Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor.

Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid-onset dementia and death. However, elimination of endogenous PrP halts prion disease progression. In this study, we describe Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM), a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates endogenous DNA methyltransferases, thereby reducing transgene size and cytotoxicity. When delivered to the mouse brain by systemic injection of adeno-associated virus (AAV), Prnp-targeted CHARM ablates PrP expression across the brain. Furthermore, we have temporally limited editor expression by implementing a kinetically tuned self-silencing approach. CHARM potentially represents a broadly applicable strategy to suppress pathogenic proteins, including those implicated in other neurodegenerative diseases.

Idiopathic Intracranial Hypertension.

Idiopathic intracranial hypertension is defined by headaches and a decline in visual acuity due to increased intracranial pressure. Treatment options historically included weight loss, acetazolamide, and/or cerebrospinal fluid diversion surgery. Recent understanding of the contributions of dural venous sinus hypertension and stenosis has led to venous sinus stenting as a treatment option.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

The Accessory Limb Model Regenerative Assay and Its Derivatives.

When the Accessory Limb Model (ALM) regenerative assay was first published by Endo, Bryant, and Gardiner in 2004, it provided a robust system for testing the cellular and molecular contributions during each of the basic steps of regeneration: the formation of the wound epithelium, neural induction of the apical epithelial cap, and the formation of a positional disparity between blastema cells. The basic ALM procedure was developed in the axolotl and involves deviating a limb nerve into a lateral wound and grafting skin from the opposing side of the limb axis into the site of injury. In this chapter, we will review the studies that lead to the conception of the ALM, as well as the studies that have followed the development of this assay. We will additionally describe in detail the standard ALM surgery and how to perform this surgery on different limb positions.

Multidimensional control of therapeutic human cell function with synthetic gene circuits.

Synthetic gene circuits that precisely control human cell function could expand the capabilities of gene- and cell-based therapies. However, platforms for developing circuits in primary human cells that drive robust functional changes in vivo and have compositions suitable for clinical use are lacking. Here, we developed synthetic zinc finger transcription regulators (synZiFTRs), which are compact and based largely on human-derived proteins. As a proof of principle, we engineered gene switches and circuits that allow precise, user-defined control over therapeutically relevant genes in primary T cells using orthogonal, US Food and Drug Administration-approved small-molecule inducers. Our circuits can instruct T cells to sequentially activate multiple cellular programs such as proliferation and antitumor activity to drive synergistic therapeutic responses. This platform should accelerate the development and clinical translation of synthetic gene circuits in diverse human cell types and contexts.

Pannexin 1 drives efficient epithelial repair after tissue injury.

Epithelial tissues such as lung and skin are exposed to the environment and therefore particularly vulnerable to damage during injury or infection. Rapid repair is therefore essential to restore function and organ homeostasis. Dysregulated epithelial tissue repair occurs in several human disease states, yet how individual cell types communicate and interact to coordinate tissue regeneration is incompletely understood. Here, we show that pannexin 1 (Panx1), a cell membrane channel activated by caspases in dying cells, drives efficient epithelial regeneration after tissue injury by regulating injury-induced epithelial proliferation. Lung airway epithelial injury promotes the Panx1-dependent release of factors including ATP, from dying epithelial cells, which regulates macrophage phenotype after injury. This process, in turn, induces a reparative response in tissue macrophages that includes the induction of the soluble mitogen amphiregulin, which promotes injury-induced epithelial proliferation. Analysis of regenerating lung epithelium identified Panx1-dependent induction of Nras and Bcas2, both of which positively promoted epithelial proliferation and tissue regeneration in vivo. We also established that this role of Panx1 in boosting epithelial repair after injury is conserved between mouse lung and zebrafish tailfin. These data identify a Panx1-mediated communication circuit between epithelial cells and macrophages as a key step in promoting epithelial regeneration after injury.

Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo.

Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. We developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, we uncovered multiple qualitative and quantitative features of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo.

Role of SPTSSB-Regulated de Novo Sphingolipid Synthesis in Prostate Cancer Depends on Androgen Receptor Signaling.

Anti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.

Inhibition of Lysosomal Function Mitigates Protective Mitophagy and Augments Ceramide Nanoliposome-Induced Cell Death in Head and Neck Squamous Cell Carcinoma.

Therapies for head and neck squamous cell carcinoma (HNSCC) are, at best, moderately effective, underscoring the need for new therapeutic strategies. Ceramide treatment leads to cell death as a consequence of mitochondrial damage by generating oxidative stress and causing mitochondrial permeability. However, HNSCC cells are able to resist cell death through mitochondria repair via mitophagy. Through the use of the C6-ceramide nanoliposome (CNL) to deliver therapeutic levels of bioactive ceramide, we demonstrate that the effects of CNL are mitigated in drug-resistant HNSCC via an autophagic/mitophagic response. We also demonstrate that inhibitors of lysosomal function, including chloroquine (CQ), significantly augment CNL-induced death in HNSCC cell lines. Mechanistically, the combination of CQ and CNL results in dysfunctional lysosomal processing of damaged mitochondria. We further demonstrate that exogenous addition of methyl pyruvate rescues cells from CNL + CQ-dependent cell death by restoring mitochondrial functionality via the reduction of CNL- and CQ-induced generation of reactive oxygen species and mitochondria permeability. Taken together, inhibition of late-stage protective autophagy/mitophagy augments the efficacy of CNL through preventing mitochondrial repair. Moreover, the combination of inhibitors of lysosomal function with CNL may provide an efficacious treatment modality for HNSCC.

Interpreting an apoptotic corpse as anti-inflammatory involves a chloride sensing pathway.

Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte 'appetite' and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This 'switch' to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK-which function upstream of SLC12A2-had a similar effect on efferocytosis. Collectively, the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse.

Context-dependent compensation among phosphatidylserine-recognition receptors.

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk -/- Bai1 Tg ) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk -/- mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk +/+ and Mertk -/- mice. Prior to the onset of photoreceptor degeneration, Mertk -/- mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.

Haemophagocytic lymphohistiocytosis presenting as HELLP syndrome: a diagnostic and therapeutic challenge.

Haemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal, haematological disorder, which can be clinically challenging to diagnose and manage. We report a case of HLH in a previously healthy 33-year-old primigravida. The patient presented at 22 weeks gestation with dyspnoea, abdominal pain, anaemia, thrombocytopenia and elevated liver enzymes suggestive of HELLP syndrome.HELLP, a syndrome characterised by haemolysis, elevated liver enzymes and low platelets is considered a severe form of pre-eclampsia. Despite delivery of the fetus, her condition deteriorated over 3-4 days with high-grade fever, worsening thrombocytopenia and anaemia requiring transfusion support. A bone marrow biopsy showed haemophagocytosis and a diagnosis of HLH was made. Partial remission was achieved with etoposide-based chemotherapy and complete remission following bone marrow transplantation. Eleven months post-transplant, the disease aggressively recurred, and the patient died within 3 weeks of relapse.

The Relationship Between Optic Nerve Sheath Decompression Failure and Intracranial Pressure in Idiopathic Intracranial Hypertension.

BACKGROUND: To our knowledge, there are no studies of patients with idiopathic intracranial hypertension (IIH) that address the relationship between level of intracranial pressure (ICP) and likelihood of progressive visual loss despite uncomplicated optic nerve sheath decompression (ONSD). This study investigated whether patients with IIH undergoing ONSD had a higher risk of surgical failure if opening pressure (OP) on lumbar puncture was ≥50 cm H2O compared to those with OP <50 cm H2O. METHODS: We conducted a retrospective chart review of consecutive patients with IIH who failed maximal medical therapy and underwent ONSD between January, 1992 and November, 2014, and were followed at least 3 months postoperatively. The main outcome measure was the relationship between OP on lumbar puncture and ONSD failure. We also investigated the relationship of OP with visual acuity, visual fields, age, and gender. RESULTS: During this period, 174 patients met inclusion criteria. Of the 40 patients who had an OP ≥50 cm H2O, 6 (15%) had progressive visual loss after uncomplicated ONSD, vs 6 (4.5%) of 134 patients with an OP <50 cm H2O (P = 0.032, Fisher exact test). Patients with worse visual acuity at presentation also had a higher risk of progressive visual loss after ONSD (P < 0.001, Cochran-Armitage trend test), as did men (P = 0.048, Fisher exact test). CONCLUSIONS: Patients with IIH and an OP ≥50 cm H2O had a 3-fold increased risk of failure of ONSD to prevent progressive visual loss, requiring a shunting procedure when compared to those with OP <50 cm H2O. Visual acuity at presentation and male sex also were associated with progressive visual decline after ONSD. These risk factors merit closer follow-up in the postoperative period when signs of further visual deterioration would indicate an urgent need for neurosurgical shunting.

Astrocytes increase ATP exocytosis mediated calcium signaling in response to microgroove structures.

Following central nervous system (CNS) injury, activated astrocytes form glial scars, which inhibit axonal regeneration, leading to long-term functional deficits. Engineered nanoscale scaffolds guide cell growth and enhance regeneration within models of spinal cord injury. However, the effects of micro-/nanosize scaffolds on astrocyte function are not well characterized. In this study, a high throughput (HTP) microscale platform was developed to study astrocyte cell behavior on micropatterned surfaces containing 1 µm spacing grooves with a depth of 250 or 500 nm. Significant changes in cell and nuclear elongation and alignment on patterned surfaces were observed, compared to on flat surfaces. The cytoskeleton components (particularly actin filaments and focal adhesions) and nucleus-centrosome axis were aligned along the grooved direction as well. More interestingly, astrocytes on micropatterned surfaces showed enhanced mitochondrial activity with lysosomes localized at the lamellipodia of the cells, accompanied by enhanced adenosine triphosphate (ATP) release and calcium activities. These data indicate that the lysosome-mediated ATP exocytosis and calcium signaling may play an important role in astrocytic responses to substrate topology. These new findings have furthered our understanding of the biomechanical regulation of astrocyte cell-substrate interactions, and may benefit the optimization of scaffold design for CNS healing.

Outpatient advance care planning for patients with metastatic cancer: a pilot quality improvement initiative.

BACKGROUND: Despite American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) guidelines recommending that oncologists discuss advance care planning (ACP) with patients with stage IV cancer early in treatment, in standard practice ACP remains a late step of a terminal illness. ACP preserves comfort and dignity at the end of life, ensuring patients receive the care that they desire. METHODS AND MATERIALS: A feasibility study in patients with stage IV cancer was developed to test whether incorporating ACP immediately after a stage IV cancer diagnosis is feasible. Inclusion criteria were consecutive new gastrointestinal and thoracic oncology patients treated by one of two oncologists. The project included creation of new workflow; development of an ACP patient education guidebook; training seminars for oncology staff; and enhancements to the electronic health record (EHR) to improve ACP documentation. RESULTS: The oncologists recorded 33 of 48 (69%) advance directive notes (ADNs) and 22 of 48 (46%) code status orders (CSOs) in the EHR of patients newly diagnosed with stage IV cancer by following ACP protocol during the 6-month trial period. Twenty-one of 33 ADNs were entered within 7 days of first consultation. The median time to ADN placement was 1 day after consultation. Twenty-two of 33 patients with ADNs had CSOs placed, of which 16 were do-not-resuscitate (DNR) and 6 were full code. One year prior to the feasibility study, only 1 of 75 deceased patients of the two oncologists had outpatient ADNs and CSOs. CONCLUSIONS: Outpatient ACP is feasible early in the care of patients with stage IV cancer through systematic improvement in workflow and motivated providers. Education and infrastructure were pivotal to routine development of advance care plans.

Arthroscopic stabilization for voluntary shoulder instability.

BACKGROUND: Voluntary shoulder instability is characterized by a patient's ability to sublux their shoulder using selective muscle contraction and relaxation. High failure rates exist with open shoulder stabilization in this group of patients. The purpose of this study was to report the outcomes for patients with voluntary instability treated arthroscopically. METHODS: All patients with voluntary instability from 2006 to 2008 treated with arthroscopic stabilization were included. All patients had documentation of preoperative and postoperative American Shoulder and Elbow Surgeons (ASES) questionnaire score, visual analogue scale of pain, simple shoulder test, and range of motion. Subjective satisfaction and return to sport was also determined. RESULTS: Ten patients were identified for inclusion in the study. The average age of the 5 male and 5 female patients was 16.2 ± 2.33 years. Average clinical follow-up period was 31 ± 6.5 months. Visual analogue scale scores improved from 5.33 ± 3.50 preoperatively to 1.44 ± 2.0 postoperatively, ASES scores improved from 52.2 ± 18.7 to 85.9 ± 14.9 and simple shoulder test improved from 8.17 ± 3.19 to 11.4 ± 1.01. All of the functional evaluation scores improved postoperatively (P < 0.05). There was no case of postoperative dislocation or subluxation, all reported excellent subjective outcome, and all those who played sports returned to their previous level. CONCLUSIONS: Good and excellent outcomes can be obtained with arthroscopic stabilization for patients with voluntary instability. Improved results from previous reports may be related to improved patient selection, surgical technique, and postoperative rehabilitation. Although long-term follow-up and comparative studies are necessary, arthroscopic stabilization seems to be an acceptable treatment option for patients who fail nonoperative treatment. LEVEL OF EVIDENCE: Level IV, case series, retrospective review.