandem duplicated arrays in the genome, a configuration that is conducive to recombinations that will lead to new, chimeric genes. These rearrangements have been recognized as major sources of novel disease resistance phenotypes. Targeted chromosome cleavage by CRISPR/Cas9 can conceivably induce rearrangements and thus emergence of new resistance gene paralogues. Two NBS-LRR families of soy have been selected to demonstrate this concept: a four-copy family in the Rpp1 region (Rpp1L) and a large, complex locus, Rps1 with 22 copies. Copy-number variations suggesting large-scale, CRISPR/Cas9-mediated chromosome rearrangements in the Rpp1L and Rps1 complexes were detected in up to 58.8% of progenies of primary transformants using droplet-digital PCR. Sequencing confirmed development of novel, chimeric paralogs with intact open reading frames. These novel paralogs may confer new disease resistance specificities. This method to diversify innate immunity of plants by genome editing is readily applicable to other disease resistance genes or other repetitive loci.

Severe childhood and adulthood stress associates with neocortical layer-specific reductions of mature spines in psychiatric disorders.

Severe stress exposure causes the loss of dendritic spines on cortical pyramidal neurons and induces psychiatric-like symptoms in rodent models. These effects are strongest following early-life stress and are most persistent on apical dendrites. However, the long-term impacts and temporal effects of stress exposure on the human brain remain poorly understood. Using a novel postmortem cohort of psychiatric cases with severe stress experienced in childhood, adulthood, or no severe stress, and matched controls, we aimed to determine the impact of stress timing on pyramidal neuron structure in the human orbitofrontal cortex (OFC). We performed Golgi Cox staining and manually measured the morphology and density of over 22,000 dendritic spines on layer-specific pyramidal neuron apical dendrites. We also quantified glucocorticoid receptor mRNA and protein as a marker of stress dysregulation. Both childhood and adulthood stress were associated with large reductions in mature mushroom spine density (up to 56% loss) in both the superficial (II/III) and deeper layers (V) of the OFC. However, childhood stress caused more substantial reductions to both total and mature mushroom spines. No difference in glucocorticoid receptor mRNA and protein were seen between groups, although both negatively correlated with total spine density within the whole cohort. These findings indicate that severe stress, especially when experienced during childhood, persistently affects the fine morphological properties of neurons in the human OFC. This may impact on cell connectivity in this brain area, and at least partly explain the social and emotional symptoms that originate in the OFC in psychiatric disorders.

Antihypertensive medications ameliorate Alzheimer's disease pathology by slowing its propagation.

INTRODUCTION: Mounting evidence supports an association between antihypertensive medication use and reduced risk of Alzheimer's disease (AD). Consensus on possible pathological mechanisms remains elusive. METHODS: Human brain tissue from a cohort followed to autopsy that included 96 cases of AD (46 medicated for hypertension) and 53 pathological controls (33 also medicated) matched for cerebrovascular disease was available from the New South Wales Brain Banks. Quantified frontal cortex amyloid beta (Aß) and tau proteins plus Alzheimer's neuropathologic change scores were analyzed. RESULTS: Univariate analyses found no difference in amounts of AD proteins in the frontal cortex between medication users, but multivariate analyses showed that antihypertensive medication use was associated with a less extensive spread of AD proteins throughout the brain. DISCUSSION: The heterogeneous nature of the antihypertensive medications is consistent with downstream beneficial effects of blood pressure lowering and/or management being associated with the reduced spreading of AD pathology observed.

Azteca ants maintain unique microbiomes across functionally distinct nest chambers.

The microbiome of built structures has considerable influence over an inhabitant's well-being, yet the vast majority of research has focused on human-built structures. Ants are well-known architects, capable of constructing elaborate dwellings, the microbiome of which is underexplored. Here, we explore the bacterial and fungal microbiomes in functionally distinct chambers within and outside the nests of Azteca alfari ants in Cecropia peltata trees. We predicted that A. alfari colonies (1) maintain distinct microbiomes within their nests compared to the surrounding environment, (2) maintain distinct microbiomes among nest chambers used for different functions, and (3) limit both ant and plant pathogens inside their nests. In support of these predictions, we found that internal and external nest sampling locations had distinct microbial communities, and A. alfari maintained lower bacterial richness in their 'nurseries'. While putative animal pathogens were suppressed in chambers that ants actively inhabited, putative plant pathogens were not, which does not support our hypothesis that A. alfari defends its host trees against microbial antagonists. Our results show that ants influence microbial communities inside their nests similar to studies of human homes. Unlike humans, ants limit the bacteria in their nurseries and potentially prevent the build-up of insect-infecting pathogens. These results highlight the importance of documenting how indoor microbiomes differ among species, which might improve our understanding of how to promote indoor health in human dwellings.

Attenuation of N-glycosylation causes polarity and adhesion defects in the C. elegans embryo.

The Caenorhabditiselegans early embryo is highly polarized, requiring sequestration of cytoplasmic polarity factors at the plasma membrane. This compartmentalization aids asymmetric distribution of lipids and proteins, which is partially responsible for the fates of the daughter cells. Since most plasma membrane proteins are glycosylated, we determined the effect of attenuation of N-glycosylation on cell polarity. While polarity establishment was not perturbed, the size difference between the two cells formed in first cell division (AB and P1) was more variable in embryos with reduced N-glycosylation than in the mock-treated embryos. In addition, among other deficiencies, we observed spindle orientation defects in two-cell embryos. Moreover, cell-cell adhesion was specifically lost at the two-cell stage when N-glycosylation was reduced. This loss-of-adhesion phenotype was rescued by interfering with polarity establishment, indicating that polarity establishment enforces plasma membrane compartmentalization. Consistent with this idea, the decreased plasma membrane levels of the adhesion proteins E-cadherin and MAGI-1 in ribo-1(RNAi) embryos were restored in the absence of functional PAR-2. Our data suggest a general role for N-glycosylation in plasma membrane compartmentalization and cell polarity.

Correction: Serum- and Glucocorticoid-Inducible Kinase-1 (SGK-1) Plays a Role in Membrane Trafficking in Caenorhabditis elegans.

[This corrects the article DOI: 10.1371/journal.pone.0130778.].

A role for septin 2 in Drp1-mediated mitochondrial fission.

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin-like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1-dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP-induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.

Symbiosis in the Soil: Citizen Microbiology in Middle and High School Classrooms.

Microorganisms are vital to environmental health, yet their association with disease often overshadows these benefits. Building citizen-science activities around the positive role of microorganisms and an understanding of their ubiquity can begin to dispel misconceptions while simultaneously engaging the public in research. Here, we describe a citizen-science microbiology project geared toward implementation in middle and high school classrooms. Students culture environmental microorganisms and document microbial diversity of plant root systems compared with adjacent bulk soil. Results contribute data toward research on microbiome recruitment of weeds and other successful plants while addressing core topics in science education.

Discovery of Novel, Potent, and Specific Cell-Death Inducers in the Jurkat Acute Lymphoblastic Leukemia Cell Line.

The limited clinical efficacy of many cancer therapeutics has initiated intense research efforts toward the discovery of novel chemical entities in this field. In this study, 31 hit candidates were selected from nearly 800,000 database compounds in a ligand-based virtual screening campaign. In turn, three of these hits were found to have (sub)micromolar potencies in proliferation assays with the Jurkat acute lymphatic leukemic cell line. In this assay, the three hits were found to exhibit higher potency than clinically tested cell-death inducers (GDC-0152, AT-406, and birinapant). Importantly, antiproliferative activity toward non-cancer peripheral blood mononuclear cells (PBMCs) was found to be marginal. Further biological characterization demonstrated the cell-death-inducing properties of these compounds. Biological testing of hit congeners excluded a nonspecific, toxic effect of the novel structures. Altogether, these findings may have profound relevance for the development of clinical candidates in tumor therapy.

Serum- and Glucocorticoid-Inducible Kinase-1 (SGK-1) Plays a Role in Membrane Trafficking in Caenorhabditis elegans.

The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in C. elegans sgk-1 null mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 null animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps.

A simple fragment of cyclic acyldepsipeptides is necessary and sufficient for ClpP activation and antibacterial activity.

The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.