Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease.

The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease.

Assessment of a Small Molecule Synthetic Lignan in Enhancing Oxidative Balance and Decreasing Lipid Accumulation in Human Retinal Pigment Epithelia.

Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 ß release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 ß release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.

Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment epithelia utilizes microtubule-associated protein 1 light chain 3B (LC3B).

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange, and waste removal. Each day the RPE removes the oldest ~10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation, and cell death. OSs are rich in very long chain fatty acids, which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblot analysis, and catalase activity assays, we investigated peroxisome abundance and activity at 6 AM, 7 AM (light onset), 8 AM, and 3 PM, in wild-type (WT) mice and mice lacking microtubule-associated protein 1 light chain 3B (Lc3b), which have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 PM, in RPE of WT, but not Lc3b-/-, mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice; however, numbers were elevated overall in Lc3b-/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.

Microtubule-Associated Protein 1 Light Chain 3 (LC3) Isoforms in RPE and Retina.

Microtubule-associated protein 1 light chain 3 (MAP1LC3), a human homologue of yeast Atg8, is an essential component of autophagy. LC3 plays a critical role in hybrid degradation pathways in which some but not all components of autophagy are coupled with phagocytosis in a process known as LC3-associated phagocytosis (LAP). LC3 exists as three highly homologous isoforms in human (LC3A, LC3B, and LC3C) with two of these (LC3A and LC3B) in mouse. LC3B predominated in both fetal and adult human retinal pigment epithelium (RPE) relative to LC3A and LC3C, while in mouse RPE and neural retina, LC3A and LC3B were expressed at approximately equivalent levels. In situ hybridization studies localized LC3A and LC3B transcripts in the retina and RPE. LC3B protein was detected in C57Bl6/J RPE and retinal lysates and was absent in the LC3BKO mouse.

Peer review of the pesticide risk assessment of the active substance lenacil.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Belgium, and co-rapporteur Member State, Austria, for the pesticide active substance lenacil are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of lenacil as a herbicide on sugar and fodder beet (field use). The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Updated peer review of the pesticide risk assessment of the active substance pydiflumetofen.

The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authority of the rapporteur Member State, France, and co-rapporteur Member State, Austria, for the pesticide active substance pydiflumetofen and the assessment of applications for maximum residue levels (MRLs) are reported. The context of the peer review was that required by Regulation (EC) No 1107/2009 of the European Parliament and of the Council. The conclusions were reached on the basis of the evaluation of the representative uses of pydiflumetofen as a fungicide field application on pome fruits, grapes, potato, fruiting vegetables, cucurbits and Brassica vegetables and updated following the request from Commission to consider additional information submitted and review the risk assessment. The reliable endpoints, appropriate for use in regulatory risk assessment and the proposed MRLs, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Updated peer review of the pesticide risk assessment of the active substance dichlorprop-P and variant dichlorprop-P-2-ethylhexyl.

The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Ireland, and co-rapporteur Member State, Poland, for the pesticide active substance dichlorprop-P and the variant dichlorprop-P-2-ethylhexyl and the assessment of applications for maximum residue levels (MRLs) are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of dichlorprop-P as a herbicide on cereals, grassland and grass seed crops and of the variant dichlorprop-P-2-ethylhexyl as a plant growth regulator on citrus. MRLs were assessed in mandarin and lemon. The conclusions from 2018 were updated in 2024 following the request from the European Commission with regard to the endocrine-disrupting properties. The reliable end points, appropriate for use in regulatory risk assessment and the proposed MRLs, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Peer review of the pesticide risk assessment of the active substance quinolin-8-ol.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Spain, and co-rapporteur Member State, the Netherlands, for the pesticide active substance quinolin-8-ol are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative use of quinolin-8-ol as a fungicide and bactericide against soil-borne pathogens in tomato cultivation in permanent greenhouses applied by drip irrigation. The reliable end points, appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Peer review of the pesticide risk assessment of the active substance clove oil.

The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authority of the rapporteur Member State, Malta, for the pesticide active substance clove oil are reported. The context of the peer review was that required by Regulation (EC) No 1107/2009 of the European Parliament and of the Council. The conclusions for the amendment of approval were reached on the basis of the evaluation of the representative use of clove oil as a preharvest nematicide on tomatoes and cucumbers (permanent greenhouse use). The representative use evaluated for the renewal of approval of clove oil was as post-harvest fungicide and bactericide on apples, pears and peaches (indoor uses). The reliable endpoints appropriate for use in regulatory risk assessment are presented. Endpoints not relevant to the scope of the proposed amendment of approval conditions will be addressed in the context of the renewal of approval procedure of clove oil running in parallel (AIR IV, EFSA Q-2016-00809). Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Efficient optimization of time-varying inputs in a fed-batch cell culture process using design of dynamic experiments.

In cell culture process development, we rely largely on an iterative, one-factor-at-a-time procedure based on experiments that explore a limited process space. Design of experiments (DoE) addresses this issue by allowing us to analyze the effects of process inputs on process responses systematically and efficiently. However, DoE cannot be applied directly to study time-varying process inputs unless an impractically large number of bioreactors is used. Here, we adopt the methodology of design of dynamic experiments (DoDE) and incorporate dynamic feeding profiles efficiently in late-stage process development of the manufacture of therapeutic monoclonal antibodies. We found that, for the specific cell line used in this article, (1) not only can we estimate the effect of nutrient feed amount on various product attributes, but we can also estimate the effect, develop a statistical model, and use the model to optimize the slope of time-trended feed rates; (2) in addition to the slope, higher-order dynamic characteristics of time-trended feed rates can be incorporated in the design but do not have any significant effect on the responses we measured. Based on the DoDE data, we developed a statistical model and used the model to optimize several process conditions. Our effort resulted in a tangible improvement in productivity-compared with the baseline process without dynamic feeding, this optimized process in a 200-L batch achieved a 27% increase in titer and > 92% viability. We anticipate our application of DoDE to be a starting point for more efficient workflows to optimize dynamic process conditions in process development.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Peer review of the pesticide risk assessment of the active substance ethephon.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, the Netherlands and co-rapporteur Member State, the United Kingdom, for the pesticide active substance ethephon are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative use of ethephon as a plant growth regulator on cereals (winter and spring barley, winter and spring wheat, winter rye, winter triticale, spelt, durum wheat). The reliable endpoints appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Peer review of the pesticide risk assessment of the active substance fat distillation residues.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Czech Republic, and co-rapporteur Member State, France, for the pesticide active substance fat distillation residues are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of fat distillation residues as a repellent on seedlings of coniferous and deciduous trees. The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns were not identified.

Peer review of the pesticide risk assessment of the active substance S-metolachlor excluding the assessment of the endocrine disrupting properties.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State Germany and co-rapporteur Member State France for the pesticide active substance S-metolachlor are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. In September 2022, the European Commission asked EFSA to deliver its conclusion on the available outcomes of the assessments in all areas excluding the full assessment of endocrine disrupting properties as several critical areas of concern related to the protection of the environment were identified. The conclusions were reached on the basis of the evaluation of the representative uses of S-metolachlor as a herbicide on maize and sunflower. The reliable end points, appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. The concerns identified are presented.

Peer review of the pesticide risk assessment of the active substance glyphosate.

The conclusions of EFSA following the peer review of the initial risk assessments carried out by the Assessment Group on Glyphosate (AGG), consisting of the competent authorities of France, the Netherlands, Sweden and Hungary, acting jointly as rapporteur Member State for the pesticide active substance glyphosate are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of glyphosate as a herbicide as proposed by the applicants, covering uses pre-sowing, pre-planting and pre-emergence plus post-harvest in vegetables and sugar beet; post-emergence of weeds in orchards, vineyards, row vegetables, railway tracks against emerged annual, biennial and perennial weeds. Moreover, uses as spot treatment against invasive species in agricultural and non-agricultural areas, and in vegetables and sugar beet against couch grass are also included. The reliable endpoints, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Peer review of the pesticide risk assessment of the active substance sulfur.

The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State France and co-rapporteur Member State Slovenia for the pesticide active substance sulfur and the considerations as regards the inclusion of the substance in Annex IV of Regulation (EC) No 396/2005 are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of sulfur as a fungicide and acaricide on grapevine and cereals (wheat, barley, oat, rye, triticale). The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Peer review of the pesticide risk assessment of the active substance metiram.

The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State Italy and co-rapporteur Member State the United Kingdom for the pesticide active substance metiram are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of metiram as a fungicide on grapes (wine table) and potatoes (all being field uses). It was concluded that following the guidance on this topic, metiram has endocrine disrupting potential, which is a cut-off criterion for non-approval of an active substance. Considerations are also provided on whether exposure to humans and the environment from the representative use of metiram on potatoes can be considered negligible, taking into account information from the applicant and the European Commission's draft technical guidance on this topic. The information available indicated this exposure was not negligible. The reliable end points, appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified. An evaluation of data concerning the necessity of metiram as a fungicide to control a serious danger to plant health which cannot be contained by other available means, including non-chemical methods is also presented.

Peer review of the pesticide risk assessment of the active substance metrafenone.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State Latvia and co-rapporteur Member State Slovakia for the pesticide active substance metrafenone are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of metrafenone as a fungicide on wheat, rye, triticale, oats, barley and grapes (field use). The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Peer review of the pesticide risk assessment of the active substance (3E)-dec-3-en-2-one.

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authority of the rapporteur Member State, the Netherlands, for the pesticide active substance (3E)-dec-3-en-2-one and the considerations as regards the inclusion of the substance in Annex IV of Regulation (EC) No 396/2005 are reported. The context of the peer review was that required by Regulation (EC) No 1107/2009 of the European Parliament and of the Council. The conclusions were reached on the basis of the evaluation of the representative post-harvest use of (3E)-dec-3-en-2-one on potato as a sprouting inhibitor applied by hot fogging in potato storage rooms. The reliable endpoints, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.