The proteomic landscape of synaptic diversity across brain regions and cell types.

Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered ∼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

Cryo-EM Structure of the TOM Core Complex from Neurospora crassa.

The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the ß-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery.

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.

Efficacy and Safety of Acoramidis in Transthyretin Amyloid Cardiomyopathy.

BACKGROUND: Transthyretin amyloid cardiomyopathy is characterized by the deposition of misfolded monomeric transthyretin (TTR) in the heart. Acoramidis is a high-affinity TTR stabilizer that acts to inhibit dissociation of tetrameric TTR and leads to more than 90% stabilization across the dosing interval as measured ex vivo. METHODS: In this phase 3, double-blind trial, we randomly assigned patients with transthyretin amyloid cardiomyopathy in a 2:1 ratio to receive acoramidis hydrochloride at a dose of 800 mg twice daily or matching placebo for 30 months. Efficacy was assessed in the patients who had an estimated glomerular filtration rate of at least 30 ml per minute per 1.73 m2 of body-surface area. The four-step primary hierarchical analysis included death from any cause, cardiovascular-related hospitalization, the change from baseline in the N-terminal pro-B-type natriuretic peptide (NT-proBNP) level, and the change from baseline in the 6-minute walk distance. We used the Finkelstein-Schoenfeld method to compare all potential pairs of patients within strata to generate a P value. Key secondary outcomes were death from any cause, the 6-minute walk distance, the score on the Kansas City Cardiomyopathy Questionnaire-Overall Summary, and the serum TTR level. RESULTS: A total of 632 patients underwent randomization. The primary analysis favored acoramidis over placebo (P<0.001); the corresponding win ratio was 1.8 (95% confidence interval [CI], 1.4 to 2.2), with 63.7% of pairwise comparisons favoring acoramidis and 35.9% favoring placebo. Together, death from any cause and cardiovascular-related hospitalization contributed more than half the wins and losses to the win ratio (58% of all pairwise comparisons); NT-proBNP pairwise comparisons yielded the highest ratio of wins to losses (23.3% vs. 7.0%). The overall incidence of adverse events was similar in the acoramidis group and the placebo group (98.1% and 97.6%, respectively); serious adverse events were reported in 54.6% and 64.9% of the patients. CONCLUSIONS: In patients with transthyretin amyloid cardiomyopathy, the receipt of acoramidis resulted in a significantly better four-step primary hierarchical outcome containing components of mortality, morbidity, and function than placebo. Adverse events were similar in the two groups. (Funded by BridgeBio Pharma; ATTRibute-CM ClinicalTrials.gov number, NCT03860935.).

Vutrisiran in Patients with Transthyretin Amyloidosis with Cardiomyopathy.

BACKGROUND: Transthyretin amyloidosis with cardiomyopathy (ATTR-CM) is a progressive, fatal disease. Vutrisiran, a subcutaneously administered RNA interference therapeutic agent, inhibits the production of hepatic transthyretin. METHODS: In this double-blind, randomized trial, we assigned patients with ATTR-CM in a 1:1 ratio to receive vutrisiran (25 mg) or placebo every 12 weeks for up to 36 months. The primary end point was a composite of death from any cause and recurrent cardiovascular events. Secondary end points included death from any cause, the change from baseline in the distance covered on the 6-minute walk test, and the change from baseline in the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS) score. The efficacy end points were assessed in the overall population and in the monotherapy population (the patients who were not receiving tafamidis at baseline) and were tested hierarchically. RESULTS: A total of 655 patients underwent randomization; 326 were assigned to receive vutrisiran and 329 to receive placebo. Vutrisiran treatment led to a lower risk of death from any cause and recurrent cardiovascular events than placebo (hazard ratio in the overall population, 0.72; 95% confidence interval [CI], 0.56 to 0.93; P = 0.01; hazard ratio in the monotherapy population, 0.67; 95% CI, 0.49 to 0.93; P = 0.02) and a lower risk of death from any cause through 42 months (hazard ratio, 0.65; 95% CI, 0.46 to 0.90; P = 0.01). A primary end-point event occurred in 163 patients in the vutrisiran group and in 202 in the placebo group. In the overall population, treatment with vutrisiran resulted in less of a decline in the distance covered on the 6-minute walk test than placebo (least-squares mean difference, 26.5 m; 95% CI, 13.4 to 39.6; P<0.001) and less of a decline in the KCCQ-OS score (least-squares mean difference, 5.8 points; 95% CI, 2.4 to 9.2; P<0.001). Similar benefits were observed in the monotherapy population. The incidence of adverse events was similar in the two groups (99% in the vutrisiran group and 98% in the placebo group); serious adverse events occurred in 62% of the patients in the vutrisiran group and in 67% of those in the placebo group. CONCLUSIONS: Among patients with ATTR-CM, treatment with vutrisiran led to a lower risk of death from any cause and cardiovascular events than placebo and preserved functional capacity and quality of life. (Funded by Alnylam Pharmaceuticals; HELIOS-B ClinicalTrials.gov number, NCT04153149.).

4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter.

We introduce MINFLUX localization with interferometric illumination through opposing objective lenses for maximizing the attainable precision in 3D-localization of single inelastic scatterers, such as fluorophores. Our 4Pi optical configuration employs three sequentially tilted counter-propagating beam pairs for illumination, each providing a narrow interference minimum of illumination intensity at the focal point. The localization precision is additionally improved by adding the inelastically scattered or fluorescence photons collected through both objective lenses. Our 4Pi configuration yields the currently highest precision per detected photon among all localization schemes. Tracking gold nanoparticles as non-blinking inelastic scatterers rendered a position uncertainty <0.4 nm3 in volume at a localization frequency of 2.9 kHz. We harnessed the record spatio-temporal precision of our 4Pi MINFLUX approach to examine the diffusion of single fluorophores and fluorescent nanobeads in solutions of sucrose in water, revealing local heterogeneities at the nanoscale. Our results show the applicability of 4Pi MINFLUX to study molecular nano-environments of diffusion and its potential for quantifying rapid movements of molecules in cells and other material composites.

Patisiran Treatment in Patients with Transthyretin Cardiac Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is associated with accumulation of ATTR amyloid deposits in the heart and commonly manifests as progressive cardiomyopathy. Patisiran, an RNA interference therapeutic agent, inhibits the production of hepatic transthyretin. METHODS: In this phase 3, double-blind, randomized trial, we assigned patients with hereditary, also known as variant, or wild-type ATTR cardiac amyloidosis, in a 1:1 ratio, to receive patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks for 12 months. A hierarchical procedure was used to test the primary and three secondary end points. The primary end point was the change from baseline in the distance covered on the 6-minute walk test at 12 months. The first secondary end point was the change from baseline to month 12 in the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS) score (with higher scores indicating better health status). The second secondary end point was a composite of death from any cause, cardiovascular events, and change from baseline in the 6-minute walk test distance over 12 months. The third secondary end point was a composite of death from any cause, hospitalizations for any cause, and urgent heart failure visits over 12 months. RESULTS: A total of 360 patients were randomly assigned to receive patisiran (181 patients) or placebo (179 patients). At month 12, the decline in the 6-minute walk distance was lower in the patisiran group than in the placebo group (Hodges-Lehmann estimate of median difference, 14.69 m; 95% confidence interval [CI], 0.69 to 28.69; P = 0.02); the KCCQ-OS score increased in the patisiran group and declined in the placebo group (least-squares mean difference, 3.7 points; 95% CI, 0.2 to 7.2; P = 0.04). Significant benefits were not observed for the second secondary end point. Infusion-related reactions, arthralgia, and muscle spasms occurred more often among patients in the patisiran group than among those in the placebo group. CONCLUSIONS: In this trial, administration of patisiran over a period of 12 months resulted in preserved functional capacity in patients with ATTR cardiac amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO-B ClinicalTrials.gov number, NCT03997383.).

Complete responses in AL amyloidosis are unequal: the impact of free light chain mass spectrometry in AL amyloidosis.

ABSTRACT: Amyloidogenic serum free light chains (sFLCs) drive disease progression in AL amyloidosis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry-based FLC assay (FLC-MS) has greater sensitivity than conventional sFLC assays allowing for the detection of serological residual disease. We report the utility of FLC-MS in a large series of patients with AL amyloidosis assessing the impact of FLC-MS negativity after treatment on overall survival (OS) and organ response rates. Serum samples were analyzed using FLC-MS at diagnosis and at 6 and 12 months after treatment. The impact of FLC-MS negativity over standard hematologic responses on survival and organ response was assessed. A total of 487 patients were included; 290 (59%) and 349 (71.5%) had cardiac and renal involvement, respectively. There was 100% concordance between the light chain (LC) fibril type and LC isotype identified by FLC-MS. At 6 and 12 months, 81 (16.6%) and 101 (20.7%) were FLC-MS negative. Of those achieving a conventional hematologic complete response (CR) at 6 and 12 months, 45 (27.7%) and 64 (39%) were FLC-MS negative. At 12 months, median OS for CR + FLC-MS negative was not reached vs 108 months in CR + FLC-MS positive (P = .024). At 12 months, 70% of patients with FLC-MS negativity (vs 50% FLC-MS positive) achieved a cardiac response (P = .015). In a multivariate analysis, FLC-MS negativity at 12 months was an independent predictor of better outcomes. FLC-MS can detect persistent monoclonal light chains in a significant proportion of patients in a conventional hematologic CR. FLC-MS assessment promises to be a new standard for response assessment in AL amyloidosis.

ABC-HuMi: the Atlas of Biosynthetic Gene Clusters in the Human Microbiome.

The human microbiome has emerged as a rich source of diverse and bioactive natural products, harboring immense potential for therapeutic applications. To facilitate systematic exploration and analysis of its biosynthetic landscape, we present ABC-HuMi: the Atlas of Biosynthetic Gene Clusters (BGCs) in the Human Microbiome. ABC-HuMi integrates data from major human microbiome sequence databases and provides an expansive repository of BGCs compared to the limited coverage offered by existing resources. Employing state-of-the-art BGC prediction and analysis tools, our database ensures accurate annotation and enhanced prediction capabilities. ABC-HuMi empowers researchers with advanced browsing, filtering, and search functionality, enabling efficient exploration of the resource. At present, ABC-HuMi boasts a catalog of 19 218 representative BGCs derived from the human gut, oral, skin, respiratory and urogenital systems. By capturing the intricate biosynthetic potential across diverse human body sites, our database fosters profound insights into the molecular repertoire encoded within the human microbiome and offers a comprehensive resource for the discovery and characterization of novel bioactive compounds. The database is freely accessible at https://www.ccb.uni-saarland.de/abc_humi/.

Clinical and Prognostic Implications of Right Ventricular Uptake on Bone Scintigraphy in Transthyretin Amyloid Cardiomyopathy.

BACKGROUND: The extent of myocardial bone tracer uptake with technetium pyrophosphate, hydroxymethylene diphosphonate, and 3,3-diphosphono-1,2-propanodicarboxylate in transthyretin amyloid cardiomyopathy (ATTR-CM) might reflect cardiac amyloid burden and be associated with outcome. METHODS: Consecutive patients with ATTR-CM who underwent diagnostic bone tracer scintigraphy with acquisition of whole-body planar and cardiac single-photon emission computed tomography (SPECT) images from the National Amyloidosis Centre and 4 Italian centers were included. Cardiac uptake was defined according to the Perugini classification: 0=absent cardiac uptake; 1=mild uptake less than bone; 2=moderate uptake equal to bone; and 3=high uptake greater than bone. Extent of right ventricular (RV) uptake was defined as focal (basal segment of the RV free wall only) or diffuse (extending beyond basal segment) on the basis of SPECT imaging. The primary outcome was all-cause mortality. RESULTS: Among 1422 patients with ATTR-CM, RV uptake accompanying left ventricular uptake was identified by SPECT imaging in 100% of cases at diagnosis. Median follow-up in the whole cohort was 34 months (interquartile range, 21 to 50 months), and 494 patients died. By Kaplan-Meier analysis, diffuse RV uptake on SPECT imaging (n=936) was associated with higher all-cause mortality compared with focal (n=486) RV uptake (77.9% versus 22.1%; P<0.001), whereas Perugini grade was not associated with survival (P=0.27 in grade 2 versus grade 3). On multivariable analysis, after adjustment for age at diagnosis (hazard ratio [HR], 1.03 [95% CI, 1.02-1.04]; P<0.001), presence of the p.(V142I) TTR variant (HR, 1.42 [95% CI, 1.20-1.81]; P=0.004), National Amyloidosis Centre stage (each category, P<0.001), stroke volume index (HR, 0.99 [95% CI, 0.97-0.99]; P=0.043), E/e' (HR, 1.02 [95% CI, 1.007-1.03]; P=0.004), right atrial area index (HR, 1.05 [95% CI, 1.02-1.08]; P=0.001), and left ventricular global longitudinal strain (HR, 1.06 [95% CI, 1.03-1.09]; P<0.001), diffuse RV uptake on SPECT imaging (HR, 1.60 [95% CI, 1.26-2.04]; P<0.001) remained an independent predictor of all-cause mortality. The prognostic value of diffuse RV uptake was maintained across each National Amyloidosis Centre stage and in both wild-type and hereditary ATTR-CM (P<0.001 and P=0.02, respectively). CONCLUSIONS: Diffuse RV uptake of bone tracer on SPECT imaging is associated with poor outcomes in patients with ATTR-CM and is an independent prognostic marker at diagnosis.

GatekeepR: an R Shiny application for the identification of nodes with high dynamic impact in Boolean networks.

MOTIVATION: Boolean networks can serve as straightforward models for understanding processes such as gene regulation, and employing logical rules. These rules can either be derived from existing literature or by data-driven approaches. However, in the context of large networks, the exhaustive search for intervention targets becomes challenging due to the exponential expansion of a Boolean network's state space and the multitude of potential target candidates, along with their various combinations. Instead, we can employ the logical rules and resultant interaction graph as a means to identify targets of specific interest within larger-scale models. This approach not only facilitates the screening process but also serves as a preliminary filtering step, enabling the focused investigation of candidates that hold promise for more profound dynamic analysis. However, applying this method requires a working knowledge of R, thus restricting the range of potential users. We, therefore, aim to provide an application that makes this method accessible to a broader scientific community. RESULTS: Here, we introduce GatekeepR, a graphical, web-based R Shiny application that enables scientists to screen Boolean network models for possible intervention targets whose perturbation is likely to have a large impact on the system's dynamics. This application does not require a local installation or knowledge of R and provides the suggested targets along with additional network information and visualizations in an intuitive, easy-to-use manner. The Supplementary Material describes the underlying method for identifying these nodes along with an example application in a network modeling pancreatic cancer. AVAILABILITY AND IMPLEMENTATION: https://www.github.com/sysbio-bioinf/GatekeepR https://abel.informatik.uni-ulm.de/shiny/GatekeepR/.

Mitochondrial sodium/calcium exchanger (NCLX) regulates basal and starvation-induced autophagy through calcium signaling.

Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporters and efflux by Na+ /Ca2+ exchangers (NCLX). Here, we describe a novel relationship among NCLX, intracellular Ca2+ , and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells.

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Current developments in antibiotic discovery: Global microbial diversity as a source for evolutionary optimized anti-bacterials: Global microbial diversity as a source for evolutionary optimized anti-bacterials.

In light of the pending antibiotic resistance crisis, we need to go back to nature and search for novel anti-bacterial compounds.

Fighting antibiotic resistance-strategies and (pre)clinical developments to find new antibacterials.

Antibacterial resistance is one of the greatest threats to human health. The development of new therapeutics against bacterial pathogens has slowed drastically since the approvals of the first antibiotics in the early and mid-20th century. Most of the currently investigated drug leads are modifications of approved antibacterials, many of which are derived from natural products. In this review, we highlight the challenges, advancements and current standing of the clinical and preclinical antibacterial research pipeline. Additionally, we present novel strategies for rejuvenating the discovery process and advocate for renewed and enthusiastic investment in the antibacterial discovery pipeline.

Location-specific strategies for eliminating US national racial-ethnic [Formula: see text] exposure inequality.

Air pollution levels in the United States have decreased dramatically over the past decades, yet national racial-ethnic exposure disparities persist. For ambient fine particulate matter ([Formula: see text]), we investigate three emission-reduction approaches and compare their optimal ability to address two goals: 1) reduce the overall population average exposure ("overall average") and 2) reduce the difference in the average exposure for the most exposed racial-ethnic group versus for the overall population ("national inequalities"). We show that national inequalities in exposure can be eliminated with minor emission reductions (optimal: ~1% of total emissions) if they target specific locations. In contrast, achieving that outcome using existing regulatory strategies would require eliminating essentially all emissions (if targeting specific economic sectors) or is not possible (if requiring urban regions to meet concentration standards). Lastly, we do not find a trade-off between the two goals (i.e., reducing overall average and reducing national inequalities); rather, the approach that does the best for reducing national inequalities (i.e., location-specific strategies) also does as well as or better than the other two approaches (i.e., sector-specific and meeting concentration standards) for reducing overall averages. Overall, our findings suggest that incorporating location-specific emissions reductions into the US air quality regulatory framework 1) is crucial for eliminating long-standing national average exposure disparities by race-ethnicity and 2) can benefit overall average exposures as much as or more than the sector-specific and concentration-standards approaches.

The alpha-1A adrenergic receptor regulates mitochondrial oxidative metabolism in the mouse heart.

AIMS: The sympathetic nervous system regulates numerous critical aspects of mitochondrial function in the heart through activation of adrenergic receptors (ARs) on cardiomyocytes. Mounting evidence suggests that α1-ARs, particularly the α1A subtype, are cardioprotective and may mitigate the deleterious effects of chronic ß-AR activation by shared ligands. The mechanisms underlying these adaptive effects remain unclear. Here, we tested the hypothesis that α1A-ARs adaptively regulate cardiomyocyte oxidative metabolism in both the uninjured and infarcted heart. METHODS: We used high resolution respirometry, fatty acid oxidation (FAO) enzyme assays, substrate-specific electron transport chain (ETC) enzyme assays, transmission electron microscopy (TEM) and proteomics to characterize mitochondrial function comprehensively in the uninjured hearts of wild type and α1A-AR knockout mice and defined the effects of chronic ß-AR activation and myocardial infarction on selected mitochondrial functions. RESULTS: We found that isolated cardiac mitochondria from α1A-KO mice had deficits in fatty acid-dependent respiration, FAO, and ETC enzyme activity. TEM revealed abnormalities of mitochondrial morphology characteristic of these functional deficits. The selective α1A-AR agonist A61603 enhanced fatty-acid dependent respiration, fatty acid oxidation, and ETC enzyme activity in isolated cardiac mitochondria. The ß-AR agonist isoproterenol enhanced oxidative stress in vitro and this adverse effect was mitigated by A61603. A61603 enhanced ETC Complex I activity and protected contractile function following myocardial infarction. CONCLUSIONS: Collectively, these novel findings position α1A-ARs as critical regulators of cardiomyocyte metabolism in the basal state and suggest that metabolic mechanisms may underlie the protective effects of α1A-AR activation in the failing heart.

The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide.

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Metabolic dysfunction induced by HFD + L-NAME preferentially affects hippocampal mitochondria, impacting spatial memory in rats.

High-fat diet-induced metabolic changes are not restricted to the onset of cardiovascular diseases, but also include effects on brain functions related to learning and memory. This study aimed to evaluate mitochondrial markers and function, as well as cognitive function, in a rat model of metabolic dysfunction. Eight-week-old male Wistar rats were subjected to either a control diet or a two-hit protocol combining a high fat diet (HFD) with the nitric oxide synthase inhibitor L-NAME in the drinking water. HFD plus L-NAME induced obesity, hypertension, and increased serum cholesterol. These rats exhibited bioenergetic dysfunction in the hippocampus, characterized by decreased oxygen (O2) consumption related to ATP production, with no changes in H2O2 production. Furthermore, OPA1 protein expression was upregulated in the hippocampus of HFD + L-NAME rats, with no alterations in other morphology-related proteins. Consistently, HFD + L-NAME rats showed disruption of performance in the Morris Water Maze Reference Memory test. The neocortex did not exhibit either bioenergetic changes or alterations in H2O2 production. Calcium uptake rate and retention capacity in the neocortex of HFD + L-NAME rats were not altered. Our results indicate that hippocampal mitochondrial bioenergetic function is disturbed in rats exposed to a HFD plus L-NAME, thus disrupting spatial learning, whereas neocortical function remains unaffected.