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Abstract Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.

Lung cancer surgery with curative intent has significantly developed over recent years, mainly focusing on minimally invasive approaches that do not compromise medical efficiency and ensure a decreased burden on the patient. It is directly linked with an efficient multidisciplinary team that will perform appropriate pre-operative assessment. Caution is required in complex patients with several comorbidities to ensure a meaningful and informed thoracic surgery referral leading to optimal patient outcomes.

Key Clinical Message There is no consensus regarding the therapeutic approach of breast neuroendocrine carcinomas (NECs). As most NECs are hormone receptor positive and HER‐2 negative, we suggest that endocrine‐based strategies may play a leading role. Here, we report a new treatment strategy by incorporating CDK4/6 inhibitors in the therapeutic armamentarium. Abstract Primary neuroendocrine neoplasms of the breast constitute a rare entity. They are characterized by predominant neuroendocrine differentiation and are further divided into well‐differentiated neuroendocrine tumors and poorly differentiated (high‐grade) neuroendocrine carcinomas (NECs). Regarding their therapeutic approach, there are no standardized guidelines. Herein, we present the first case ever reported, concerning a female patient with de novo metastatic breast NEC who received hormonal therapy, a combination of a CDK4/6 inhibitor palbociclib with letrozole and triptorelin, as first‐line treatment with significant clinical and radiological response. As most NECs are estrogen receptor and/or progesterone receptor positive and HER‐2 negative, we suggest that hormonal therapy may play a leading role even in the first‐line setting. The present report provides a new treatment strategy by incorporating CDK4/6 inhibitors in the therapeutic armamentarium of breast NECs.

Recent evidence indicates that exposure to environmental toxins, both short-term and long-term, can increase the risk of developing neurological disorders, including neurodegenerative diseases (i.e., Alzheimer’s disease and other dementias) and acute brain injury (i.e., stroke). For stroke, the latest systematic analysis revealed that exposure to ambient particulate matter is the second most frequent stroke risk after high blood pressure. However, preclinical and clinical stroke investigations on the deleterious consequences of environmental pollutants are scarce. This review examines recent evidence of how environmental toxins, absorbed along the digestive tract or inhaled through the lungs, affect the host cellular response. We particularly address the consequences of environmental toxins on the immune response and the microbiome at the gut and lung barrier sites. Additionally, this review highlights findings showing the potential contribution of environmental toxins to an increased risk of stroke. A better understanding of the biological mechanisms underlying exposure to environmental toxins has the potential to mitigate stroke risk and other neurological disorders.

Abstract The gut contains the largest reservoir of microorganisms of the human body, termed as the gut microbiota which emerges as a key pathophysiological factor in health and disease. The gut microbiota has been demonstrated to influence various brain functions along the “gut-brain axis”. Stroke leads to intestinal dysmotility and leakiness of the intestinal barrier which are associated with change of the gut microbiota composition and its interaction with the human host. Growing evidence over the past decade has demonstrated an important role of these post-stroke changes along the gut-brain axis to contribute to stroke pathology and be potentially druggable targets for future therapies. The impact of the gut microbiota on brain health and repair after stroke might be attributed to the diverse functions of gut bacteria in producing neuroactive compounds, modulating the host’s metabolism and immune status. Therefore, a better understanding on the gut-brain axis after stroke and its integration in a broader concept of stroke pathology could open up new avenues for stroke therapy. Here, we discuss current concepts from preclinical models and human studies on the bi-directional communication along the microbiota-gut-brain axis in stroke.

Summary: Investigations on the microbiota in neurological diseases such as stroke are increasingly common; however, stroke researchers may have limited experience with designing such studies. Here, we describe a protocol to conduct a stroke microbiota study in mice, from experimental stroke surgery and sample collection to data analysis. We provide details on sample processing and sequencing and provide a reproducible data analysis pipeline. In doing so, we hope to enable researchers to conduct robust studies and facilitate identification of stroke-associated microbial signatures.For complete details on the use and execution of this protocol, please refer to Sorbie et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism of how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations – with TH1 cells inducing a type I INF signaling in microglia and regulatory T cells (TREG) cells promoting microglial genes associated with chemotaxis. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. Whereas microglia polarization by T cell subsets did not affect the acute development of the infarct volume, these findings substantiate the role of T cells in stroke by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.

Key Clinical Message: There is no consensus regarding the therapeutic approach of breast neuroendocrine carcinomas (NECs). As most NECs are hormone receptor positive and HER‐2 negative, we suggest that endocrine‐based strategies may play a leading role. Here, we report a new treatment strategy by incorporating CDK4/6 inhibitors in the therapeutic armamentarium. Primary neuroendocrine neoplasms of the breast constitute a rare entity. They are characterized by predominant neuroendocrine differentiation and are further divided into well‐differentiated neuroendocrine tumors and poorly differentiated (high‐grade) neuroendocrine carcinomas (NECs). Regarding their therapeutic approach, there are no standardized guidelines. Herein, we present the first case ever reported, concerning a female patient with de novo metastatic breast NEC who received hormonal therapy, a combination of a CDK4/6 inhibitor palbociclib with letrozole and triptorelin, as first‐line treatment with significant clinical and radiological response. As most NECs are estrogen receptor and/or progesterone receptor positive and HER‐2 negative, we suggest that hormonal therapy may play a leading role even in the first‐line setting. The present report provides a new treatment strategy by incorporating CDK4/6 inhibitors in the therapeutic armamentarium of breast NECs. [ABSTRACT FROM AUTHOR]

Summary: Homeostasis of gut microbiota is crucial in maintaining human health. Alterations, or “dysbiosis,” are increasingly implicated in human diseases, such as cancer, inflammatory bowel diseases, and, more recently, neurological disorders. In ischemic stroke patients, gut microbial profiles are markedly different compared to healthy controls, whereas manipulation of microbiota in animal models of stroke modulates outcome, further implicating microbiota in stroke pathobiology. Despite this, evidence for the involvement of specific microbes or microbial products and microbial signatures have yet to be identified, likely owing to differences in methodology, data analysis, and confounding variables between different studies. Here, we provide a set of guidelines to enable researchers to conduct high-quality, reproducible, and transparent microbiota studies, focusing on 16S rRNA sequencing in the emerging subfield of the stroke-microbiota. In doing so, we aim to facilitate novel and reproducible associations between the microbiota and brain diseases, including stroke, and translation into clinical practice.

The SuperPATH approach is a direct superior portal-assisted approach for total hip arthroplasty (THA) that utilizes the interval between the gluteus minimus and the piriformis to access the hip capsule. Patients and Methods: SuperPATH arthroplasty was performed by a single surgeon between December of 2016 and December of 2021 in 344 cases. The technique described by Chow was performed in all cases. The average length of stay was 1.3 days, and all patients were discharged for home. All patients were mobilized on the day of the operation. Six cases presented complications: four intraoperative femur fractures and two peroneal nerve palsies. No infection and no hip dislocation were noticed in any case. Modifications: We recommend that the patient be placed as far from the surgeon as possible as well as the use of a sterilized standard linen pack to elevate the foot and internally rotate the hip. In addition, regarding instrumentation, we recommend the use of a 4.5 mm drill for the first guidance femur drilling and a standard corkscrew for femoral head removal. Finally, we propose a different reduction technique using a hook. The use of the SuperPATH approach allows for maximal tissue sparing through preservation of external rotators and minimizing stretching of the gluteus medius. There is no range of motion restrictions postoperatively and patients can achieve a high level of function with a very low dislocation risk and reduced inpatient stay. Furthermore, an incision extension is possible if needed in complex cases. For surgeons familiar with the standard posterolateral approach, the SuperPATH approach is a reliable and safe method with promising results for the patient. In order to improve the surgical effect and facilitate some steps in the procedure, we share our experience and recommend some modifications.

Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer’s disease (AD) progression. However, the mechanisms of microbiome–brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aβ deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aβ plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aβ plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aβ plaques upon SCFA supplementation, microglia contained less intracellular Aβ. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aβ deposition likely via modulation of the microglial phenotype.

Stroke is a major health burden as it is a leading cause of morbidity and mortality worldwide. Blood flow restoration, through thrombolysis or endovascular thrombectomy, is the only effective treatment but is restricted to a limited proportion of patients due to time window constraint and accessibility to technology. Over the past two decades, research has investigated the basic mechanisms that lead to neuronal death following cerebral ischemia. However, the use of neuroprotective paradigms in stroke has been marked by failure in translation from experimental research to clinical practice. In the past few years, much attention has focused on the immune response to acute cerebral ischemia as a major factor to the development of brain lesions and neurological deficits. Key inflammatory processes after stroke include the activation of resident glial cells as well as the invasion of circulating leukocytes. Recent research on anti-inflammatory strategies for stroke has focused on limiting the transendothelial migration of peripheral immune cells from the compromised vasculature into the brain parenchyma. However, recent trials testing the blockage of cerebral leukocyte infiltration in patients reported inconsistent results. This emphasizes the need to better scrutinize how immune cells are regulated at the blood–brain interface and enter the brain parenchyma, and particularly to also consider alternative cerebral infiltration routes for leukocytes, including the meninges and the choroid plexus. Understanding how immune cells migrate to the brain via these alternative pathways has the potential to develop more effective approaches for anti-inflammatory stroke therapies.

Microglia are the resident immune cells of the brain and react quickly to changes in their environment with transcriptional regulation and morphological changes. Brain tissue injury such as ischemic stroke induces a local inflammatory response encompassing microglial activation. The change in activation status of a microglia is reflected in its gradual morphological transformation from a highly ramified into a less ramified or amoeboid cell shape. For this reason, the morphological changes of microglia are widely utilized to quantify microglial activation and studying their involvement in virtually all brain diseases. However, the currently available methods, which are mainly based on manual rating of immunofluorescent microscopic images, are often inaccurate, rater biased, and highly time consuming. To address these issues, we created a fully automated image analysis tool, which enables the analysis of microglia morphology from a confocal Z-stack and providing up to 59 morphological features. We developed the algorithm on an exploratory dataset of microglial cells from a stroke mouse model and validated the findings on an independent data set. In both datasets, we could demonstrate the ability of the algorithm to sensitively discriminate between the microglia morphology in the peri-infarct and the contralateral, unaffected cortex. Dimensionality reduction by principal component analysis allowed to generate a highly sensitive compound score for microglial shape analysis. Finally, we tested for concordance of results between the novel automated analysis tool and the conventional manual analysis and found a high degree of correlation. In conclusion, our novel method for the fully automatized analysis of microglia morphology shows excellent accuracy and time efficacy compared to traditional analysis methods. This tool, which we make openly available, could find application to study microglia morphology using fluorescence imaging in a wide range of brain disease models.

Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17-positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal [IL-17.sup.+] γδ T cells on ischemic injury., Ischemic stroke is a highly prevalent disease with limited therapeutic options (1). Inflammation is a key component in the pathophysiology of cerebral ischemia (2), and numerous experimental approaches have explored [...]

The serine protease thrombin plays a role in signalling ischemic neuronal death in the brain. Paradoxically, endogenous neuroprotective mechanisms can be triggered by preconditioning with thrombin (thrombin preconditioning, TPC), leading to tolerance to cerebral ischemia. Here we studied the role of thrombin’s endogenous potent inhibitor, protease nexin-1 (PN-1), in ischemia and in tolerance to cerebral ischemia induced by TPC. Cerebral ischemia was modelled in vitro in organotypic hippocampal slice cultures from rats or genetically engineered mice lacking PN-1 or with the reporter gene lacZ knocked into the PN-1 locus PN-1HAPN-1-lacZ/HAPN-1-lacZ (PN-1 KI) exposed to oxygen and glucose deprivation (OGD). We observed increased thrombin enzyme activity in culture homogenates 24 h after OGD. Lack of PN-1 increased neuronal death in the CA1, suggesting that endogenous PN-1 inhibits thrombin-induced neuronal damage after ischemia. OGD enhanced β-galactosidase activity, reflecting PN-1 expression, at one and 24 h, most strikingly in the stratum radiatum, a glial cell layer adjacent to the CA1 layer of ischemia sensitive neurons. TPC, 24 h before OGD, additionally increased PN-1 expression 1 h after OGD, compared to OGD alone. TPC failed to induce tolerance in cultures from PN-1−/− mice confirming PN-1 as an important TPC target. PN-1 upregulation after TPC was blocked by the c-Jun N-terminal kinase (JNK) inhibitor, L-JNKI1, known to block TPC. This work suggests that PN-1 is an endogenous neuroprotectant in cerebral ischemia and a potential target for neuroprotection.