MDA5-autoimmunity and interstitial pneumonitis contemporaneous with the COVID-19 pandemic (MIP-C).

BACKGROUND: Anti-MDA5 (Melanoma differentiation-associated protein-5) positive dermatomyositis (MDA5+-DM) is characterised by rapidly progressive interstitial lung disease (ILD) and high mortality. MDA5 is an RNA sensor and a key pattern recognition receptor for the SARS-CoV-2 virus. METHODS: This is a retrospective observational study of a surge in MDA5 autoimmunity, as determined using a 15 muscle-specific autoantibodies (MSAs) panel, between Janurary 2018 and December 2022 in Yorkshire, UK. MDA5-positivity was correlated with clinical features and outcome, and regional SARS-CoV-2 positivity and vaccination rates. Gene expression patterns in COVID-19 were compared with autoimmune lung disease and idiopathic pulmonary fibrosis (IPF) to gain clues into the genesis of the observed MDA5+-DM outbreak. FINDINGS: Sixty new anti-MDA5+, but not other MSAs surged between 2020 and 2022, increasing from 0.4% in 2019 to 2.1% (2020), 4.8% (2021) and 1.7% (2022). Few (8/60) had a prior history of confirmed COVID-19, peak rates overlapped with regional SARS-COV-2 community positivity rates in 2021, and 58% (35/60) had received anti-SARS-CoV-2 vaccines. 25/60 cases developed ILD which rapidly progression with death in 8 cases. Among the 35/60 non-ILD cases, 14 had myositis, 17 Raynaud phenomena and 10 had dermatomyositis spectrum rashes. Transcriptomic studies showed strong IFIH1 (gene encoding for MDA5) induction in COVID-19 and autoimmune-ILD, but not IPF, and IFIH1 strongly correlated with an IL-15-centric type-1 interferon response and an activated CD8+ T cell signature that is an immunologic hallmark of progressive ILD in the setting of systemic autoimmune rheumatic diseases. The IFIH1 rs1990760TT variant blunted such response. INTERPRETATION: A distinct pattern of MDA5-autoimmunity cases surged contemporaneously with circulation of the SARS-COV-2 virus during COVID-19. Bioinformatic insights suggest a shared immunopathology with known autoimmune lung disease mechanisms. FUNDING: This work was supported in part by the National Institute for Health Research (NIHR) Leeds Biomedical Research Centre (BRC), and in part by the National Institutes of Health (NIH) grant R01-AI155696 and pilot awards from the UC Office of the President (UCOP)-RGPO (R00RG2628, R00RG2642 and R01RG3780) to P.G. S.S was supported in part by R01-AI141630 (to P.G) and in part through funds from the American Association of Immunologists (AAI) Intersect Fellowship Program for Computational Scientists and Immunologists.

Silicon Ether Ionizable Lipids Enable Potent mRNA Lipid Nanoparticles with Rapid Tissue Clearance.

The advent of mRNA for nucleic acid (NA) therapeutics has unlocked many diverse areas of research and clinical investigation. However, the shorter intracellular half-life of mRNA compared with other NAs may necessitate more frequent dosing regimens. Because lipid nanoparticles (LNPs) are the principal delivery system used for mRNA, this could lead to tolerability challenges associated with an accumulated lipid burden. This can be addressed by introducing enzymatically cleaved carboxylic esters into the hydrophobic domains of lipid components, notably, the ionizable lipid. However, enzymatic activity can vary significantly with age, disease state, and species, potentially limiting the application in humans. Here we report an alternative approach to ionizable lipid degradability that relies on nonenzymatic hydrolysis, leading to a controlled and highly efficient lipid clearance profile. We identify highly potent examples and demonstrate their exceptional tolerability in multiple preclinical species, including multidosing in nonhuman primates (NHP).

Novel Iron-Chelating Prodrug Significantly Enhanced Fluorescence-Mediated Detection of Glioma Cells Experimentally In Vitro.

(1) Background: The protoporphyrin IX (PpIX)-mediated fluorescence-guided resection and interoperative photodynamic therapy (PDT) of remaining cells may be effective adjuvants to the resection of glioma. Both processes may be enhanced by increasing intracellular PpIX concentrations, which can be achieved through iron chelation. AP2-18 is a novel combinational drug, which ester-links a PpIX precursor (aminolaevulinic acid; ALA) to an iron-chelating agent (CP94). (2) Methods: Human glioma U-87 MG cells were cultured in 96-well plates for 24 h and incubated for 3 or 6 h with various test compound combinations: ALA (±) CP94, methyl aminolevulinate (MAL) (±) CP94 and AP2-18. PpIX fluorescence was measured at 0, 3 or 6 h with a Bio-tek Synergy HT plate reader, as well as immediately after irradiation with a 635 nm red light (Aktilite CL16 LED array), representing the PDT procedure. Cell viability post-irradiation was assessed using the neutral red assay. (3) Results: AP2-18 significantly increased PpIX fluorescence compared to all other test compounds. All treatment protocols effectively achieved PDT-induced cytotoxicity, with no significant difference between test compound combinations. (4) Conclusions: AP2-18 has potential to improve the efficacy of fluorescence-guided resection either with or without the subsequent intraoperative PDT of glioma. Future work should feature a more complex in vitro model of the glioma microenvironment.

MDA5-autoimmunity and Interstitial Pneumonitis Contemporaneous with the COVID-19 Pandemic (MIP-C).

Background: Anti-MDA5 (Melanoma differentiation-associated protein-5) positive dermatomyositis (MDA5 + -DM) is characterised by rapidly progressive interstitial lung disease (ILD) and high mortality. MDA5 senses single-stranded RNA and is a key pattern recognition receptor for the SARS-CoV-2 virus. Methods: This is a retrospective observational study of a surge in MDA5 autoimmunity, as determined using a 15 muscle-specific autoantibodies (MSAs) panel, between Janurary 2018-December 2022 in Yorkshire, UK. MDA5-positivity was correlated with clinical features and outcome, and regional SARS-CoV-2 positivity and vaccination rates. Gene expression patterns in COVID-19 were compared with autoimmune lung disease and idiopathic pulmonary fibrosis (IPF) to gain clues into the genesis of the observed MDA5 + -DM outbreak. Results: Sixty new anti-MDA5+, but not other MSAs surged between 2020-2022, increasing from 0.4% in 2019 to 2.1% (2020), 4.8% (2021) and 1.7% (2022). Few (8/60) had a prior history of confirmed COVID-19, peak rates overlapped with regional SARS-COV-2 community positivity rates in 2021, and 58% (35/60) had received anti-SARS-CoV-2 RNA vaccines. Few (8/60) had a prior history of COVID-19, whereas 58% (35/60) had received anti-SARS-CoV-2 RNA vaccines. 25/60 cases developed ILD which rapidly progression with death in 8 cases. Among the 35/60 non-ILD cases, 14 had myositis, 17 Raynaud phenomena and 10 had dermatomyositis spectrum rashes. Transcriptomic studies showed strong IFIH1 (gene encoding for MDA5) induction in COVID-19 and autoimmune-ILD, but not IPF, and IFIH1 strongly correlated with an IL-15-centric type-1 interferon response and an activated CD8+ T cell signature that is an immunologic hallmark of progressive ILD in the setting of systemic autoimmune rheumatic diseases. The IFIH1 rs1990760TT variant blunted such response. Conclusions: A distinct pattern of MDA5-autoimmunity cases surged contemporaneously with circulation of the SARS-COV-2 virus during COVID-19. Bioinformatic insights suggest a shared immunopathology with known autoimmune lung disease mechanisms.

Unsaturated, Trialkyl Ionizable Lipids are Versatile Lipid-Nanoparticle Components for Therapeutic and Vaccine Applications.

Lipid nanoparticles (LNPs) have proven a successful platform for the delivery of nucleic acid (NA)-based therapeutics and vaccines, with the ionizable lipid component playing a key role in modulating potency and tolerability. Here, a library of 16 novel ionizable lipids is screened hypothesizing that short, branched trialkyl hydrophobic domains can improve LNP fusogenicity or endosomal escape, and potency. LNPs formulated with the top-performing trialkyl lipid (Lipid 10) encapsulating transthyretin siRNA elicit significantly greater gene silencing and are better tolerated than those with the benchmark Onpattro lipid DLin-MC3-DMA. Lipid 10 also demonstrates superior liver delivery of mRNA when compared to other literature ionizable lipids, is well tolerated, and successfully repeat-doses in nonhuman primates. In a prime-boost hemagglutinin rodent vaccine model, intramuscular administration of Lipid-10 LNP elicits comparable or better antibody titers to the SM-102 and ALC-0315 lipid compositions used in the U.S. Food and Drug Administration approved mRNA COVID vaccines. These data suggest that Lipid 10 is a particularly versatile ionizable lipid, well-suited for both systemic therapeutic and intramuscular vaccine applications and able to successfully deliver diverse NA payloads.

Combination treatment of mannose and GalNAc conjugated small interfering RNA protects against lethal Marburg virus infection.

Marburg virus (MARV) infection results in severe viral hemorrhagic fever with mortalities up to 90%, and there is a pressing need for effective therapies. Here, we established a small interfering RNA (siRNA) conjugate platform that enabled successful subcutaneous delivery of siRNAs targeting the MARV nucleoprotein. We identified a hexavalent mannose ligand with high affinity to macrophages and dendritic cells, which are key cellular targets of MARV infection. This ligand enabled successful siRNA conjugate delivery to macrophages both in vitro and in vivo. The delivered hexa-mannose-siRNA conjugates rendered substantial target gene silencing in macrophages when supported by a mannose functionalized endosome release polymer. This hexa-mannose-siRNA conjugate was further evaluated alongside our hepatocyte-targeting GalNAc-siRNA conjugate, to expand targeting of infected liver cells. In MARV-Angola-infected guinea pigs, these platforms offered limited survival benefit when used as individual agents. However, in combination, they achieved up to 100% protection when dosed 24 h post infection. This novel approach, using two different ligands to simultaneously deliver siRNA to multiple cell types relevant to infection, provides a convenient subcutaneous route of administration for treating infection by these dangerous pathogens. The mannose conjugate platform has potential application to other diseases involving macrophages and dendritic cells.

Paediatric inflammatory multisystem syndrome: temporally associated with SARS-CoV-2 (PIMS-TS) - A mimic of intra-abdominal malignancy.

We report the case of a school-aged boy who presented with clinical features suggesting acute appendicitis. However, further imaging which included CT, demonstrated an inflammatory mass involving the transverse colon raising the suspicion of lymphoma. He then developed intestinal obstruction, and in view of the rapid progression of the disease, he was thought to have non-Hodgkin's lymphoma. He underwent an open excisional biopsy, which revealed a necroinflammatory process and no suggestion of lymphoma or an alternative malignancy or specific diagnosis. His steroid treatment was stopped, and he made a good recovery postoperatively. Positive COVID-19 antibodies, positive response to steroids, results and clinical features were consistent with paediatric inflammatory multisystem syndrome (PIMS-TS), with extensive investigation not offering an alternative diagnosis.While PIMS-TS is a relatively new entity, we believe that this case highlights the importance of it being considered a differential diagnosis of a child presenting with an inflammatory mass.

Understanding Engagement With Forensic Smartphone Apps: The Service Design Engagement Model.

Justice services have begun to integrate the use of mobile applications into treatment, support, and rehabilitative programs for forensic clients. One such application that been adopted to support forensic clients is "eRecovery": a smartphone application that provides clients recovering from a substance addiction with support for managing relapse. In this article, we report on evaluation findings from a trial of eRecovery in an Australian Community Justice Centre, and reflect on several issues relating to fostering and sustaining client engagement with similar applications within forensic and justice settings. We propose the Service Design Engagement Model to organize, visualize, and describe the stages and factors important to adoption, appropriation, and on-going routine use of the software by forensic clients. The model recognizes the role of contextual and environmental factors in supporting users through the early stages of engagement, and the importance of user agency in longer-term engagement with therapeutic apps.

Vasorelaxant Activity of AP39, a Mitochondria-Targeted H2S Donor, on Mouse Mesenteric Artery Rings In Vitro.

Mitochondria-targeted hydrogen sulfide (H2S) donor compounds, such as compound AP39, supply H2S into the mitochondrial environment and have shown several beneficial in vitro and in vivo effects in cardiovascular conditions such as diabetes and hypertension. However, the study of their direct vascular effects has not been addressed to date. Thus, the objective of the present study was to analyze the effects and describe the mechanisms of action of AP39 on the in vitro vascular reactivity of mouse mesenteric artery. Protein and gene expressions of the H2S-producing enzymes (CBS, CSE, and 3MPST) were respectively analyzed by Western blot and qualitative RT-PCR, as well the in vitro production of H2S by mesenteric artery homogenates. Gene expression of CSE and 3MPST in the vessels has been evidenced by RT-PCR experiments, whereas the protein expression of all the three enzymes was demonstrated by Western blotting experiments. Nonselective inhibition of H2S-producing enzymes by AOAA abolished H2S production, whereas it was partially inhibited by PAG (a CSE selective inhibitor). Vasorelaxation promoted by AP39 and its H2S-releasing moiety (ADT-OH) were significantly reduced after endothelium removal, specifically dependent on NO-cGMP signaling and SKCa channel opening. Endogenous H2S seems to participate in the mechanism of action of AP39, and glibenclamide-induced KATP blockade did not affect the vasorelaxant response. Considering the results of the present study and the previously demonstrated antioxidant and bioenergetic effects of AP39, we conclude that mitochondria-targeted H2S donors may offer a new promising perspective in cardiovascular disease therapeutics.

Mitochondria-Targeted Hydrogen Sulfide Delivery Molecules Protect Against UVA-Induced Photoaging in Human Dermal Fibroblasts, and in Mouse Skin In Vivo.

Aims: Oxidative stress and mitochondrial dysfunction play a role in the process of skin photoaging via activation of matrix metalloproteases (MMPs) and the subsequent degradation of collagen. The activation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor controlling antioxidant and cytoprotective defense systems, might offer a pharmacological approach to prevent skin photoaging. We therefore investigated a pharmacological approach to prevent skin photoaging, and also investigated a protective effect of the novel mitochondria-targeted hydrogen sulfide (H2S) delivery molecules AP39 and AP123, and nontargeted control molecules, on ultraviolet A light (UVA)-induced photoaging in normal human dermal fibroblasts (NHDFs) in vitro and the skin of BALB/c mice in vivo. Results: In NHDFs, AP39 and AP123 (50-200 nM) but not nontargeted controls suppressed UVA (8 J/cm2)-mediated cytotoxicity and induction of MMP-1 activity, preserved cellular bioenergetics, and increased the expression of collagen and nuclear levels of Nrf2. In in vivo experiments, topical application of AP39 or AP123 (0.3-1 µM/cm2; but not nontargeted control molecules) to mouse skin before UVA (60 J/cm2) irradiation prevented skin thickening, MMP induction, collagen loss of oxidative stress markers 8-hydroxy-2'-deoxyguanosine (8-OHdG), increased Nrf2-dependent signaling, as well as increased manganese superoxide dismutase levels and levels of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α). Innovation and Conclusion: Targeting H2S delivery to mitochondria may represent a novel approach for the prevention and treatment of skin photoaging, as well as being useful tools for determining the role of mitochondrial H2S in skin disorders and aging. Antioxid. Redox Signal. 36, 1268-1288.

Experimental investigation of a combinational iron chelating protoporphyrin IX prodrug for fluorescence detection and photodynamic therapy.

Photodynamic therapy (PDT) is an oxygen-dependent, light-activated, and locally destructive drug treatment of cancer. Protoporphyrin IX (PpIX)-induced PDT exploits cancer cells' own innate heme biosynthesis to hyper-accumulate the naturally fluorescent and photoactive precursor to heme, PpIX. This occurs as a result of administering heme precursors (e.g., aminolevulinic acid; ALA) because the final step of the pathway (the insertion of ferrous iron into PpIX by ferrochelatase to form heme) is relatively slow. Separate administration of an iron chelating agent has previously been demonstrated to significantly improve dermatological PpIX-PDT by further limiting heme production. A newly synthesized combinational iron chelating PpIX prodrug (AP2-18) has been assessed experimentally in cultured primary human cells of bladder and dermatological origin, as an alternative photosensitizing agent to ALA or its methyl or hexyl esters (MAL and HAL respectively) for photodetection/PDT. Findings indicated that the technique of iron chelation (either through the separate administration of the established hydroxypyridinone iron chelator CP94 or the just as effective combined AP2-18) did not enhance either PpIX fluorescence or PDT-induced (neutral red assessed) cell death in human primary normal and malignant bladder cells. However, 500 µM AP2-18 significantly increased PpIX accumulation and produced a trend of increased cell death within epithelial squamous carcinoma cells. PpIX accumulation destabilized the actin cytoskeleton in bladder cancer cells prior to PDT and resulted in caspase-3 cleavage/early apoptosis afterwards. AP2-18 iron chelation should continue to be investigated for the enhancement of dermatological PpIX-PDT applications but not bladder photodetection/PDT.

Primary Crime Prevention Apps: A Typology and Scoping Review.

In the last decade, an array of smartphone apps have been designed to prevent crime, violence, and abuse. The evidence base of these apps has, however, yet to analyzed systematically. To rectify this, the aims of this review were (1) to establish the extent, range, and nature of research into smartphone apps with a primary crime prevention function; (2) to locate gaps in the primary crime prevention app literature; and (3) to develop a typology of primary crime prevention apps. Employing a scoping review methodology and following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, studies were identified via Web of Science, EBSCOhost, and Google Scholar. We included English-language research published between 2008 and 2020 that examined smartphone applications designed explicitly for primary crime prevention. Sixty-one publications met our criteria for review, out of an initial sample of 151 identified. Our review identified six types of crime prevention app examined in these publications: self-surveillance apps, decision aid apps, child-tracking apps, educational apps, crime-mapping/alert apps, and crime reporting apps. The findings of our review indicate that most of these forms of primary crime prevention apps have yet to be rigorously evaluated and many are not evidence-based in their design. Consequently, our review indicates that recent enthusiasm over primary crime prevention apps is not supported by an adequate evidence base.

Hydrogen Sulfide Is a Novel Protector of the Retinal Glycocalyx and Endothelial Permeability Barrier.

Significantly reduced levels of the anti-inflammatory gaseous transmitter hydrogen sulfide (H2S) are observed in diabetic patients and correlate with microvascular dysfunction. H2S may protect the microvasculature by preventing loss of the endothelial glycocalyx. We tested the hypothesis that H2S could prevent or treat retinal microvascular endothelial dysfunction in diabetes. Bovine retinal endothelial cells (BRECs) were exposed to normal (NG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L) ± the slow-release H2S donor NaGYY4137 in vitro. Glycocalyx coverage (stained with WGA-FITC) and calcein-labeled monocyte adherence were measured. In vivo, fundus fluorescein angiography (FFA) was performed in normal and streptozotocin-induced (STZ) diabetic rats. Animals received intraocular injection of NaGYY4137 (1 µM) or the mitochondrial-targeted H2S donor AP39 (100 nM) simultaneously with STZ (prevention) or on day 6 after STZ (treatment), and the ratio of interstitial to vascular fluorescence was used to estimate apparent permeability. NaGYY4137 prevented HG-induced loss of BREC glycocalyx, increased monocyte binding to BRECs (p ≤ 0.001), and increased overall glycocalyx coverage (p ≤ 0.001). In rats, the STZ-induced increase in apparent retinal vascular permeability (p ≤ 0.01) was significantly prevented by pre-treatment with NaGYY4137 and AP39 (p < 0.05) and stabilized by their post-STZ administration. NaGYY4137 also reduced the number of acellular capillaries (collagen IV + /IB4-) in the diabetic retina in both groups (p ≤ 0.05). We conclude that NaGYY4137 and AP39 protected the retinal glycocalyx and endothelial permeability barrier from diabetes-associated loss of integrity and reduced the progression of diabetic retinopathy (DR). Hydrogen sulfide donors that target the glycocalyx may therefore be a therapeutic candidate for DR.

Ligand conjugate SAR and enhanced delivery in NHP.

N-Acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) are a leading RNA interference (RNAi) platform allowing targeted inhibition of disease-causing genes in hepatocytes. More than a decade of development has recently resulted in the first approvals for this class of drugs. While substantial effort has been made to improve nucleic acid modification patterns for better payload stability and efficacy, relatively little attention has been given to the GalNAc targeting ligand. In addition, the lack of an intrinsic endosomal release mechanism has limited potency. Here, we report a stepwise analysis of the structure activity relationships (SAR) of the components comprising these targeting ligands. We show that there is relatively little difference in biological performance between bi-, tri-, and tetravalent ligand structures while identifying other features that affect their biological activity more significantly. Further, we demonstrate that subcutaneous co-administration of a GalNAc-functionalized, pH responsive endosomal release agent markedly improved the activity and duration of effect for siRNA conjugates, without compromising tolerability, in non-human primates. These findings could address a significant bottleneck for future siRNA ligand conjugate development.

Anti-tumour necrosis factor treatment for the prevention of ischaemic events in patients with deficiency of adenosine deaminase 2 (DADA2).

OBJECTIVE: To evaluate the impact of anti-Tumour Necrosis Factor-α (anti-TNF) treatment on the occurrence of vasculitic ischaemic events in patients with deficiency of adenosine deaminase 2 (DADA2). METHODS: A retrospective analysis of DADA2 patients referred from six centres to Great Ormond Street Hospital for Children was conducted. Ischaemic events, vasculitic disease activity, biochemical, immunological, and radiological features were compared, before and after anti-TNF treatment. RESULTS: A total of 31 patients with genetically confirmed DADA2 were included in the study. The median duration of active disease activity prior to anti-TNF treatment was 73 months (inter-quartile range [IQR] 27.5-133.5 months). Twenty seven/31 patients received anti-TNF treatment for a median of 32 months (IQR 12.0-71.5 months). The median event rate of central nervous system (CNS) and non-CNS ischemic events before anti-TNF treatment was 2.37 per 100 patient-months (IQR 1.25-3.63); compared with 0.00 per 100 patient-months (IQR 0.0-0.0) post-treatment (p< 0.0001). Paediatric vasculitis activity score (PVAS) was also significantly reduced: median score of 20/63 (IQR 13.0-25.8/63) pre-treatment vs. 2/63 (IQR 0.0-3.8/63) following anti-TNF treatment (p< 0.0001), with mild livedoid rash being the main persisting feature. Anti-TNF treatment was not effective for severe immunodeficiency or bone marrow failure, which required haematopoietic stem cell transplantation (HSCT). CONCLUSION: Anti-TNF treatment significantly reduced the incidence of ischaemic events and other vasculitic manifestations of DADA2, but was not effective for immunodeficiency or bone marrow failure.

A lipophilic cation protects crops against fungal pathogens by multiple modes of action.

The emerging resistance of crop pathogens to fungicides poses a challenge to food security and compels discovery of new antifungal compounds. Here, we show that mono-alkyl lipophilic cations (MALCs) inhibit oxidative phosphorylation by affecting NADH oxidation in the plant pathogens Zymoseptoria tritici, Ustilago maydis and Magnaporthe oryzae. One of these MALCs, consisting of a dimethylsulfonium moiety and a long alkyl chain (C18-SMe2+), also induces production of reactive oxygen species at the level of respiratory complex I, thus triggering fungal apoptosis. In addition, C18-SMe2+ activates innate plant defense. This multiple activity effectively protects cereals against Septoria tritici blotch and rice blast disease. C18-SMe2+ has low toxicity in Daphnia magna, and is not mutagenic or phytotoxic. Thus, MALCs hold potential as effective and non-toxic crop fungicides.

Investigating ROS, RNS, and H2S-Sensitive Signaling Proteins.

The modification of proteins is a key way to alter their activity and function. Often thiols, cysteine residues, on proteins are attractive targets for such modification. Assuming that the thiol group is accessible then reactions may take place with a range of chemicals found in cells. These may include reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), reactive nitrogen species such as nitric oxide (NO), hydrogen sulfide (H2S), or glutathione. Such modifications often are instrumental to important cellular signaling processes, which ultimately result in modification of physiology of the organism. Therefore, there is a need to be able to identify such modifications. There are a variety of techniques to find proteins which may be altered in this way but here the focus is on two approaches: firstly, the use of fluorescent thiol derivatives and the subsequent use of mass spectrometry to identify the thiols involved; secondly the confirmation of such changes using biochemical assays and genetic mutants. The discussion will be based on the use of two model organisms: firstly the plant Arabidopsis thaliana (both as cell cultures and whole plants) and secondly the nematode worm Caenorhabditis elegans. However, these tools, as described, may be used in a much wider range of biological systems, including human and human tissue cultures.

Effects of measurement timing on subgroup identification using growth mixture modeling: An empirical application to alcohol use.

Growth mixture modeling (GMM) identifies latent classes exhibiting distinct longitudinal patterns on an outcome. Subgroups identified by GMM may be artifactually influenced by measurement timing (e.g., timing of the initial assessment, length of the interval from the first to the last assessment, and total number of assessments) as well as the theoretically posited developmental patterns of the behavior. The current study investigated this possibility using alcohol data from the 1997 National Longitudinal Survey of Youth (n = 2686; 49.44% female; 71.84% White). Three assessment configurations were examined: all 12 waves, first 6 waves, and last 7 waves. Five subgroups were identified using all 12 waves: Normative (71.33%), Low-Increasing (8.45%), Low-Steady (8.97%), High-Slowly Decreasing (7.67%), and Extreme-Sharply Decreasing (3.57%). When comparing participants' subgroup membership for all 12 waves to the first six waves, 14% of the sample was differentially classified. When comparing all 12 waves to the last seven waves, 62% of the sample was differentially classified. Alterations in the timing of the initial assessment had a substantial impact on latent class estimation, underscoring the importance of selecting the developmental window a priori based on theory and empirical knowledge. The time-bounded nature of mixture modeling solutions (i.e., a selected developmental window within the course of a phenomenon) suggests that the latent subgroups should not be interpreted as representing subgroups that are present in the population. Future directions and strategies for testing alternative interpretations are presented. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Improving in vitro photodynamic therapy through the development of a novel iron chelating aminolaevulinic acid prodrug.

BACKGROUND: Photodynamic therapy (PDT) is a light activated drug therapy that can be used to treat a number of cancers and precancers. It is particularly useful in its topical form in dermatology but improvement of efficacy is required to widen its application. METHODS: An ester between aminolaevulinic acid (ALA) and CP94 was synthesised (AP2-18) and experimentally evaluated to determine whether protoporphyrin IX (PpIX)-induced PDT effectiveness could be improved. A biological evaluation of AP2-18 was conducted in cultured human primary cells with both PpIX fluorescence and cell viability (as determined via the neutral red assay) being assessed in comparison to the PpIX prodrugs normally utilised in clinical practice (aminolaevulinic acid (ALA) or its methyl ester (MAL)) either administered alone or with the comparator iron chelator, CP94. RESULTS: No significant dark toxicity was observed in human lung fibroblasts but AP2-18 significantly increased PpIX accumulation above and beyond that achieved with ALA or MAL administration +/- CP94 in both human dermal fibroblasts and epithelial squamous carcinoma cells. On light exposure, the combined hydroxypyridinone iron chelating ALA prodrug AP2-18 generated significantly greater cytotoxicity than any of the other treatment parameters investigated when the lowest concentration (250 µM) was employed. CONCLUSIONS: Newly synthesised AP2-18 is therefore concluded to be an efficacious prodrug for PpIX-induced PDT in these dermatologically relevant human cells, achieving enhanced effects at lower concentrations than currently possible with existing pharmaceuticals.