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Conflicting views have been put forward on the derivation of the distribution of solubilized molecules among micelles. This stems from failure to consider the arrangement of the solubilized molecules in the micelles. In the treatment presented enthalpy effects are ignored as they are not amenable to a simple general theory. (Author/BB)

INTRODUCTION Evidence strongly suggests that soluble oligomers of amyloid beta protein (oAβ) help initiate the pathogenic cascade of Alzheimer's disease (AD). To date, there have been no validated assays specific for detecting and quantifying oAβ in human blood. METHODS We developed an ultrasensitive oAβ immunoassay using a novel capture antibody (71A1) with N-terminal antibody 3D6 for detection that specifically quantifies soluble oAβ in the human brain, cerebrospinal fluid (CSF), and plasma. RESULTS Two new antibodies (71A1; 1G5) are oAβ-selective, label Aβ plaques in non-fixed AD brain sections, and potently neutralize the synaptotoxicity of AD brain-derived oAβ. The 71A1/3D6 assay showed excellent dilution linearity in CSF and plasma without matrix effects, good spike recovery, and specific immunodepletion. DISCUSSION We have created a sensitive, high throughput, and inexpensive method to quantify synaptotoxic oAβ in human plasma for analyzing large cohorts of aged and AD subjects to assess the dynamics of this key pathogenic species and response to therapy.

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains—Lewy bodies and Lewy neurites—are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs β-synuclein (βS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-β component of plaques (NAC) domain, constituting amino acids 61–95, has been identified to be critical for aggregation in vitro. This domain is partially absent in βS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that βS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased βS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

BACKGROUND AND PURPOSE: Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) for cGMP production, but in disease, sGC becomes insensitive towards NO activation. What changes occur to sGC during its inactivation in cells is not clear. EXPERIMENTAL APPROACH: We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its haem content, heterodimer status and sGCβ protein partners when the cells were given the oxidant ODQ or the NO donor NOC12 to inactivate sGC. Haem content of sGCβ was monitored in live cells through use of a fluorescent-labelled sGCβ construct, whereas sGC heterodimer status and protein interactions were studied by Western blot analysis. Experiments with purified proteins were also performed. KEY RESULTS: ODQ- or NOC12-driven inactivation of sGC in HEK293 cells was associated with haem oxidation (by ODQ), S-nitrosation of the sGCβ subunit (by NOC12), sGC heterodimer breakup and association of the freed sGCβ subunit with cell chaperone Hsp90. These changes occurred without detectable loss of haem from the sGCβ reporter construct. Treating a purified ferrous haem-containing sGCβ with ODQ or NOC12 caused it to bind with Hsp90 without showing any haem loss. CONCLUSION AND IMPLICATIONS: Oxidative (ODQ) or nitrosative (NOC12) inactivation of cell sGC involves sGC heterodimer dissociation and rearrangement of the sGCβ protein partners without any haem loss from sGCβ. Clarifying what changes do and do not occur to sGC during its inactivation in cells may direct strategies to preserve or recover NO-dependent cGMP signalling in health and disease. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc

The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25–400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ∼4 log10 spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ∼4 log10 cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.

Interfaces between molecular organic architectures and oxidic substrates are a central feature of biosensors and applications of biomimetics in science and technology. For phospholipid bilayers, the large range of pH- and ionic strength-dependent surface charge densities adopted by titanium dioxide and other oxidic surfaces leads to a rich landscape of phenomena that provides exquisite control of membrane interactions with such substrates. Using neutron reflectometry measurements, we report sharp, reversible transitions that occur between closely surface-associated and weakly coupled states. We show that these states arise from a complex interplay of the tunable length scale of electrostatic interactions with the length scale arising from other forces that are independent of solution conditions. A generalized free energy potential, with its inputs only derived from established measurements of surface and bilayer properties, quantitatively describes these and previously reported observations concerning the unbinding of bilayers from supporting substrates.

Objective Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression. Methods The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes. Results Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes. Conclusions Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

Background: Cytotoxic chemotherapy (CT) in combination with trastuzumab and pertuzumab (HP) is standard of care for patients (pts) diagnosed with HER2-positive early breast cancer (EBC). While highly effective, the toxicity associated with CT is challenging. In KRISTINE/TRIO-021, neoadjuvant T-DM1 was combined with pertuzumab (KP) and compared to standard TCHP. Pts. received six cycles of neoadjuvant treatment followed by adjuvant therapy (KP or HP). Pts. in the KP arm were allowed to receive standard adjuvant CT. Pathologic complete response (pCR) rate was significantly lower with KP versus TCHP and more pts. had disease progression prior to surgery with KP, resulting in a meaningfully lower event-free survival rate vs. the control (85.3% vs 94.2%). However, 3-year invasive disease-free survival was numerically similar in both arms. Neoadjuvant KP demonstrated less toxicity than standard CT, although treatment discontinuation was higher post-surgery. Association of KP and TCHP with treatment-related amenorrhea (TRA) in premenopausal EBC pts. has not been ascertained. Methods: All pts. with premenopausal status at study entry (those not meeting the menopause definition based on National Comprehensive Cancer Network Guidelines v3, 2012) and with menstrual period documented within 3 months of randomization, were independently evaluated for presence or absence of TRA by two reviewers. TRA, a prespecified exploratory endpoint of KRISTINE, was defined as cessation of menstruation for >12 months in the absence of treatment with ovarian suppression or other interventions that can induce amenorrhea. Pts. were followed from the time of study entry through the 3-year follow up period after surgery. For cases with inconsistent determination between the two reviewers, a third reviewer adjudicated. TRA rates were calculated per arm, hormone-receptor (HR) status, adjuvant CT and age group. Proportions were compared by estimating the odds ratio (OR) and the 95% confidence interval. Results: Of 444 pts. enrolled, 205 were excluded based on being post-menopausal per NCCN guidelines. Of 239 pts. remaining, 56 were excluded due to insufficient data. The median age of pts. included was 40 years (range: 22-53) for TCHP and 42.5 (range: 23-52) for KP. TRA was observed in 55% (50/91) of pts. treated with TCHP compared to 30% (28/92) treated with KP (OR=2.79; 95% CI 1.52-5.12). In pts. with HR-positive EBC, TRA occurred in 62% with TCHP vs 35% for KP (OR=2.998; 95% CI 1.44-6.25). In those with HR-negative EBC, TRA was observed in 42% with TCHP vs. 21% with KP (OR=2.77; 95% CI 0.88-8.72). In the KP arm, TRA was observed in 38% (8/21) of pts. treated with standard adjuvant CT vs. 28% (20/71) of those that did not (OR 1.57; 95% CI 0.57-4.36). For women age ≤ 40, the rate of TRA was 38% with TCHP vs. 17% with KP (OR=3.00; 95% CI 1.05-8.60). For those > 40 years, TRA was observed in 74% treated with TCHP vs. 39% of those with KP (OR=4.50; 95% CI 1.88-10.73). Conclusion: The rate of TRA with standard TCHP is nearly double that observed with KP, suggesting that targeted CT with an antibody-drug conjugate regimen is associated with less gonadal toxicity. Rates of TRA are higher in women over the age of 40 for each treatment arm however KP is associated with lower rate of TRA in each age group. Association of TRA with efficacy outcomes (pCR, iDFS) will be presented. Citation Format: Sara A Hurvitz, Rodrigo Fresco, Karen Afenjar, Daniil Stroyakovskiy, Chiun-Sheng Huang, Hans Wildiers, Kyung Hae Jung, Jean-François Boileau, Mario Campone, Miguel Martín, Vicente Valero, Joseph A. Sparano, W. Fraser Symmans, Peter A. Fasching, Alastair M. Thompson, Nadia Harbeck, Vanesa López-Valverde, Chunyan Song, Thomas Boulet, Eleonora Restuccia, Dennis J. Slamon. Treatment-related amenorrhea with T-DM1 plus pertuzumab (KP) is lower than with docetaxel/carboplatin/trastuzumab/pertuzumab (TCHP) in the phase III neoadjuvant KRISTINE trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD12-06.

Lithium poisoning remains common. Symptomatic lithium poisonings often require hemodialysis, especially when the patient has impaired renal function. An effective and non-invasive treatment to remo...

Protein–Protein interactions (PPIs) are involved in a myriad of cellular processes in all living organisms and the modulation of PPIs is already under investigation for the development of new drugs targeting cancers, autoimmune diseases and viruses. PPIs are also involved in the regulation of vital functions in bacteria and, therefore, targeting bacterial PPIs offers an attractive strategy for the development of antibiotics with novel modes of action. The latter are urgently needed to tackle multidrug-resistant and multidrug-tolerant bacteria. In this review, we describe recent developments in the modulation of PPIs in pathogenic bacteria for antibiotic development, including advanced small molecule and peptide inhibitors acting on bacterial PPIs involved in division, replication and transcription, outer membrane protein biogenesis, with an additional focus on toxin–antitoxin systems as upcoming drug targets., This review describes recent efforts towards the modulation of protein–protein interactions in infectious bacteria.

Objective Parkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease-modifying treatments are approved. A key therapeutic target in PD is α-synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl-coenzyme A desaturase (SCD) inhibitor ("5b") that prevented αS-positive vesicular inclusions and cytotoxicity in cultured human neurons. Methods Oral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild-type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD-like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD-related lipid changes were quantified in 5b- versus placebo-treated mice. Outcomes were compared to effects of crossing 3K to SCD1-/- mice. Results 5b treatment reduced αS hyperphosphorylation in E46K-expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD-like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K-resistant lipid-rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation. Interpretation Prolonged reduction of brain SCD activity prevented PD-like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α-synucleinopathies. ANN NEUROL 2021;89:74-90.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

The catalytic condensation of ethanol to n-butanol and higher alcohols continues to attract attention as new outlets are sought for this bio-renewable feedstock. Results are presented here for the ...

Soluble guanylyl cyclase (sGC) is a key component of NO–cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCβ involves GAPDH. We utilized an sGCβ reporter construct, tetra-Cys sGCβ, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCβ in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCβ correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCβ associates with GAPDH in cells and dissociates when heme binds sGCβ, and that the purified GAPDH–heme complex binds to apo-sGCβ and transfers its heme to sGCβ. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCβ to accomplish heme delivery to sGCβ. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein-protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein-protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS-PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.

It is increasingly accepted that early cognitive impairment in Alzheimer's disease results in considerable part from synaptic dysfunction caused by the accumulation of a range of oligomeric assemblies of amyloid β-protein (Aβ). Most studies have used synthetic Aβ peptides to explore the mechanisms of memory deficits in rodent models, but recent work suggests that Aβ assemblies isolated from human (AD) brain tissue are far more potent and disease-relevant. Although reductionist experiments show Aβ oligomers to impair synaptic plasticity and neuronal viability, the responsible mechanisms are only partly understood. Glutamatergic receptors, GABAergic receptors, nicotinic receptors, insulin receptors, the cellular prion protein, inflammatory mediators, and diverse signaling pathways have all been suggested. Studies using AD brain-derived soluble Aβ oligomers suggest that only certain bioactive forms (principally small, diffusible oligomers) can disrupt synaptic plasticity, including by binding to plasma membranes and changing excitatory-inhibitory balance, perturbing mGluR, PrP, and other neuronal surface proteins, down-regulating glutamate transporters, causing glutamate spillover, and activating extrasynaptic GluN2B-containing NMDA receptors. We synthesize these emerging data into a mechanistic hypothesis for synaptic failure in Alzheimer's disease that can be modified as new knowledge is added and specific therapeutics are developed.

The development of multiple PARP inhibitors in BRCA mutant cancers has been one of the most successful implementations of molecularly targeted therapies in oncology. The recent approval of niraparib in BRCA WT Ovarian cancers demonstrates the potential for expansion of PARP inhibitors to other BRCA WT indications. The successful development of PARP inhibitors into new cancer indications will depend on using molecular biomarkers to identify tumors with a unique dependency on PARP1/2 activity. We screened a large (N=523), diverse panel of human cancer cell lines for response to three approved PARP inhibitors: olaparib, niraparib, and talazoparib. Despite differences in relative potency, there was a strong correlation between sensitive and resistant cell lines to all three compounds. In vitro experimentation revealed the inhibitors share a similar biologic mechanism of response, namely a global decrease in PARylation, as well as PARP-trapping at DNA nicks leading to G2 arrest and ultimately cell death. A suite of baseline genomic and proteomic characteristics were found to be strongly associated with response to PARP1/2 inhibition, and were largely consistent between different cancer types. Special attention was paid to BRCA1/2 variants, which represent the only approved patient selection biomarkers. Notably, neither mutations in BRCA1 nor BRCA2 were found to be associated with response to PARP1/2 inhibition in our panel, even when restricting to those with confirmed deleterious ClinVar scores. The molecular predictors of response identified in our screen may ultimately be used to develop diagnostic tools for enrollment into biomarker-enriched clinical trials to expand the use of this promising class of drug into areas of high unmet need. Triple-negative breast cancer represents one of the most intriguing spaces for development. The clinical failures in this space may be partially explained by the lack of empirical predictive biomarkers in the trial design. Our diagnostics may finally allow for the efficacious deployment of PARP inhibitors to triple-negative breast cancer. In particular we have identified a panel of biomarkers that can predict for sensitivity to PARP inhibitors in breast cancer that could be used as patient selection criteria for the expanded clinical development of these compounds into PARP-naïve patient populations with high unmet need. Citation Format: Martina SJ McDermott, Dylan F Conklin, Neil A O'Brien, Kevin Chau, Dennis J Slamon. Pan-cancer analysis of PARP inhibition reveals a suite of biomarkers that correlate with PARP1/2 activity in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-07.

Background: Neoadjuvant HER2-targeted therapy in combination with chemotherapy is a standard treatment approach for early-stage HER2-positive breast cancer. Proposed biomarkers to predict pathologic complete response (pCR), and thereby inform which patients may benefit from de-escalation of therapy, include expression-based subtyping and immune enrichment scores. Little is known about how tumors and their microenvironment may change with HER2-targeted therapy alone, and whether these changes may predict outcome. Methods: The TRIO-US B07 phase II trial randomized 128 participants with stage I-III HER2-positive breast cancer to trastuzumab (N=34), lapatinib (N=36), or the combination (N=58) for three weeks, followed by six cycles of docetaxel and carboplatin with continued HER2-targeted therapy. Fresh-frozen core biopsies of the tumor prior to therapy (N=110) and after 14-21 days of HER2-targeted therapy alone (N=89) were collected, and RNA was extracted and subjected to Agilent Whole Human Genome 44K 2-color chip. The pre-treatment tumor RNA was normalized against a mixed breast tumor reference, and the on-treatment tumor RNA against the matched pre-treatment sample. Absolute intrinsic molecular subtyping was used to determine intrinsic subtype, the iC10 expression-based classifier to determine integrative subtype, gene set enrichment analysis (GSEA) to assess signature changes across treatment, single-sample GSEA to compare individual gene signature scores between tumors, and CIBERSORT to quantify immune cell populations before and on treatment. Results: Primary trial results have been reported previously and showed a pCR rate of 47% with trastuzumab, 25% with lapatinib, and 52% with the combination. Prior to treatment, 56% of tumors classified as the HER2-enriched intrinsic subtype and 78% as the iC5 integrative subtype. HER2-enriched tumors trended toward a higher rate of pCR relative to other intrinsic subtypes (50% vs 33%, P=0.12), as did iC5 tumors relative to other integrative subtypes (48% vs 25%, P=0.08). However, in multivariate analysis, HER2 FISH ratio (P=0.04) and hormone receptor status (P=0.02), each associated themselves with intrinsic and integrative subtype, proved the most valuable in predicting pCR, with little information added by expression-based subtyping. Immune cell signatures correlated with pCR in the trastuzumab-containing arms only. Of 65 gene signatures tested, 47 changed across HER2-targeted therapy with false discovery rate < 0.1, driven by decreasing tumor proliferation, increasing immune cell signatures, and increasing stromal cell/epithelial mesenchymal transition signatures. Quantification of immune cell populations suggested the immune changes were both anti-tumor (CD8+ T cells) and pro-tumor (M2 macrophages). Intrinsic subtype changed in 54% of tumors (79% of these converting to normal-like) and integrative subtype changed in 26%. Change in subtype, proliferation, or immune infiltration with targeted therapy did not correlate with pCR. A higher proportion of tumors treated with trastuzumab alone maintained their proliferation (42%), compared with lapatinib alone (20%; P=0.16) or the combination (16%; P=0.04). Conclusions: In the TRIO-US B07 study, the biomarkers most predictive of response to neoadjuvant HER2-targeted therapy were hormone receptor status in combination with HER2 FISH ratio. Multiple changes in the tumor and its microenvironment occurred with HER2-targeted therapy, but these changes did not predict pCR. Tumors treated with lapatinib tended to decrease proliferation more than tumors treated with trastuzumab, despite trastuzumab being more effective in preventing recurrence, an observation with implications for window of opportunity studies. Citation Format: Jennifer L. Caswell-Jin, Katherine L. McNamara, Judy Dering, Hsiao-Wang Chen, Robert Dichmann, Alejandra Perez, Ravindranath Patel, Eran Kotler, Jason J. Zoeller, Joan S. Brugge, Michael F. Press, Dennis J. Slamon, Christina Curtis, Sara A. Hurvitz. Tumor expression and microenvironment in HER2-positive breast cancer before and on HER2-targeted therapy: Analysis of microarray expression data from the TRIO-US B07 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-07-01.

Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr–calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis. In the fungal pathogen Cryptococcus neoformans, Bim1 is a copper-binding lytic polysaccharide monooxygenase-like protein that participates in copper uptake in concert with the Ctr1 importer to drive virulence mechanisms during fungal meningitis.

Introduction of a bis(isopropylidene)-protected galactopyranosyl moiety in s-triazine-based boron-rich carboxylic acids and amines results in soluble and suitable coupling partners for tumour-selective biomolecules with applications as selective agents for boron neutron capture therapy (BNCT). Bearing either a carboxylic acid or primary amine as a functional group, these compounds are highly versatile and thus largely extend the possible coupling strategies with suitable biomolecules. Modification of the gastrin-releasing peptide receptor (GRPR) selective agonist [D-Phe6, β-Ala11, Ala13, Nle14]Bn(6–14) with the carboxylic acid derivative yielded a bioconjugate with an optimal receptor activation and internalisation profile. This demonstrates the great potential of this approach for the development of novel boron delivery agents.

The soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate signaling pathway evokes vascular smooth muscle relaxation; whether this pathway mediates airway smooth muscle relaxation remains controversial. We posit that sGC activators are equi-effective as β-agonists in reversing contractile agonist-induced airway smooth muscle shortening. To provide clarity, we tested the efficacy of sGC stimulator and activator drugs, BAY 41-2272 and BAY 60-2270, respectively, in reversing bronchoconstriction of human small airways using human precision-cut lung slices (hPCLS). Both BAY drugs reversed carbachol-induced bronchoconstriction to a maximal degree comparable to that of formoterol. Moreover, the sGC drugs remained effective bronchodilators despite formoterol-induced desensitization of the airways. Analysis of the hPCLS after their activation by sGC or β2-adrenergic receptor agonist showed distinct cyclic nucleotide accumulation in the hPCLS. Collectively, these data suggest that cAMP and cyclic guanosine monophosphate pathways are equi-effective for reversing carbachol-induced bronchoconstriction in the human airway via separate and distinct second messenger pathways. This should open the door for future studies to test whether sGC-targeted drugs alone or in combination can serve as effective bronchodilators in asthma and chronic obstructive pulmonary disease.

Analysis of clinically relevant amino acids using ion-exchange chromatography coupled to photometric detection has been an indispensable component in the detection of inborn errors of metabolism for six decades. Detection of amino acids using mass spectrometry offers advantages in speed and analytic specificity. Employing methanol extraction and controlled butylation, C8 reversed-phase chromatography, and MS/MS detection, 32 amino acids are quantified in 20 min with clinically appropriate imprecision in plasma, urine, and CSF. Quantitation is linear to 1,000 micromol/L and limits of detection are at least 1.0 micromol/L. Important isobaric amino acids are distinguished by chromatography or by unique patterns of fragmentation following collision-induced dissociation. The technique employs commercially available reagents and may be expanded and customized for specific clinical or research settings.

Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells. Recently, programmable endonucleases such as clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas) and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions, enabling their detection with deep next generation sequencing (NGS). However, the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage. To overcome these shortcomings, we conceived a new assay (Programmable Enzyme-Assisted Selective Exponential Amplification, PASEA) that combines the cleavage of wild type alleles with concurrent polymerase amplification. While PASEA increases the numbers of both wild type and mutant alleles, the numbers of mutant alleles increase at much greater rates, allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles. By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification, we converted samples with 0.01% somatic mutant allele fractions (MAFs) to products with 70% MAFs in a single step within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Furthermore, PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes. Real-time PASEA' performance was proved equivalent to clinical amplification refractory mutation system (ARMS)-PCR and NGS when testing over hundred cancer patients' samples. This strategy has the potential to reduce the cost and time of cancer screening and genotyping, and to enable targeted therapies in resource-limited settings.

[Image: see text] The discovery of new targets for the treatment of malaria, in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this paper presents initial structure–activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization, and cell-based antiparasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity, and cell-based antiparasitic activity against multiple Plasmodium species that appears to be correlated with the in vitro potency.

The discovery of new targets for treatment of malaria and in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this manuscript presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization and cell-based anti-parasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity and cell-based anti-parasitic activity against multiple Plasmodium species that appears to correlate with in vitro potency.

Sustained neuronal activity demands quick resupply of synaptic vesicles in order to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by sub-micromolar presynaptic Ca2+ signals by an as yet unidentified high-affinity Ca2+ sensor1-4. Here we identify a novel presynaptic role for the high-affinity Ca2+ sensor Synaptotagmin-3 (SYT3)5 in driving vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibit enhanced short-term depression, and recovery is slower and insensitive to presynaptic residual Ca2+. During sustained neuronal firing, SYT3 speeds vesicle replenishment and increases the size of the readily releasable pool of vesicles. SYT3 also mediates a second form of short-term enhancement called facilitation, under conditions of low vesicle release probability. Models of vesicle trafficking suggest that SYT3 could combat synaptic depression by accelerating vesicle docking at active zones. Our results reveal a critical role for presynaptic SYT3 in maintaining reliable high-frequency synaptic transmission in neural circuits.

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.

The discovery of new targets for treatment of malaria advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, we recently reported the discovery and optimization of novel, potent isoxazole-based PfPKG inhibitors that lacked any obvious safety warnings. This manuscript presents representative in vitro ADME, hERG characterization and cell-based antiparasitic activity of these PfPKG inhibitors. We also report the discovery and structure-activity relationships of a new series with good potency, low hERG activity and cell-based anti-parasitic activity comparable to a literature standard.

Significance The mechanisms responsible for brain α-synuclein (αS) dyshomeostasis, caused by Gaucher’s GBA1 mutations that increase Parkinson’s disease (PD) risk, are largely unknown. We previously showed that abrogating physiological αS tetramers by a familial PD-E46K–amplified 3K mutation produces PD-like syndrome in mice and that treatment with stearoyl-CoA desaturase inhibitors increased a portion of the αS tetramers, benefitting the motor phenotypes. Here, we show that—similar to previous findings in GBA1-mutant PD culture—GCase elevation prolonged the stabilization of wild-type and 3K mutant αS tetramers in wtGBA1–transduced mouse brains, improving lysosomal integrity and motor and cognitive phenotypes. These data help elucidating lipid modulators that impact the αS physiological state in vivo and the development of PD therapeutic approaches., Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson’s disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient–derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer–monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD–causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)–human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets.

Background Total serum calcium (tCa) concentrations are poorly predictive of ionized calcium (iCa) status in dogs. Hypothesis There is an optimal threshold of tCa concentration that is highly predictive of ionized hypercalcemia and this threshold is higher in hyperphosphatemic dogs as compared to nonhyperphosphatemic dogs. Animals Nonhyperphosphatemic (n = 1593) and hyperphosphatemic (n = 250) adult dogs. Methods Retrospective medical record review of paired tCa and iCa concentration measurements in dogs presented to a university teaching hospital over a 5‐year period. Positive and negative predictive values, sensitivity, and specificity were calculated for tCa concentration thresholds of 11.0‐15.0 mg/dL (upper limit of laboratory reference interval = 11.5 mg/dL) in nonhyperphosphatemic and hyperphosphatemic groups. Results In nonhyperphosphatemic dogs, an optimal tCa concentration threshold of 12.0 mg/dL resulted in a positive predictive value of 93% (95% confidence interval [CI], 84%‐98%) and sensitivity of 52% (95% CI, 43%‐61%) for ionized hypercalcemia. An optimal tCa concentration threshold was not identified for hyperphosphatemic dogs. The nonhyperphosphatemic dogs had a higher prevalence of ionized hypercalcemia than the hyperphosphatemic dogs (7 versus 3%, P = .04) and a lower prevalence of ionized hypocalcemia (23 versus 62%, respectively; P

G-protein-coupled receptors like the human Y1 receptor (hY1R) are promising targets in cancer therapy due to their high overexpression on cancer cells and their ability to internalize together with...

We report that placental growth factor (PlGF) negatively affects the endothelial cell (EC) barrier function through a novel regulatory mechanism. The PlGF mAb promotes (but recombinant protein disrupts) EC barrier function, thus affecting the barrier-forming protein levels, membrane distribution, and EC monolayer impedance by the electrical cell-impedance sensing system, Western blot, and immunofluorescence staining. RNA sequencing–based transcriptome analysis identified the up-regulation of the pentose phosphate pathway (PPP) and the antioxidant defense protein by PlGF blockade. The PlGF and PlGF/VEGF dimers (but not VEGF-A) down-regulated the protein expression of glucose-6-phosphate dehydrogenase (G6PD) and peroxiredoxin (PRDX). G6PD inhibition and gene silencing (small interfering RNA) abolished the beneficial effects of PlGF inhibition on EC barrier function and PRDX3/6 protein expression. VEGF receptor (VEGFR)1 or VEGFR2 blockade prevented the inhibitory effect of PlGF on G6PD protein expression and EC barrier function. The PRDX6 played dual roles in EC barrier function through glutathione peroxidase and phospholipase A(2) activity. In sum, PlGF negatively regulates EC barrier function through the activation of VEGFR1 and VEGFR2 and the suppression of the G6PD/PPP and the antioxidant pathways.—Huang, H., Lennikov, A., Saddala, M. S., Gozal, D., Grab, D. J., Khalyfa, A., Fan, L. Placental growth factor negatively regulates endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems.

The Rio Grande Cone is a major fanlike depositional feature in the continental slope of the Pelotas Basin, Southern Brazil. Two representative sediment cores collected in the Cone area were retrieved using a piston core device. In this work, the organic matter (OM) in the sediments was characterized for a continental vs. marine origin using chemical proxies to help constrain the origin of gas in hydrates. The main contribution of OM was from marine organic carbon based on the stable carbon isotope (δ13C-org) and total organic carbon/total nitrogen ratio (TOC:TN) analyses. In addition, the 14C data showed important information about the origin of the OM and we suggest some factors that could modify the original organic matter and therefore mask the “real” 14C ages: (1) biological activity that could modify the carbon isotopic composition of bulk terrestrial organic matter values, (2) the existence of younger sediments from mass wasting deposits unconformably overlying older sediments, and (3) the deep-sediment-sourced methane contribution due to the input of “old” (>50 ka) organic compounds from migrating fluids.

An effect of ultraviolet exposure over the inhomogeneous structure of ionomer was investigated by using small-angle X-ray scattering (SAXS). Ionomer sheet laminated in between two sheets of glass was artificially weathered using a xenon lamp in an Atlas Ci4000 weatherometer. Though no significant changes were detected by chemical, thermal and optical analysis approach, SAXS identified changes at a low-angle upturn in the region of q

PURPOSE The KRISTINE study compared neoadjuvant trastuzumab emtansine plus pertuzumab (T-DM1+P) with docetaxel, carboplatin, trastuzumab plus P (TCH+P) for the treatment human epidermal growth factor receptor 2–positive stage II to III breast cancer. T-DM1+P led to a lower pathologic complete response rate (44.4% v 55.7%; P = .016), but fewer grade 3 or greater and serious adverse events (AEs). Here, we present 3-year outcomes from KRISTINE. METHODS Patients were randomly assigned to neoadjuvant T-DM1+P or TCH+P every 3 weeks for six cycles. Patients who received T-DM1+P continued adjuvant T-DM1+P, and patients who received TCH+P received adjuvant trastuzumab plus pertuzumab. Secondary end points included event-free survival (EFS), overall survival, patient-reported outcomes (measured from random assignment), and invasive disease-free survival (IDFS; measured from surgery). RESULTS Of patients, 444 were randomly assigned (T-DM1+P, n = 223; TCH+P, n = 221). Median follow-up was 37 months. Risk of an EFS event was higher with TDM-1+P (hazard ratio [HR], 2.61 [95% CI, 1.36 to 4.98]) with more locoregional progression events before surgery (15 [6.7%] v 0). Risk of an IDFS event after surgery was similar between arms (HR, 1.11 [95% CI, 0.52 to 2.40]). Pathologic complete response was associated with a reduced risk of an IDFS event (HR, 0.24 [95% CI, 0.09 to 0.60]) regardless of treatment arm. Overall, grade 3 or greater AEs (31.8% v 67.7%) were less common with T-DM1+P. During adjuvant treatment, grade 3 or greater AEs (24.5% v 9.9%) and AEs leading to treatment discontinuation (18.4% v 3.8%) were more common with T-DM1+P. Patient-reported outcomes favored T-DM1+P during neoadjuvant treatment and were similar to trastuzumab plus pertuzumab during adjuvant treatment. CONCLUSION Compared with TCH+P, T-DM1+P resulted in a higher risk of an EFS event owing to locoregional progression events before surgery, a similar risk of an IDFS event, fewer grade 3 or greater AEs during neoadjuvant treatment, and more AEs leading to treatment discontinuation during adjuvant treatment.

Introduction There is keen interest in elucidating the biological mechanisms underlying recent failures of β-site amyloid precursor protein–cleaving enzyme-1 (BACE1) inhibitors in Alzheimer's disease trials. Methods We developed a highly sensitive and specific immunoassay for BACE1 in cell lines and iPSC-derived human neurons to systematically analyze the effects of eight clinically relevant BACE1 inhibitors. Results Seven of 8 inhibitors elevated BACE1 protein levels. Among protease inhibitors tested, the elevation was specific to BACE1 inhibitors. The inhibitors did not increase BACE1 transcription but extended the protein's half-life. BACE1 became elevated at concentrations below the IC50 for amyloid β (Aβ). Discussion Elevation of BACE1 by 7 of 8 BACE1 inhibitors raises new concerns about advancing such β-secretase inhibitors for AD. Chronic elevation could lead to intermittently uninhibited BACE1 when orally dosed inhibitors reach trough levels, abnormally increasing substrate processing. Compounds such as roburic acid that lower Aβ by dissociating β/γ secretase complexes are better candidates because they neither inhibit β- and γ-secretase nor increase BACE1 levels.

Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H](+) ion at m/z 509.3351, previously designated as lysoSM-509. The structure of N-palmitoyl-O-phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among N-acyl-O-phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.

Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3. These channels transduce light information into electrical signals carried by both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2), which results in attenuation or loss of color vision, daylight blindness, and reduced visual acuity. Classical techniques to measure CNG channel function utilize patch clamp electrophysiology measuring Na currents in the absence of divalent cations, yet intracellular Ca2+ regulates both light and dark adaptation in photoreceptors. We developed a fluorescence-based, high-throughput Ca2+ flux assay using yellow fluorescent protein (YFP) tagged CNGA3 channels expressed in HEK293 cells which allow monitoring for folding defects in mutant channels. The cell permeant cGMP analog, 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), was used to activate Ca2+ flux. The assay was validated using wild-type CNGA3 homomeric and heteromeric channels and ACHM2-associated homomeric mutant CNG channels, CNGA3-R427C, CNGA3-E590K, and CNGA3-L633P. Additionally, we examined two naturally occurring canine mutations causing day-blindness previously studied by patch clamp. We compared the CPT-cGMP K0.5 values of the channels with patch clamp values from previous studies. The assay provides a screen for modulation of gating and/or rescue of trafficking and/or misfolding defects in ACHM2-associated CNG channels. Importantly, the calcium flux assay is advantageous compared to patch clamp as it allows the ability to monitor CNG channel activity in the presence of calcium.