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Generating mammalian cells with specific mitochondrial DNA (mtDNA)–nuclear DNA (nDNA) combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed ‘MitoPunch’, a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneousl y . MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA–nDNA combinations to enable fundamental studies and potential translational applications. Mitochondria are specialized structures within cells that generate vital energy and biological building blocks. Mitochondria have a double membrane and contain many copies of their own circular DNA (mitochondrial DNA), which include the blueprints to create just thirteen essential mitochondrial proteins. Like all genetic material, mitochondrial DNA can become damaged or mutated, and these changes can be passed on to offspring. Some of these alterations are linked to severe and debilitating diseases. Both the double membrane of the mitochondria and their high number of DNA copies make treating such diseases difficult. A successful therapy must be capable of correcting almost every copy of mitochondrial DNA. However, the multiple copies of mitochondrial DNA create a problem for genetic research as current techniques are unable to reliably introduce particular mitochondrial mutations to all types of human cells to investigate how they may alter cell function. Sercel, Patananan et al. have developed a method to deliver new mitochondria into thousands of cells at the same time. This technique, called MitoPunch, uses a pressure-driven device to propel mitochondria taken from donor cells into recipient cells without mitochondrial DNA to reestablish their function. Using human cancer cells and healthy skin cells that lack mitochondrial DNA, Sercel, Patananan et al. showed that cells that received mitochondria retained the new mitochondrial DNA. The technique uses readily accessible parts, meaning it can be performed quickly and inexpensively in any laboratory. It further only requires a small amount of donor starting material, meaning that even precious samples with limited material could be used as mitochondrial donors. This new technique has several important potential applications for mitochondrial DNA research. It could be used in the lab to create large numbers of cell lineswith known mutations in the mitochondrial DNA to establish new systems that test drugs or probe the interaction between mitochondrial and nuclear DNA. It could be used to study a broad spectrum of biological questions since mitochondrial function is essential for several processes required for life. Critically, it could also be used as a starting point to develop next-generation therapies capable of treating inherited mitochondrial genetic diseases in severely affected patients.

ABSTRACT Generating mammalian cells with specific mtDNA-nDNA combinations is desirable but difficult to achieve and would be enabling for studies of mitochondria-nucleus communication and coordination in controlling cell fates and functions. We developed ‘MitoPunch’, a force-actuated mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, an alternative force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications. Competing Interest Statement M.A.T. and P.-Y.C. are co-founders, board members, shareholders, and consultants for NanoCav, LLC, a private start-up company working on mitochondrial transfer techniques and applications. T.-H.W. was an employee of NanoCav, LLC, and is currently employed by ImmunityBio, Inc. S.R. and K.R.N. are board members of NanoCav, LLC, and employed by ImmunityBio, Inc. The other authors do not have any conflicting interests to declare.

Hippocampal synaptic plasticity is important for learning and memory formation. Homeostatic synaptic plasticity is a specific form of synaptic plasticity that is induced upon prolonged changes in neuronal activity to maintain network homeostasis. While astrocytes are important regulators of synaptic transmission and plasticity, it is largely unclear how they interact with neurons to regulate synaptic plasticity at the circuit level. Here, we show that neuronal activity blockade selectively increases the expression and secretion of IL-33 (interleukin-33) by astrocytes in the hippocampal cornu ammonis 1 (CA1) subregion. This IL-33 stimulates an increase in excitatory synapses and neurotransmission through the activation of neuronal IL-33 receptor complex and synaptic recruitment of the scaffold protein PSD-95. We found that acute administration of tetrodotoxin in hippocampal slices or inhibition of hippocampal CA1 excitatory neurons by optogenetic manipulation increases IL-33 expression in CA1 astrocytes. Furthermore, IL-33 administration in vivo promotes the formation of functional excitatory synapses in hippocampal CA1 neurons, whereas conditional knockout of IL- 33 in CA1 astrocytes decreases the number of excitatory synapses therein. Importantly, blockade of IL-33 and its receptor signaling in vivo by intracerebroventricular administration of its decoy receptor inhibits homeostatic synaptic plasticity in CA1 pyramidal neurons and impairs spatial memory formation in mice. These results collectively reveal an important role of astrocytic IL-33 in mediating the negative-feedback signaling mechanism in homeostatic synaptic plasticity, providing insights into how astrocytes maintain hippocampal network homeostasis.

Alzheimer’s disease (AD) is a devastating condition with no known effective treatment. AD is characterized by memory loss as well as impaired locomotor ability, reasoning, and judgment. Emerging evidence suggests that the innate immune response plays a major role in the pathogenesis of AD. In AD, the accumulation of β-amyloid (Aβ) in the brain perturbs physiological functions of the brain, including synaptic and neuronal dysfunction, microglial activation, and neuronal loss. Serum levels of soluble ST2 (sST2), a decoy receptor for interleukin (IL)-33, increase in patients with mild cognitive impairment, suggesting that impaired IL-33/ST2 signaling may contribute to the pathogenesis of AD. Therefore, we investigated the potential therapeutic role of IL-33 in AD, using transgenic mouse models. Here we report that IL-33 administration reverses synaptic plasticity impairment and memory deficits in APP/PS1 mice. IL-33 administration reduces soluble Aβ levels and amyloid plaque deposition by promoting the recruitment and Aβ phagocytic activity of microglia; this is mediated by ST2/p38 signaling activation. Furthermore, IL-33 injection modulates the innate immune response by polarizing microglia/macrophages toward an antiinflammatory phenotype and reducing the expression of proinflammatory genes, including IL-1β, IL-6, and NLRP3, in the cortices of APP/PS1 mice. Collectively, our results demonstrate a potential therapeutic role for IL-33 in AD.

Background Hometime, the total number of days a person is living in the community (not in a healthcare institution) in a defined period of time after a hospitalization, is a patient-centred outcome metric increasingly used in healthcare research. Hometime exhibits several properties which make its statistical analysis difficult: it has a highly non-normal distribution, excess zeros, and is bounded by both a lower and upper limit. The optimal methodology for the analysis of hometime is currently unknown. Methods Using administrative data we identified adult patients diagnosed with stroke between April 1, 2010 and December 31, 2017 in Ontario, Canada. 90-day hometime and clinically relevant covariates were determined through administrative data linkage. Fifteen different statistical and machine learning models were fit to the data using a derivation sample. The models’ predictive accuracy and bias were assessed using an independent validation sample. Results Seventy-five thousand four hundred seventy-five patients were identified (divided into a derivation set of 49,402 and a test set of 26,073). In general, the machine learning models had lower root mean square error and mean absolute error than the statistical models. However, some statistical models resulted in lower (or equal) bias than the machine learning models. Most of the machine learning models constrained predicted values between the minimum and maximum observable hometime values but this was not the case for the statistical models. The machine learning models also allowed for the display of complex non-linear interactions between covariates and hometime. No model captured the non-normal bucket shaped hometime distribution. Conclusions Overall, no model clearly outperformed the others. However, it was evident that machine learning methods performed better than traditional statistical methods. Among the machine learning methods, generalized boosting machines using the Poisson distribution as well as random forests regression were the best performing. No model was able to capture the bucket shaped hometime distribution and future research on factors which are associated with extreme values of hometime that are not available in administrative data is warranted.

Temporary cardiac pacemakers used in periods of need during surgical recovery involve percutaneous leads and externalized hardware that carry risks of infection, constrain patient mobility and may damage the heart during lead removal. Here we report a leadless, battery-free, fully implantable cardiac pacemaker for postoperative control of cardiac rate and rhythm that undergoes complete dissolution and clearance by natural biological processes after a defined operating timeframe. We show that these devices provide effective pacing of hearts of various sizes in mouse, rat, rabbit, canine and human cardiac models, with tailored geometries and operation timescales, powered by wireless energy transfer. This approach overcomes key disadvantages of traditional temporary pacing devices and may serve as the basis for the next generation of postoperative temporary pacing technology. A biodegradable pacemaker without external leads improves the safety of temporary cardiac pacing.

Dendrites, branched structures extending from neuronal cell soma, are specialized for processing information from other neurons. The morphogenesis of dendritic structures is spatiotemporally regulated by well-orchestrated signaling cascades. Dysregulation of these processes impacts the wiring of neuronal circuit and efficacy of neurotransmission, which contribute to the pathogeneses of neurological disorders. While Cdk5 (cyclin-dependent kinase 5) plays a critical role in neuronal dendritic development, its underlying molecular control is not fully understood. In this study, we show that p39, one of the two neuronal Cdk5 activators, is a key regulator of dendritic morphogenesis. Pyramidal neurons deficient in p39 exhibit aberrant dendritic morphology characterized by shorter length and reduced arborization, which is comparable to dendrites in Cdk5-deficient neurons. RNA sequencing analysis shows that the adaptor protein, WDFY1 (WD repeat and FYVE domain-containing 1), acts downstream of Cdk5/p39 to regulate dendritic morphogenesis. While WDFY1 is elevated in p39-deficient neurons, suppressing its expression rescues the impaired dendritic arborization. Further phosphoproteomic analysis suggests that Cdk5/p39 mediates dendritic morphogenesis by modulating various downstream signaling pathways, including PI3K/Akt-, cAMP-, or small GTPase-mediated signaling transduction pathways, thereby regulating cytoskeletal organization, protein synthesis, and protein trafficking.

Obesity and elevated circulating cholesterol are risk factors for breast cancer recurrence, while the use of statins, cholesterol biosynthesis inhibitors widely used for treating hypercholesterolemia, is associated with improved disease-free survival. Here, we show that cholesterol mediates the metastatic effects of a high-fat diet via its oxysterol metabolite, 27-hydroxycholesterol. Ablation or inhibition of CYP27A1, the enzyme responsible for the rate-limiting step in 27-hydroxycholesterol biosynthesis, significantly reduces metastasis in relevant animal models of cancer. The robust effects of 27-hydroxycholesterol on metastasis requires myeloid immune cell function, and it was found that this oxysterol increases the number of polymorphonuclear-neutrophils and γδ-T cells at distal metastatic sites. The pro-metastatic actions of 27-hydroxycholesterol requires both polymorphonuclear-neutrophils and γδ-T cells, and 27-hydroxycholesterol treatment results in a decreased number of cytotoxic CD8 + T lymphocytes. Therefore, through its actions on γδ-T cells and polymorphonuclear-neutrophils, 27-hydroxycholesterol functions as a biochemical mediator of the metastatic effects of hypercholesterolemia. High cholesterol is a risk factor for breast cancer recurrence. Here the authors show that cholesterol promotes breast cancer metastasis via its metabolite 27-hydroxycholesterol (27HC) that acts on immune myeloid cells residing at the distal metastatic sites, thus promoting an immune suppressive environment.

AbstractObjectiveTo examine risks of severe adverse patient outcomes shortly after a left‐without‐being‐seen emergency department (LWBS ED) visit since 2020. MethodsIn this retrospective study using linked administrative data, we examined temporal trends in monthly rates of ED and LWBS visits for adults in Ontario, Canada, 2014‒2023. In patient‐level analyses restricted to the first eligible LWBS ED visit, we used modified Poisson regression to compare the composite outcome of 7‐day all‐cause mortality or hospitalization following a LWBS ED visit for April 1, 2022‒March 31, 2023 (recent period) to April 1, 2014‒March 31, 2020 (baseline period), adjusted for age, sex, and Charlson comorbidity index. ResultsDespite fewer monthly ED visits since 2020, temporal trends revealed sustained increases in monthly LWBS rates. LWBS ED visits after April 1, 2020 exceeded the baseline period's single‐month LWBS maximum of 4.0% in 15 out of 36 months. The composite outcome of 7‐day all‐cause mortality or hospitalization was 3.4% in the recent period versus 2.9% in the baseline period (adjusted risk ratio [aRR] 1.14, 95% confidence interval [CI] 1.11‒1.18) and remained elevated at 30 days (6.2% vs. 5.8%, respectively; aRR 1.05, 95% CI 1.03‒1.07), despite similar rates of post‐ED outpatient visits (7‐day recent and baseline: 38.9% and 39.7%, respectively, p = 0.38; 30‐day: 59.4% and 59.7%, respectively, p = 0.05). ConclusionsThe rate of short‐term mortality or hospitalization after a LWBS ED visit has recently increased, despite fewer ED visits/month and similar proportion of post‐ED outpatient encounters. This concerning signal should prompt interventions to address system‐ and population‐level causes.

Background Ninety-day hometime, the number of days a patient is living in the community in the first 90 after stroke, exhibits a non-normal bucket-shaped distribution, with lower and upper constraints making its analysis difficult. In this proof-of-concept study we evaluated the performance of random forests regression in the analysis of hometime. Methods Using administrative data we identified stroke hospitalizations between 2010 and 2017 in Ontario, Canada. We used random forests regression to predict 90-day hometime using 15 covariates. Model accuracy was determined using the r-squared statistic. Variable importance in prediction and the marginal effects of each covariate were explored. Results We identified 75,745 eligible patients. Median 90-day hometime was 59 days (Q1: 2, Q3: 83). Random forests predicted hometime with reasonable accuracy (adjusted r-squared 0.3462); no implausible values were predicted but extreme values were predicted with low accuracy. Frailty, stroke severity, and age exhibited inverse non-linear relationships with hometime and patients arriving by ambulance had less hometime than those who did not. Conclusions Random forests may be a useful method for analyzing 90-day hometime and capturing the complex non-linear relationships which exist between predictors and hometime. Future work should compare random forests to other models and focus on improving the accuracy of predictions of extreme values of hometime.