AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS neurons.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative disorder caused by intronic biallelic, nonreference CCCTT/AAGGG repeat expansions within RFC1. To investigate how these repeats cause disease, we generated patient induced pluripotent stem cell-derived neurons (iNeurons). CCCTT/AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway function. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins. However, these proteins and repeat RNA foci were not detected in iNeurons, and overexpression of these repeats failed to induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded AAGGG allele. These deficits were neither replicated by RFC1 knockdown in control iNeurons nor rescued by RFC1 reprovision in CANVAS iNeurons. These findings support a repeat-dependent but RFC1 protein-independent cause of neuronal dysfunction in CANVAS, with implications for therapeutic development in this currently untreatable condition.

Molecular Visualization of Neuronal TDP43 Pathology In Situ.

Nuclear exclusion and cytoplasmic accumulation of the RNA-binding protein TDP43 are characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite this, the origin and ultrastructure of cytosolic TDP43 deposits remain unknown. Accumulating evidence suggests that abnormal RNA homeostasis can drive pathological TDP43 mislocalization, enhancing RNA misprocessing due to loss of nuclear TDP43 and engendering a cycle that ends in cell death. Here, we show that adding small monovalent oligonucleotides successfully recapitulates pathological TDP43 mislocalization and aggregation in iPSC-derived neurons (iNeurons). By employing a multimodal in situ cryo-correlative light and electron microscopy pipeline, we examine how RNA influences the localization and aggregation of TDP43 in near-native conditions. We find that mislocalized TDP43 forms ordered fibrils within lysosomes and autophagosomes in iNeurons as well as in patient tissue, and provide the first high-resolution snapshots of TDP43 aggregates in situ. In so doing, we provide a cellular model for studying initial pathogenic events underlying ALS, FTLD, and related TDP43-proteinopathies.

Performance of Risk Assessment Models for VTE in Patients Who Are Critically Ill Receiving Pharmacologic Thromboprophylaxis: A Post Hoc Analysis of the Pneumatic Compression for Preventing VTE Trial.

BACKGROUND: The diagnostic performance of the available risk assessment models for VTE in patients who are critically ill receiving pharmacologic thromboprophylaxis is unclear. RESEARCH QUESTION: For patients who are critically ill receiving pharmacologic thromboprophylaxis, do risk assessment models predict who would develop VTE or who could benefit from adjunctive pneumatic compression for thromboprophylaxis? STUDY DESIGN AND METHODS: In this post hoc analysis of the Pneumatic Compression for Preventing VTE (PREVENT) trial, different risk assessment models for VTE (ICU-VTE, Kucher, Intermountain, Caprini, Padua, and International Medical Prevention Registry on VTE [IMPROVE] models) were evaluated. Receiver-operating characteristic curves were constructed, and the sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios were calculated. In addition, subgroup analyses were performed evaluating the effect of adjunctive pneumatic compression vs none on the study primary outcome. RESULTS: Among 2,003 patients receiving pharmacologic thromboprophylaxis, 198 (9.9%) developed VTE. With multivariable logistic regression analysis, the independent predictors of VTE were Acute Physiology and Chronic Health Evaluation II score, prior immobilization, femoral central venous catheter, and invasive mechanical ventilation. All risk assessment models had areas under the curve < 0.60 except for the Caprini model (0.64; 95% CI, 0.60-0.68). The Caprini, Padua, and Intermountain models had high sensitivity (> 85%) but low specificity (< 20%) for predicting VTE, whereas the ICU-VTE, Kucher, and IMPROVE models had low sensitivities (< 15%) but high specificities (> 85%). The positive predictive value was low (< 20%) for all studied cutoff scores, whereas the negative predictive value was mostly > 90%. Using the risk assessment models to stratify patients into high- vs low-risk subgroups, the effect of adjunctive pneumatic compression vs pharmacologic prophylaxis alone did not differ across the subgroups (Pinteraction > .05). INTERPRETATION: The risk assessment models for VTE performed poorly in patients who are critically ill receiving pharmacologic thromboprophylaxis. None of the models identified a subgroup of patients who might benefit from adjunctive pneumatic compression. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov; No.: NCT02040103; URL: www. CLINICALTRIALS: gov. ISRCTN44653506.

TorsinA is essential for neuronal nuclear pore complex localization and maturation.

As lifelong interphase cells, neurons face an array of unique challenges. A key challenge is regulating nuclear pore complex (NPC) biogenesis and localization, the mechanisms of which are largely unknown. Here we identify neuronal maturation as a period of strongly upregulated NPC biogenesis. We demonstrate that the AAA+ protein torsinA, whose dysfunction causes the neurodevelopmental movement disorder DYT-TOR1A dystonia and co-ordinates NPC spatial organization without impacting total NPC density. We generated an endogenous Nup107-HaloTag mouse line to directly visualize NPC organization in developing neurons and find that torsinA is essential for proper NPC localization. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized nascent NPCs, and the formation of complete NPCs is delayed. Our work demonstrates that NPC spatial organization and number are independently determined and identifies NPC biogenesis as a process vulnerable to neurodevelopmental disease insults.

Development of an engineered extracellular vesicles-based vaccine platform for combined delivery of mRNA and protein to induce functional immunity.

mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EVX-M+P vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs, natural nanoparticle carriers shed by all cells, to contain ovalbumin mRNA and protein (EVOvaM+P vaccine) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors. EVOvaM+P administration to mice with established melanoma tumors resulted in tumor regression associated with effective humoral and adaptive immune responses. As a second example, we generated engineered EVs that contain Spike (S) mRNA and protein to serve as a combined mRNA and protein vaccine (EVSpikeM+P vaccine) against SARS-CoV-2 infection. EVSpikeM+P vaccine administration in mice and baboons elicited robust production of neutralizing IgG antibodies against RBD (receptor binding domain) of S protein and S protein specific T cell responses. Our proof-of-concept study describes a new platform with an ability for rapid development of combination mRNA and protein vaccines employing EVs for deployment against cancer and other diseases.

Stress granule formation helps to mitigate neurodegeneration.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

Rates, predictors, and causes of readmission after transcatheter aortic valve replacement in patients with chronic kidney disease.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is a revolutionary procedure for severe aortic stenosis. The coexistence of chronic kidney disease (CKD) and TAVR introduces a challenge that significantly impacts patient outcomes. AIM: To define readmission rates, predictors, and causes after TAVR procedure in CKD stage 1-4 patients. METHODS: We used the national readmission database 2018 and 2020 to look into readmission rates, causes and predictors after TAVR procedure in patients with CKD stage 1-4. RESULTS: Out of 24758 who underwent TAVR and had CKD, 7892 (32.4%) patients were readmitted within 90 days, and had higher adjusted odds of being females (adjusted odds ratio: 1.17, 95%CI: 1.02-1.31, P = 0.02) with longer length of hospital stay > 6 days, and more comorbidities including but not limited to diabetes mellitus, anemia, and congestive heart failure (CHF). CONCLUSION: Most common causes of readmission included CHF (18.0%), sepsis, and complete atrioventricular block. Controlling readmission predictors with very close follow-up is warranted to prevent such high rate of readmission.

TDP43 autoregulation gives rise to shortened isoforms that are tightly controlled by both transcriptional and post-translational mechanisms.

The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.

Plastic and terrestrial organic matter degradation by the humic lake microbiome continues throughout the seasons.

Boreal freshwaters go through four seasons, however, studies about the decomposition of terrestrial and plastic compounds often focus only on summer. We compared microbial decomposition of 13C-polyethylene, 13C-polystyrene, and 13C-plant litter (Typha latifolia) by determining the biochemical fate of the substrate carbon and identified the microbial decomposer taxa in humic lake waters in four seasons. For the first time, the annual decomposition rate including separated seasonal variation was calculated for microplastics and plant litter in the freshwater system. Polyethylene decomposition was not detected, whereas polystyrene and plant litter were degraded in all seasons. In winter, decomposition rates of polystyrene and plant litter were fivefold and fourfold slower than in summer, respectively. Carbon from each substrate was mainly respired in all seasons. Plant litter was utilized efficiently by various microbial groups, whereas polystyrene decomposition was limited to Alpha- and Gammaproteobacteria. The decomposition was not restricted only to the growth season, highlighting that the decomposition of both labile organic matter and extremely recalcitrant microplastics continues throughout the seasons.

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

Counter-regulation of RNA stability by UPF1 and TDP43.

RNA quality control is crucial for proper regulation of gene expression. Disruption of nonsense mediated mRNA decay (NMD), the primary RNA decay pathway responsible for the degradation of transcripts containing premature termination codons (PTCs), can disrupt development and lead to multiple diseases in humans and other animals. Similarly, therapies targeting NMD may have applications in hematological, neoplastic and neurological disorders. As such, tools capable of accurately quantifying NMD status could be invaluable for investigations of disease pathogenesis and biomarker identification. Toward this end, we assemble, validate, and apply a next-generation sequencing approach (NMDq) for identifying and measuring the abundance of PTC-containing transcripts. After validating NMDq performance and confirming its utility for tracking RNA surveillance, we apply it to determine pathway activity in two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) characterized by RNA misprocessing and abnormal RNA stability. Despite the genetic and pathologic evidence implicating dysfunctional RNA metabolism, and NMD in particular, in these conditions, we detected no significant differences in PTC-encoding transcripts in ALS models or disease. Contrary to expectations, overexpression of the master NMD regulator UPF1 had little effect on the clearance of transcripts with PTCs, but rather restored RNA homeostasis through differential use and decay of alternatively poly-adenylated isoforms. Together, these data suggest that canonical NMD is not a significant contributor to ALS/FTD pathogenesis, and that UPF1 promotes neuronal survival by regulating transcripts with abnormally long 3'UTRs.

Preparing for the future offspring: European perch (Perca fluviatilis) biosynthesis of physiologically required fatty acids for the gonads happens already in the autumn.

Long-chain polyunsaturated fatty acids (PUFA) are critical for reproduction and thermal adaptation. Year-round variability in the expression of fads2 (fatty acid desaturase 2) in the liver of European perch (Perca fluviatilis) in a boreal lake was tested in relation to individual variation in size, sex, and maturity, together with stable isotopes values as well as fatty acids (FA) content in different tissues and prey items. ARA and DHA primary production was restricted to the summer months, however, perch required larger amounts of these PUFA during winter, as their ARA and DHA muscle content was higher compared to summer. The expression of fads2 in perch liver increased during winter and was higher in mature females. Mature females stored DHA in their gonads already in late summer and autumn, long before the upcoming spring spawning period in May. Lower δ13CDHA values in the gonads in September suggest that these females actively synthesized DHA as part of this reproductive investment. Lower δ13CARA values in the liver of all individuals during winter suggest that perch were synthesizing essential FA to help cope with over-wintering conditions. Perch seem able to modulate its biosynthesis of physiologically required PUFA in situations of stress (fasting or cold temperatures) or in situations of high energetic demand (gonadal development). Biosynthesis of physiologically required PUFA may be an important part of survival and reproduction in aquatic food webs with long cold periods.

Stress granule formation helps to mitigate neurodegeneration.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP (rin in Drosophila) and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

Outcomes in patients with COVID-19 and new onset heart blocks: Insight from the National Inpatient Sample database.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a worldwide health crisis since it first appeared. Numerous studies demonstrated the virus's predilection to cardiomyocytes; however, the effects that COVID-19 has on the cardiac conduction system still need to be fully understood. AIM: To analyze the impact that COVID-19 has on the odds of major cardiovascular complications in patients with new onset heart blocks or bundle branch blocks (BBB). METHODS: The 2020 National Inpatient Sample (NIS) database was used to identify patients admitted for COVID-19 pneumonia with and without high-degree atrioventricular blocks (HDAVB) and right or left BBB utilizing ICD-10 codes. The patients with pre-existing pacemakers, suggestive of a prior diagnosis of HDAVB or BBB, were excluded from the study. The primary outcome was inpatient mortality. Secondary outcomes included total hospital charges (THC), the length of hospital stay (LOS), and other major cardiac outcomes detailed in the Results section. Univariate and multivariate regression analyses were used to adjust for confounders with Stata version 17. RESULTS: A total of 1058815 COVID-19 hospitalizations were identified within the 2020 NIS database, of which 3210 (0.4%) and 17365 (1.6%) patients were newly diagnosed with HDAVB and BBB, respectively. We observed a significantly higher odds of in-hospital mortality, cardiac arrest, cardiogenic shock, sepsis, arrythmias, and acute kidney injury in the COVID-19 and HDAVB group. There was no statistically significant difference in the odds of cerebral infarction or pulmonary embolism. Encounters with COVID-19 pneumonia and newly diagnosed BBB had a higher odds of arrythmias, acute kidney injury, sepsis, need for mechanical ventilation, and cardiogenic shock than those without BBB. However, unlike HDAVB, COVID-19 pneumonia and BBB had no significant impact on mortality compared to patients without BBB. CONCLUSION: In conclusion, there is a significantly higher odds of inpatient mortality, cardiac arrest, cardiogenic shock, sepsis, acute kidney injury, supraventricular tachycardia, ventricular tachycardia, THC, and LOS in patients with COVID-19 pneumonia and HDAVB as compared to patients without HDAVB. Likewise, patients with COVID-19 pneumonia in the BBB group similarly have a higher odds of supraventricular tachycardia, atrial fibrillation, atrial flutter, ventricular tachycardia, acute kidney injury, sepsis, need for mechanical ventilation, and cardiogenic shock as compared to those without BBB. Therefore, it is essential for healthcare providers to be aware of the possible worse predicted outcomes that patients with new-onset HDAVB or BBB may experience following SARS-CoV-2 infection.

Roadmap for C9ORF72 in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis: Report on the C9ORF72 FTD/ALS Summit.

A summit held March 2023 in Scottsdale, Arizona (USA) focused on the intronic hexanucleotide expansion in the C9ORF72 gene and its relevance in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS; C9ORF72-FTD/ALS). The goal of this summit was to connect basic scientists, clinical researchers, drug developers, and individuals affected by C9ORF72-FTD/ALS to evaluate how collaborative efforts across the FTD-ALS disease spectrum might break down existing disease silos. Presentations and discussions covered recent discoveries in C9ORF72-FTD/ALS disease mechanisms, availability of disease biomarkers and recent advances in therapeutic development, and clinical trial design for prevention and treatment for individuals affected by C9ORF72-FTD/ALS and asymptomatic pathological expansion carriers. The C9ORF72-associated hexanucleotide repeat expansion is an important locus for both ALS and FTD. C9ORF72-FTD/ALS may be characterized by loss of function of the C9ORF72 protein and toxic gain of functions caused by both dipeptide repeat (DPR) proteins and hexanucleotide repeat RNA. C9ORF72-FTD/ALS therapeutic strategies discussed at the summit included the use of antisense oligonucleotides, adeno-associated virus (AAV)-mediated gene silencing and gene delivery, and engineered small molecules targeting RNA structures associated with the C9ORF72 expansion. Neurofilament light chain, DPR proteins, and transactive response (TAR) DNA-binding protein 43 (TDP-43)-associated molecular changes were presented as biomarker candidates. Similarly, brain imaging modalities (i.e., magnetic resonance imaging [MRI] and positron emission tomography [PET]) measuring structural, functional, and metabolic changes were discussed as important tools to monitor individuals affected with C9ORF72-FTD/ALS, at both pre-symptomatic and symptomatic disease stages. Finally, summit attendees evaluated current clinical trial designs available for FTD or ALS patients and concluded that therapeutics relevant to FTD/ALS patients, such as those specifically targeting C9ORF72, may need to be tested with composite endpoints covering clinical symptoms of both FTD and ALS. The latter will require novel clinical trial designs to be inclusive of all patient subgroups spanning the FTD/ALS spectrum.

The C9ORF72 Summit was held in March 2023 in Scottsdale, Arizona (USA). Some people who have the disease frontotemporal dementia or the disease amyotrophic lateral sclerosis have a change in one of their genes; the name of the gene is C9ORF72. People who carry this genetic difference usually inherited it from a parent. Researchers are improving their understanding of how the change in the C9ORF72 gene affects people, and efforts are being made to use this knowledge to develop treatments for amyotrophic lateral sclerosis and frontotemporal dementia. In addition to studying the cellular and molecular mechanisms of how the C9ORF72 mutation leads to cellular dysfunction and frontotemporal dementia and amyotrophic lateral sclerosis clinical symptoms, a large effort of the research community is aimed at developing measurements, called biomarkers, that could enhance therapy development efforts in multiple ways. Examples include monitoring of disease activity, identifying those at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, predicting which people might benefit from a particular treatment, and showing that a drug has had a biological effect. Markers that identify healthy people who are at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia could be used to test treatments that would start before a person shows any symptoms and hopefully would delay or even prevent their onset.

Environmental drivers alter PUFA content in littoral macroinvertebrate assemblages via changes in richness and abundance.

Shallow littoral areas in lakes are productive and highly diverse ecotonal zones, providing habitats for both invertebrate and vertebrate species. We developed a Bayesian modeling framework to elucidate the relationships between environmental drivers (lake typology, habitat, water chemistry, and latitude) and taxon richness, abundance, as well as the content of polyunsaturated fatty acids (PUFAs) in littoral macroinvertebrate communities in 95 boreal lakes. PUFAs, particularly arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are critical micronutrients to maintain normal physiological functions in consumers. Lake typology was a significant predictor for PUFA content in the invertebrate assemblages, which was connected to taxon richness and/or abundance. Benthic communities in large humus-poor or nutrient-rich lakes displayed higher abundance, taxon richness, and more PUFA-rich taxa, whereas those in medium- and large-sized humic (color 30-90 mg Pt/L) and humus-rich lakes (color >90 mg Pt/L) were characterized by decreased abundance and subsequently low PUFA content. The abundance, taxon richness, and nutritional quality of the communities were also strongly related to latitude. Lakes with lower pH were characterized by lower benthic invertebrate diversity and low frequency of taxa with high somatic EPA and DHA content. The complexity of littoral habitats dominated by various macrophyte assemblages creates an environment that favors higher benthic abundance and increased presence of taxonomic groups with high PUFA content. Nutritional quality of benthic invertebrates for upper trophic levels can be modulated by a complex interplay between external stressors and abiotic factors that typically shape the structure of littoral benthic communities. Supplementary Information: The online version contains supplementary material available at 10.1007/s00027-023-00996-2.

Eutrophication effect on production and transfer of omega-3 fatty acids in boreal lake food webs.

Eutrophication, i.e. increasing level of nutrients and primary production, is a central environmental change of lakes globally with wide effects on food webs. However, how eutrophication affects the synthesis of physiologically essential biomolecules (omega-3 fatty acids) and their transfer to higher trophic levels at the whole food web level is not well understood. We assessed food web (phytoplankton, zooplankton, and fish) biomass, community structure and fatty acid content (eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]), together with fatty acid specific primary production in 12 Finnish boreal lakes covering the total nutrient gradient from oligotrophic to highly eutrophic lakes (4-140 µg TP l-1; 413-1814 µg TN l-1). Production was measured as the incorporation of 13C-NaHCO3 into phytoplankton fatty acids and differentiated into volumetric production (production per litre of water) and productivity (production per phytoplankton biomass). Increases in nutrients led to higher biomass of phytoplankton, zooplankton and fish communities while also affecting community composition. Eutrophication negatively influenced the contribution of phytoplankton biomass preferentially grazed by zooplankton (<35 µm). Total volumetric production saturated at high phytoplankton biomass while EPA volumetric production presented a logarithmic relationship with nutrient increase. Meanwhile, total and EPA productivity had unimodal responses to this change in nutrients. DHA volumetric production and productivity presented large variation with increases in total phosphorus, but a unimodal model best described DHA changes with eutrophication. Results showed that eutrophication impaired the transfer of EPA and DHA into zooplankton and fish, showing a clear negative impact in some species (e.g. perch) while having no effect in other species (e.g. roach, ruffe). Results show non-linear trends in fatty acid production and productivity peaking at nutrient concentrations 22-35 µg l-1 TP followed by a gradual decrease.

KRASG12D inhibition reprograms the microenvironment of early and advanced pancreatic cancer to promote FAS-mediated killing by CD8+ T cells.

The KRASG12D mutation is present in nearly half of pancreatic adenocarcinomas (PDAC). We investigated the effects of inhibiting the KRASG12D mutant protein with MRTX1133, a non-covalent small molecule inhibitor of KRASG12D, on early and advanced PDAC and its influence on the tumor microenvironment. Employing 16 different models of KRASG12D-driven PDAC, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector T cells, decreases myeloid infiltration, and reprograms cancer-associated fibroblasts. MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ T cells and immune checkpoint blockade (ICB) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of KRASG12D in advanced PDAC and human patient derived organoids induces FAS expression in cancer cells and facilitates CD8+ T cell-mediated death. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with ICB in clinical trials.

Temperature, phosphorus and species composition will all influence phytoplankton production and content of polyunsaturated fatty acids.

Temperature increases driven by climate change are expected to decrease the availability of polyunsaturated fatty acids in lakes worldwide. Nevertheless, a comprehensive understanding of the joint effects of lake trophic status, nutrient dynamics and warming on the availability of these biomolecules is lacking. Here, we conducted a laboratory experiment to study how warming (18-23°C) interacts with phosphorus (0.65-2.58 µM) to affect phytoplankton growth and their production of polyunsaturated fatty acids. We included 10 species belonging to the groups diatoms, golden algae, cyanobacteria, green algae, cryptophytes and dinoflagellates. Our results show that both temperature and phosphorus will boost phytoplankton growth, especially stimulating certain cyanobacteria species (Microcystis sp.). Temperature and phosphorus had opposing effects on polyunsaturated fatty acid proportion, but responses are largely dependent on species. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) synthesizing species did not clearly support the idea that warming decreases the production or content of these essential polyunsaturated fatty acids. Our results suggest that warming may have different effects on the polyunsaturated fatty acid availability in lakes with different nutrient levels, and that different species within the same phytoplankton group can have contrasting responses to warming. Therefore, we conclude that future production of EPA and DHA is mainly determined by species composition.