Cystatin F (Cst7) drives sex-dependent changes in microglia in an amyloid-driven model of Alzheimer's disease.

Microglial endolysosomal (dys)function is strongly implicated in neurodegenerative disease. Transcriptomic studies show that a microglial state characterised by a set of genes involved in endolysosomal function is induced in both mouse Alzheimer's disease (AD) models and human AD brain, and that the emergence of this state is emphasised in females. Cst7 (encoding cystatin F) is among the most highly upregulated genes in these microglia. However, despite such striking and robust upregulation, the function of Cst7 in neurodegenerative disease is not understood. Here, we crossed Cst7-/- mice with the AppNL-G-F mouse to test the role of Cst7 in a model of amyloid-driven AD. Surprisingly, we found that Cst7 plays a sexually dimorphic role regulating microglia in this model. In females, Cst7-/-AppNL-G-F microglia had greater endolysosomal gene expression, lysosomal burden, and amyloid beta (Aß) burden in vivo and were more phagocytic in vitro. However, in males, Cst7-/-AppNL-G-F microglia were less inflammatory and had a reduction in lysosomal burden but had no change in Aß burden. Overall, our study reveals functional roles for one of the most commonly upregulated genes in microglia across disease models, and the sex-specific profiles of Cst7-/--altered microglial disease phenotypes. More broadly, the findings raise important implications for AD including crucial questions on sexual dimorphism in neurodegenerative disease and the interplay between endolysosomal and inflammatory pathways in AD pathology.

Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

Synthetic chromosome fusion: Effects on mitotic and meiotic genome structure and function.

We designed and synthesized synI, which is ∼21.6% shorter than native chrI, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance and is now attached to synIII, yielding the first synthetic yeast fusion chromosome. Additional fusion chromosomes were constructed to study nuclear function. ChrIII-I and chrIX-III-I fusion chromosomes have twisted structures, which depend on silencing protein Sir3. As a smaller chromosome, chrI also faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of the crossover-promoting protein Red1. These effects extend over 100 kb and promote disproportionate Red1 enrichment, and thus crossover potential, on small chromosomes like chrI. These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems.

Context-dependent neocentromere activity in synthetic yeast chromosome VIII.

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae. With the completion of synthetic chromosome VIII (synVIII) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII, we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (∼500 bp) of the CDEIII-proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality.

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains.

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

Treatment with IgM-enriched intravenous immunoglobulins enhances clearance of stroke-associated bacterial lung infection.

Post-stroke infection is a common complication of stroke that is associated with poor outcome. We previously reported that stroke induces an ablation of multiple sub-populations of B cells and reduces levels of immunoglobulin M (IgM) antibody, which coincides with the development of spontaneous bacterial pneumonia. The loss of IgM after stroke could be an important determinant of infection susceptibility and highlights this pathway as a target for intervention. We treated mice with a replacement dose of IgM-enriched intravenous immunoglobulin (IgM-IVIg) prior to and 24 h after middle cerebral artery occlusion (MCAO) and allowed them to recover for 2- or 5-day post-surgery. Treatment with IgM-IVIg enhanced bacterial clearance from the lung after MCAO and improved lung pathology but did not impact brain infarct volume. IgM-IVIg treatment induced immunomodulatory effects systemically, including rescue of splenic plasma B cell numbers and endogenous mouse IgM and IgA circulating immunoglobulin concentrations that were reduced by MCAO. Treatment attenuated MCAO-induced elevation of selected pro-inflammatory cytokines in the lung. IgM-IVIg treatment did not increase the number of lung mononuclear phagocytes or directly modulate macrophage phagocytic capacity but enhanced phagocytosis of Staphylococcus aureus bioparticles in vitro. Low-dose IgM-IVIg contributes to increased clearance of spontaneous lung bacteria after MCAO likely via increasing availability of antibody in the lung to enhance opsonophagocytic activity. Immunomodulatory effects of IgM-IVIg treatment may also contribute to reduced levels of damage in the lung after MCAO. IgM-IVIg shows promise as an antibacterial and immunomodulatory agent to use in the treatment of post-stroke infection.

A conditional counterselectable Piga knockout in mouse embryonic stem cells for advanced genome writing applications.

Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.

Stroke-induced changes to immune function and their relevance to increased risk of severe COVID-19 disease.

As the COVID-19 pandemic moves towards endemic disease, it remains of key importance to identify groups of individuals vulnerable to severe infection and understand the biological factors that mediate this risk. Stroke patients are at increased risk of developing severe COVID-19, likely due to stroke-induced alterations to systemic immune function. Furthermore, immune responses associated with severe COVID-19 in patients without a history of stroke parallel many of the immune alterations induced by stroke, possibly resulting in a compounding effect that contributes to worsened disease severity. In this review, we discuss the changes to systemic immune function that likely contribute to augmented COVID-19 severity in patients with a history of stroke and the effects of COVID-19 on the immune system that may exacerbate these effects.

De novo assembly and delivery to mouse cells of a 101 kb functional human gene.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

Preceding infection and risk of stroke: An old concept revived by the COVID-19 pandemic.

Anecdotal reports and clinical observations have recently emerged suggesting a relationship between COVID-19 disease and stroke, highlighting the possibility that infected individuals may be more susceptible to cerebrovascular events. In this review we draw on emerging studies of the current pandemic and data from earlier, viral epidemics, to describe possible mechanisms by which SARS-CoV-2 may influence the prevalence of stroke, with a focus on the thromboinflammatory pathways, which may be perturbed. Some of these potential mechanisms are not novel but are, in fact, long-standing hypotheses linking stroke with preceding infection that are yet to be confirmed. The current pandemic may present a renewed opportunity to better understand the relationship between infection and stroke and possible underlying mechanisms.

Circulating and tissue specific transcription of angiopoietin-like protein 4 in human Type 2 diabetes.

AIMS: Obesity is associated with adipose tissue (AT) dysfunction marked by cellular hypertrophy, inflammation, hypoxia and fibrosis. Angiopoietin-like protein 4 (ANGPTL4) inhibits lipoprotein lipase which regulates triglyceride storage. Recently, inhibition of ANGPTL4 has been suggested as potential treatment for type 2 diabetes. Here we evaluate ANGPTL4's role in diabetes and examine ANGPTL4 in relation to markers of AT dysfunction and fatty liver disease. MATERIALS & METHODS: We obtained a unique set of paired samples from subjects undergoing weight loss surgery including subcutaneous AT (SCAT), omental AT (OMAT), liver, thigh muscle biopsies and serum including a post-surgical SCAT biopsy after 9 months. RESULTS: SCAT ANGPTL4 expression and circulating protein levels were higher in people with diabetes and correlated with glucose levels and HOMA-IR but not BMI. At post-surgical follow up, SCAT ANGPTL4 declined in subjects with diabetes to levels of those without diabetes. ANGPTL4 expression correlated with HIF1A and inflammation (MCP-1, IL-6). CONCLUSIONS: We found that SCAT ANGPTL4 was closely linked with the expression of ANGPTL4 in the liver and represented a good proxy for liver steatosis. We suggest the elevation of ANGPTL4 levels in diabetes and the association with inflammation and hypoxia is due to a compensatory mechanism to limit further AT dysfunction. A reduction of ANGPTL4 for the treatment of T2DM as previously suggested is thus unlikely to be of further benefit.

Adoption of an Electronic Medical Record Tool for Childhood Obesity by Primary Care Providers.

BACKGROUND: Primary care providers are tasked with the increasingly difficult job of addressing childhood obesity during clinic visits. Electronic medical record (EMR)-enabled decision-support tools may aid providers in this task; however, information is needed regarding whether providers perceive such tools to be useful for addressing nutrition and physical activity lifestyle behaviors. OBJECTIVES: This study aimed to evaluate the usefulness and usability of FitTastic, an EMR-enabled tool to support prevention and management of childhood obesity in primary care. METHODS: In this mixed-method study, we implemented the FitTastic tool in two primary-care clinics, then surveyed and conducted focused interviews with providers. Validated Technology Acceptance Model perceived usefulness and National Aeronautics and Space Administration (NASA) perceived usability survey questions were e-mailed to 60 providers. In-depth provider interviews with family medicine and pediatric physicians (n = 12) were used to further probe adoption of FitTastic. RESULTS: Surveys were completed by 73% of providers (n = 44). The mean score for FitTastic's usefulness was 3.3 (standard deviation [SD] = 0.54, scale 1-5, where 5 is strongly agree) and usability, 4.8 (SD = 0.86, scale 1-7, where 7 is strongly agree). Usefulness and usability scores were associated with intention to use FitTastic (correlation for both, p < 0.05). Data from provider interviews indicated that useful features of FitTastic included: standardizing the approach to childhood obesity, and facilitating conversations about weight management, without increasing cognitive workload. However, use of FitTastic required more time from nurses to input lifestyle data. CONCLUSION: FitTastic is perceived as a useful and usable EMR-based lifestyle behavior tool that standardizes, facilitates, and streamlines healthy lifestyle conversations with families. Perceived usability and usefulness scores correlated with provider intention-to-use the technology. These data suggest that EMR-based child obesity prevention and management tools can be feasible to use in the clinic setting, with potential for scalability. Usefulness can be optimized by limiting amount of time needed by staff to input data.

Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence.

Background: Blockade of the cytokine interleukin-1 (IL-1) with IL-1 receptor antagonist (IL-1Ra) is a candidate treatment for stroke entering phase II/III trials, which acts by inhibiting harmful inflammatory responses.  Infection is a common complication after stroke that significantly worsens outcome and is related to stroke-induced deficits in systemic immune function thought to be mediated by the sympathetic nervous system.  Therefore, immunomodulatory treatments for stroke, such as IL-1Ra, carry a risk of aggravating stroke-associated infection. Our primary objective was to determine if factors associated with antibody-mediated antibacterial defences were further compromised in patients treated with IL-1Ra after stroke. Methods: We assessed plasma concentrations of immunoglobulin isotypes and complement components in stroke patients treated with IL-1Ra or placebo and untreated non-stroke controls using multiplex protein assays. Activation of the sympathetic nervous system (SNS) was determined by measuring noradrenaline, a major SNS mediator. Results:  There were significantly lower plasma concentrations of IgM, IgA, IgG1 and IgG4 in stroke-patients compared to non-stroke controls, however there were no differences between stroke patients treated with placebo or IL-1Ra. Concentrations of complement components associated with the classical pathway  were increased and those associated with the alternative pathways decreased in stroke patients, neither being affected by treatment with IL-1Ra.  Noradrenaline concentrations were increased after stroke in both placebo and IL-1Ra-treated stroke patients compared to non-stroke controls.  Conclusion: These data show treatment with IL-1Ra after stroke does not alter circulating immunoglobulin and complement concentrations and is therefore unlikely to further aggravate stroke-associated infection susceptibility through reduced availability of these key anti-microbial mediators.

Experimental Stroke Differentially Affects Discrete Subpopulations of Splenic Macrophages.

Changes to the immune system after stroke are complex and can result in both pro-inflammatory and immunosuppressive consequences. Following ischemic stroke, brain resident microglia are activated and circulating monocytes are recruited to the injury site. In contrast, there is a systemic deactivation of monocytes/macrophages that may contribute to immunosuppression and the high incidence of bacterial infection experienced by stroke patients. The manipulation of macrophage subsets may be a useful therapeutic strategy to reduce infection and improve outcome in patients after stroke. Recent research has enhanced our understanding of the heterogeneity of macrophages even within the same tissue. The spleen is the largest natural reservoir of immune cells, many of which are mobilized to the site of injury after ischemic stroke and is notable for the diversity of its functionally distinct macrophage subpopulations associated with specific micro-anatomical locations. Here, we describe the effects of experimental stroke in mice on these distinct splenic macrophage subpopulations. Red pulp (RP) and marginal zone macrophages (MZM) specifically showed increases in density and alterations in micro-anatomical location. These changes were not due to increased recruitment from the bone marrow but may be associated with increases in local proliferation. Genes associated with phagocytosis and proteolytic processing were upregulated in the spleen after stroke with increased expression of the lysosome-associated protein lysosomal-associated membrane proteins specifically increased in RP and MZM subsets. In contrast, MHC class II expression was reduced specifically in these populations. Furthermore, genes associated with macrophage ability to communicate with other immune cells, such as co-stimulatory molecules and inflammatory cytokine production, were also downregulated in the spleen after stroke. These findings suggest that selective splenic macrophage functions could be impaired after stroke and the contribution of macrophages to stroke-associated pathology and infectious complications should be considered at a subset-specific level. Therefore, optimal therapeutic manipulation of macrophages to improve stroke outcome is likely to require selective targeting of functionally and spatially distinct subpopulations.

Driving Cessation in Patients Attending a Young-Onset Dementia Clinic: A Retrospective Cohort Study.

BACKGROUND: Although driving by persons with dementia is an important public health concern, little is known about driving cessation in younger people with dementia. We aimed to determine the prevalence and factors affecting driving cessation in individuals with and without dementia aged under 65 years attending a memory clinic in a European setting. METHODS: Subjects were consecutive patients assessed at a specialist memory service at a university teaching hospital between 2000 and 2010. The data collected included demographic, clinical, standardized cognitive assessments as well as information on driving. Dementia diagnosis was made using ICD-10 criteria. RESULTS: Of the 225 people who were or had been drivers, 32/79 (41%) with young-onset dementia (YOD) stopped driving compared to 25/146 (17%) patients who had cognitive impairment due to other causes. Women were more likely to cease driving and voluntarily than men (p < 0.001). Diagnosis of YOD was associated with driving cessation (1.193, 95% CI 0.570-1.815, p ≤ 0.001), and was mediated by impairment in praxis with the highest indirect mediation effect (0.754, 95% CI 0.183-1.401, p = 0.009). CONCLUSIONS: YOD diagnosis, female gender, and impairment in praxis have a higher probability for driving cessation in those under 65 years of age with cognitive impairment.

Sitagliptin and Roux-en-Y gastric bypass modulate insulin secretion via regulation of intra-islet PYY.

AIMS: The gut hormone peptide tyrosine tyrosine (PYY) is critical for maintaining islet integrity and restoring islet function following Roux-en-Y gastric bypass (RYGB). The expression of PYY and its receptors (NPYRs) in islets has been documented but not fully characterized. Modulation of islet PYY by the proteolytic enzyme dipeptidyl peptidase IV (DPP-IV) has not been investigated and the impact of DPP-IV inhibition on islet PYY function remains unexplored. Here we have addressed these gaps and their effects on glucose-stimulated insulin secretion (GSIS). We have also investigated changes in pancreatic PYY in diabetes and following RYGB. METHODS: Immunohistochemistry and gene expression analysis were used to assess PYY, NPYRs and DPP-IV expression in rodent and human islets. DPP-IV activity inhibition was achieved by sitagliptin. Secretion studies were used to test PYY and the effects of sitagliptin on insulin release, and the involvement of GLP-1. Radioimmunoassays were used to measure hormone content in islets. RESULTS: PYY and DPP-IV localized in different cell types in islets while NPYR expression was confined to the beta-cells. Chronic PYY application enhanced GSIS in rodent and diabetic human islets. DPP-IV inhibition by sitagliptin potentiated GSIS; this was mediated by locally-produced PYY, and not GLP-1. Pancreatic PYY was markedly reduced in diabetes. RYGB strongly increased islet PYY content, but did not lead to full restoration of pancreatic GLP-1 levels. CONCLUSION: Local regulation of pancreatic PYY, rather than GLP-1, by DPP-IV inhibition or RYGB can directly modulate the insulin secretory response to glucose, indicating a novel role of pancreatic PYY in diabetes and weight-loss surgery.

Lysyl oxidase and adipose tissue dysfunction.

BACKGROUND/OBJECTIVES: Lysyl oxidase (LOX) is an enzyme crucial for collagen fibre crosslinking and thus for fibrosis development. Fibrosis is characterised by a surplus of collagen fibre accumulation and is amongst others also a feature of obesity-associated dysfunctional adipose tissue (AT) which has been linked with type 2 diabetes. We hypothesised that in type 2 diabetes and obesity LOX expression and activity will be increased as a consequence of worsening AT dysfunction. This study aimed to provide a comprehensive characterisation of LOX in human AT. METHODS: LOX mRNA expression was analysed in omental and abdominal subcutaneous AT obtained during elective surgery from subjects with a wide range of BMI, with and without diabetes. In addition, LOX expression was studied in subcutaneous AT before and 9.5months after bariatric surgery. To study the mechanism of LOX changes, its expression and activity were assessed after either hypoxia, recombinant human leptin or glucose treatment of AT explants. In addition, LOX response to acute inflammation was tested after stimulation by a single injection of lipopolysaccharide versus saline solution (control) in healthy men, in vivo. Quantity of mRNA was measured by RT-qPCR. RESULTS: LOX expression was higher in obesity and correlated with BMI whilst, in vitro, leptin at high concentrations, as a potential feedback mechanism, suppressed its expression. Neither diabetes status, nor hyperglycaemia affected LOX. Hypoxia and lipopolysaccharide-induced acute inflammation increased LOX AT expression, latter was independent of macrophage infiltration. CONCLUSIONS: Whilst LOX may not be affected by obesity-associated complications such as diabetes, our results confirm that LOX is increased by hypoxia and inflammation as underlying mechanism for its upregulation in adipose tissue with obesity.

Corrigendum: Adrenergic-mediated loss of splenic marginal zone B cells contributes to infection susceptibility after stroke.

This corrects the article DOI: 10.1038/ncomms15051.

Systematic review of social media interventions for smoking cessation.

BACKGROUND: Popular social media could extend the reach of smoking cessation efforts. In this systematic review, our objectives were: 1) to determine whether social media interventions for smoking cessation are feasible, acceptable, and potentially effective; 2) to identify approaches for recruiting subjects; and 3) to examine the specific intervention design components and strategies employed to promote user engagement and retention. METHODS: We searched Scopus, Medline, EMBASE, Cochrane Central, PsychINFO, CINAHL, and Web of Science through July 2016 and reference lists of relevant articles. Included studies described social media interventions for smoking cessation and must have reported outcomes related to feasibility, acceptability, usability, or smoking-related outcomes. RESULTS: We identified 7 studies (all were published since 2014) that enrolled 9755 participants (median=136 [range 40 to 9042]). Studies mainly used Facebook (n=4) or Twitter (n=2), and emerged as feasible and acceptable. Five studies reported smoking-related outcomes such as greater abstinence, reduction in relapse, and an increase in quit attempts. Most studies (n=6) recruited participants using online or Facebook advertisements. Tailored content, targeted reminders, and moderated discussions were used to promote participant engagement. Three studies found that active participation through posting comments or liking content may be associated with improved outcomes. Retention ranged from 35% to 84% (median=70%) across the included studies. CONCLUSIONS: Our review highlights the feasibility, acceptability and preliminary effectiveness of social media interventions for smoking cessation. Future research should continue to explore approaches for promoting user engagement and retention, and whether sustained engagement translates to clinically meaningful smoking cessation outcomes.

Adrenergic-mediated loss of splenic marginal zone B cells contributes to infection susceptibility after stroke.

Infection is a major complication of acute stroke and causes increased mortality and morbidity; however, current interventions do not prevent infection and improve clinical outcome in stroke patients. The mechanisms that underlie susceptibility to infection in these patients are unclear. Splenic marginal zone (MZ) B cells are innate-like lymphocytes that provide early defence against bacterial infection. Here we show experimental stroke in mice induces a marked loss of MZ B cells, deficiencies in capturing blood-borne antigen and suppression of circulating IgM. These deficits are accompanied by spontaneous bacterial lung infection. IgM levels are similarly suppressed in stroke patients. ß-adrenergic receptor antagonism after experimental stroke prevents loss of splenic MZ B cells, preserves IgM levels, and reduces bacterial burden. These findings suggest that adrenergic-mediated loss of MZ B cells contributes to the infection-prone state after stroke and identify systemic B-cell disruption as a target for therapeutic manipulation.