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Background and purpose: Effective communication of radiologic findings is crucial in the acute evaluation of stroke. Conventional methods (CM) of communication can present challenges leading to delays in reporting, ultimately impacting patient outcomes. The goal of this retrospective study was to assess the utility of a HIPAA-compliant messaging system (MS) for expediting communication of CT findings by radiologists to members of the stroke team. Materials and methods: Multimodal stroke protocol CT examinations performed at a high-volume stroke center between January and December 2019 were identified as part of this IRB-approved retrospective study. Read-back times were determined based on templated documentation within individual radiologic reports. The time interval between identification of findings and notification was calculated for each report, and usage of CM versus MS was documented. In addition, an online voluntary survey was disseminated to staff to assess user satisfaction with the MS. Results and conclusions: In total, 442 CTs were included, and 126 (29%) documented use of the MS. The MS was associated with significant reductions in median read-back times, both for all reported findings (1.5 versus 16 minutes, p

Background: The level of radiation exposure received by operators performing transcatheter aortic valve replacement (TAVR) is not well investigated. The aim of this study is to measure the amount of radiation received by operators performing transfemoral TAVR and to identify various patient and procedural characteristics associated with increased radiation exposure. Methods: Primary (operator 1) and secondary (operator 2) operators' equivalent radiation doses in micro Sieverts (µSv) were calculated prospectively using real-time radiation dosimeters for a total of 140 consecutive transfemoral TAVRs. Corresponding eye and thorax radiation exposures between the operators were compared. Associations between various patient and procedural characteristics and the radiation exposure were tested using the t-test and Wilcoxon Mann-Whitney rank-sum test with Monte Carlo estimation. Multivariable regression analysis was also conducted. Results: Operator 1 had significantly higher cumulative equivalent radiation exposure than operator 2 (86 µSv vs 38 µSv, p-value:

In this study, we attempt to target both the urokinase plasminogen activator and the mitogen-activated protein kinase pathway in acute myeloid leukemia (AML) cell lines and primary AML blasts using PrAgU2/LF, a urokinase-activated anthrax lethal toxin. PrAgU2/LF was cytotoxic to five out of nine AML cell lines. Cytotoxicity of PrAgU2/LF appeared to be nonapoptotic and was associated with MAPK activation and urokinase activity because all the PrAgU2/LF-sensitive cell lines showed both uPAR expression and high levels of MEK1/2 phosphorylation. Inhibition of uPAR or desensitization of cells to MEK1/2 inhibition blocked toxicity of PrAgU2/LF, indicating requirement for both uPAR expression and MAPK activation for activity. PrAgU2/LF was also cytotoxic to primary blasts from AML patients, with blasts from four out of five patients showing a cytotoxic response to PrAgU2/LF. Cytotoxicity of primary AML blasts was also dependent on uPAR expression and phos-MEK1/2 levels. CD34+ bone marrow blasts and peripheral blood mononuclear cells lacked uPAR expression and were resistant to PrAgU2/LF, demonstrating the lack of toxicity to normal hematological cells and, therefore, the tumor selectivity of this approach. Dose escalation in mice revealed that the maximal tolerated dose of PrAgU2/LF is at least 5.7-fold higher than that of the wild-type anthrax lethal toxin, PrAg/LF, further demonstrating the increased safety of this molecule. We have shown, in this study, that PrAgU2/LF is a novel, dual-specific molecule for the selective targeting of AML.

This paper investigates whether leading indicators can help explain the cross-country incidence of the 2008-09 financial crisis. Rather than looking for indicators with specific relevance to the current crisis, the selection of variables is driven by an extensive review of more than eighty papers from the previous literature on early warning indicators. The review suggests that central bank reserves and past movements in the real exchange rate were the two leading indicators that had proven the most useful in explaining crisis incidence across different countries and crises in the past. For the 2008-09 crisis, we use six different variables to measure crisis incidence: drops in GDP and industrial production, currency depreciation, stock market performance, reserve losses, and participation in an IMF program. We find that the level of reserves in 2007 appears as a consistent and statistically significant leading indicator of who got hit by the 2008-09 crisis, in line with the conclusions of the pre-2008 literature. In addition to reserves, recent real appreciation is a statistically significant predictor of devaluation and of a measure of exchange market pressure during the current crisis. So is the exchange rate regime. We define the period of the global financial crisis as running from late 2008 to early 2009, which probably explains why we find stronger results than earlier papers such as Obstfeld, Shambaugh and Taylor (2009, 2010) and Rose and Spiegel (2009a,b) which use annual data.

Background: Robotic-assisted, endoscopic transforaminal lumbar interbody fusion (RE-TLIF) is a promising, minimally invasive surgical option for degenerative lumbar spondylosis/spondylolisthesis; however, outcomes data and efficacy are limited, especially in multilevel disease. Here, we present the first reported series of patients that underwent either single or multilevel RE-TLIF. Methods: A retrospective review was performed on 23 consecutive patients who underwent a single level or multilevel RE-TLIF by a single surgeon. Variables included demographics, perioperative results, pain scores, and functional outcome scores. Results: Eighteen patients (78.3 %) underwent single level RE-TLIF and 5 patients (21.7 %) underwent multilevel RE-TLIF. The median reduction of visual analog scale (VAS) for low back pain (LBP) of all subjects was 6 (IQR = 4.5, 6.5) with no significant difference between single level and multilevel RE-TLIF (p = 0.565). The median reduction of VAS for leg pain of all subjects 7 (IQR = 6, 8) with no significant difference between single level and multilevel RE-TLIF (p = 0.702). Median blood loss was 25 cc (IQR = 25, 25) and 50 cc (IQR = 25, 100) for single and multilevel RE-TLIF, respectively (p = 0.025), whereas median length of stay was 1 (IQR = 1, 1; mean = 1.0 ± 00.18) days and 1 (IQR = 1, 2; mean = 1.4 ± 00.54) days, respectively (p = 0.042). One major complication was observed requiring reoperation for demineralized bone matrix migration resulting in an L5 radiculopathy. Conclusions: Single and multi-level RE-TLIF appears to be a safe and efficacious approach with comparable outcomes to open and other minimally invasive approaches. Additionally, we observed favorable accuracy in robot-assisted pedicle screw, endoscope, and interbody device placement.

Summary: Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis (5-year survival rate of 30.5% in the United States). Designing cell therapies to target AML is challenging because no single tumor-associated antigen (TAA) is highly expressed on all cancer subpopulations. Furthermore, TAAs are also expressed on healthy cells, leading to toxicity risk. To address these targeting challenges, we engineer natural killer (NK) cells with a multi-input gene circuit consisting of chimeric antigen receptors (CARs) controlled by OR and NOT logic gates. The OR gate kills a range of AML cells from leukemic stem cells to blasts using a bivalent CAR targeting FLT3 and/or CD33. The NOT gate protects healthy hematopoietic stem cells (HSCs) using an inhibitory CAR targeting endomucin, a protective antigen unique to healthy HSCs. NK cells with the combined OR-NOT gene circuit kill multiple AML subtypes and protect primary HSCs, and the circuit also works in vivo.

For all clinical purposes, cornual, angular, and interstitial pregnancies are considered ectopic pregnancies that can have grave consequences for the patient. In this article, we describe and distinguish 3 types of ectopic pregnancies in the cornual region of the uterus. The authors advocate using the “cornual pregnancy” term only for ectopic pregnancies in malformed uteruses. We describe an ectopic pregnancy in a 25-year-old G2P1 patient in the cornual region of the uterus that was missed twice sonographically in the second trimester and had almost fatal consequences in the patient. Radiologists and sonographers should be aware of the sonographic diagnosis of angular, cornual and interstitial pregnancies. Whenever possible, first-trimester transvaginal ultrasound scanning is crucial for diagnosing these 3 types of ectopic pregnancies in the cornual region. In the second and third trimesters, ultrasound tends to become equivocal; hence alternate imaging, such as MRI, might add additional value to the management of the patient. A case report assessment and a comprehensive literature review comprising 61 case reports of ectopic pregnancy in the second and third trimesters are diligently undertaken in the Medline, Embase and Web of Science databases. The major strength of our study is that it is one of the few studies that describe a literature review of ectopic pregnancy in the cornual region exclusively in the second and third trimesters.

Background: Acute exacerbations of interstitial lung disease (AE-ILD) cause severe respiratory failure, and mortality is high despite treatment. Lung transplantation is an effective therapy for late-stage interstitial lung disease (ILD), but prior studies on post-transplant outcomes for patients trandsplanted in AE-ILD are conflicting. Methods: We performed a retrospective evaluation of all first-time lung transplant recipients for ILD performed at our institution between May 1, 2005, and April 1, 2019. Patients were stratified according to a published consensus definition into AE-ILD recipients, other inpatients, or outpatients. One-year survival was compared with a Cox proportional hazards model. Subset analysis was performed on those with idiopathic pulmonary fibrosis (IPF). Patients were also assessed for survival free of long-term chronic lung allograft dysfunction (CLAD). Results: We identified 717 first-time lung transplant ILD recipients: 41 inpatients in AE-ILD, 31 other inpatients, and 645 outpatients. One-year survival was 93% for AE-ILD recipients, 61% for other inpatient recipients, and 82% for outpatient recipients. Those transplanted in AE-ILD had a lower hazard of death or retransplantation compared to other inpatients (hazard ratio [HR] 0.16, 95% confidence interval [CI] 0.04-0.56) and outpatients (HR 0.29, CI 0.09-1.00). Results were similar among the subset of patients with IPF, but differences were not significant. For those transplanted during AE-ILD, rates of CLAD were not significantly different compared to other inpatients (HR 1.34, CI 0.51-3.54) or to outpatients (HR 1.05, CI 0.52-2.13). Conclusions: With careful selection, patients in AE-ILD can be transplanted and have acceptable 1-year outcomes without increased risk of long-term graft dysfunction.

Summary: Oncogene-induced senescence (OIS) is a persistent anti-proliferative response that acts as a barrier against malignant transformation. During OIS, cells undergo dynamic remodeling, which involves alterations in protein and organelle homeostasis through autophagy. Here, we show that ribosomes are selectively targeted for degradation by autophagy during OIS. By characterizing senescence-dependent alterations in the ribosomal interactome, we find that the deubiquitinase USP10 dissociates from the ribosome during the transition to OIS. This release of USP10 leads to an enhanced ribosome ubiquitination, particularly of small subunit proteins, including lysine 275 on RPS2. Both reinforcement of the USP10-ribosome interaction and mutation of RPS2 K275 abrogate ribosomal delivery to lysosomes without affecting bulk autophagy. We show that the selective recruitment of ubiquitinated ribosomes to autophagosomes is mediated by the p62 receptor. While ribophagy is not required for the establishment of senescence per se, it contributes to senescence-related metabolome alterations and facilitates the senescence-associated secretory phenotype.

Objectives: Safety-net hospitals (SNHs) provide essential services to predominantly underserved patients regardless of their ability to pay. We hypothesized that patients who underwent coronary artery bypass grafting (CABG) would have inferior observed outcomes at SNHs compared with non-SNHs but that matched cohorts would have comparable outcomes. Methods: We queried the Nationwide Readmissions Database for patients who underwent isolated CABG from 2016 to 2018. We ranked hospitals by the percentage of all admissions in which the patient was uninsured or insured with Medicaid; hospitals in the top quartile were designated as SNHs. We used propensity-score matching to mitigate the effect of confounding factors and compare outcomes between SNHs and non-SNHs. Results: A total of 525,179 patients underwent CABG, including 96,133 (18.3%) at SNHs, who had a greater burden of baseline comorbidities (median Elixhauser score 8 vs 7; P = .04) and more frequently required urgent surgery (57.1% vs 52.8%; P

Summary: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high metastasis and therapeutic resistance. Activating transcription factor 4 (ATF4), a master regulator of cellular stress, is exploited by cancer cells to survive. Prior research and data reported provide evidence that high ATF4 expression correlates with worse overall survival in PDAC. Tomatidine, a natural steroidal alkaloid, is associated with inhibition of ATF4 signaling in multiple diseases. Here, we discovered that in vitro and in vivo tomatidine treatment of PDAC cells inhibits tumor growth. Tomatidine inhibited nuclear translocation of ATF4 and reduced the transcriptional binding of ATF4 with downstream promoters. Tomatidine enhanced gemcitabine chemosensitivity in 3D ECM-hydrogels and in vivo. Tomatidine treatment was associated with induction of ferroptosis signaling validated by increased lipid peroxidation, mitochondrial biogenesis, and decreased GPX4 expression in PDAC cells. This study highlights a possible therapeutic approach utilizing a plant-derived metabolite, tomatidine, to target ATF4 activity in PDAC.

Background: Prone transpsoas (PTP) is a novel approach for performing lateral lumbar interbody fusion in a single position. This approach may be beneficial for difficult clinical scenarios. In particular, the PTP position may facilitate lateral corpectomy for certain lumbar pathologies where a circumferential approach is necessary. Case presentation: A 23-year-old woman presented to the ED with wound breakdown four months after a T10-L2 posterior spinal fusion (PSF) was performed following a motor vehicle accident. Imaging studies revealed distal hardware failure and compromise of her distal vertebral body. A lateral corpectomy and interbody fusion was performed. Discussion: We report a case of lateral lumbar corpectomy via a novel PTP approach for a patient with a significant amount of kyphosis, wound breakdown, and hardware failure. We include a review of the current literature on prone transpsoas lateral lumbar interbody fusion. Initial reports do not suggest inferiority compared to other approaches. However, given its novelty, further investigation is needed.

Background & Aims: Oncogenic Kirsten Rat Sarcoma virus (KRAS) is the hallmark mutation of human pancreatic cancer and a driver of tumorigenesis in genetically engineered mouse models of the disease. Although the tumor cell–intrinsic effects of oncogenic Kras expression have been widely studied, its role in regulating the extensive pancreatic tumor microenvironment is less understood. Methods: Using a genetically engineered mouse model of inducible and reversible oncogenic Kras expression and a combination of approaches that include mass cytometry and single-cell RNA sequencing we studied the effect of oncogenic KRAS in the tumor microenvironment. Results: We have discovered that non–cell autonomous (ie, extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, and the main source of cytokines mediating the polarization of protumorigenic macrophages while also preventing tissue repair. Conclusions: Our study provides fundamental knowledge on the mechanisms underlying the formation of the fibroinflammatory stroma in pancreatic cancer and highlights stromal pathways with the potential to be exploited therapeutically.

Summary: Background: The scale of COVID-19 and its well documented long-term sequelae support a need to understand long-term outcomes including frailty. Methods: This prospective cohort study recruited adults who had survived hospitalisation with clinically diagnosed COVID-19 across 35 sites in the UK (PHOSP-COVID). The burden of frailty was objectively measured using Fried's Frailty Phenotype (FFP). The primary outcome was the prevalence of each FFP group—robust (no FFP criteria), pre-frail (one or two FFP criteria) and frail (three or more FFP criteria)—at 5 months and 1 year after discharge from hospital. For inclusion in the primary analysis, participants required complete outcome data for three of the five FFP criteria. Longitudinal changes across frailty domains are reported at 5 months and 1 year post-hospitalisation, along with risk factors for frailty status. Patient-perceived recovery and health-related quality of life (HRQoL) were retrospectively rated for pre-COVID-19 and prospectively rated at the 5 month and 1 year visits. This study is registered with ISRCTN, number ISRCTN10980107. Findings: Between March 5, 2020, and March 31, 2021, 2419 participants were enrolled with FFP data. Mean age was 57.9 (SD 12.6) years, 933 (38.6%) were female, and 429 (17.7%) had received invasive mechanical ventilation. 1785 had measures at both timepoints, of which 240 (13.4%), 1138 (63.8%) and 407 (22.8%) were frail, pre-frail and robust, respectively, at 5 months compared with 123 (6.9%), 1046 (58.6%) and 616 (34.5%) at 1 year. Factors associated with pre-frailty or frailty were invasive mechanical ventilation, older age, female sex, and greater social deprivation. Frail participants had a larger reduction in HRQoL compared with before their COVID-19 illness and were less likely to describe themselves as recovered. Interpretation: Physical frailty and pre-frailty are common following hospitalisation with COVID-19. Improvement in frailty was seen between 5 and 12 months although two-thirds of the population remained pre-frail or frail. This suggests comprehensive assessment and interventions targeting pre-frailty and frailty beyond the initial illness are required. Funding: UK Research and Innovation and National Institute for Health Research.

Summary: Absence seizures, manifested by spike-wave discharges (SWD) in the electroencephalogram, display synchronous reciprocal excitation between the neocortex and thalamus. Recent studies have revealed that inhibitory neurons in the reticular thalamic (RT) nucleus and excitatory thalamocortical (TC) neurons are two subcortical players in generating SWD. However, the signals that drive SWD-related activity remain elusive. Here, we show that SWD predominately occurs during wakefulness in several mouse models of absence epilepsy. In more focused studies of Gnb1 mutant mice, we found that sensory input regulates SWD. Using in vivo recording, we demonstrate that TC cells are activated prior to the onset of SWD and then inhibited during SWD. On the contrary, RT cells are slightly inhibited prior to SWD, but are strongly activated during SWD. Furthermore, chemogenetic activation of TC cells leads to the enhancement of SWD. Together, our results indicate that sensory input can regulate SWD by activating the thalamocortical pathway.

Summary: Cell-mediated immunity may contribute to providing protection against SARS-CoV-2 and its variants of concern (VOC). We developed COH04S1, a synthetic multiantigen modified vaccinia Ankara (MVA)-based COVID-19 vaccine that stimulated potent spike (S) and nucleocapsid (N) antigen-specific humoral and cellular immunity in a phase 1 clinical trial in healthy adults. Here, we show that individuals vaccinated with COH04S1 or mRNA vaccine BNT162b2 maintain robust cross-reactive cellular immunity for six or more months post-vaccination. Although neutralizing antibodies induced in COH04S1- and BNT162b2-vaccinees showed reduced activity against Delta and Omicron variants compared to ancestral SARS-CoV-2, S-specific T cells elicited in both COH04S1- and BNT162b2-vaccinees and N-specific T cells elicited in COH04S1-vaccinees demonstrated potent and equivalent cross-reactivity against ancestral SARS-CoV-2 and the major VOC. These results suggest that vaccine-induced T cells to S and N antigens may constitute a critical second line of defense to provide long-term protection against SARS-CoV-2 VOC.

Objective: Recording seizures using personal seizure diaries can be challenging during everyday life and many seizures are missed or mis-reported. People living with epilepsy could benefit by having a more accurate and objective wearable EEG system for counting seizures that can be used outside of the hospital. The objective of this study was to (1) determine which seizure types can be electrographically recorded from the scalp below the hairline, (2) determine epileptologists’ ability to identify electrographic seizures from single-channels extracted from full-montage wired-EEG, and (3) determine epileptologists’ ability to identify electrographic seizures from Epilog, a wireless single-channel EEG sensor. Methods: Epilog sensors were worn concurrently during epilepsy monitoring unit (EMU) monitoring. During standard-of-care review, epileptologists were asked if the electrographic portion of the seizure was visible on single channels of wired electrodes at locations proximal to Epilog sensors, and if focal-onset, which electrode was closest to the focus. From these locations, single channels of EEG extracted from wired full-montage EEG and the proximal Epilog sensor were presented to 3 blinded epileptologists along with markers for when known seizures occurred (taken from the standard-of-care review). Control segments at inter-ictal times were included as control. The epileptologists were asked whether a seizure event was visible in the single channel EEG record at or near the marker. Results: A total of 75 seizures were recorded from 22 of 40 adults that wore Epilog during their visit to the EMU. Epileptologists were able to visualize known seizure activity on at least one of the wired electrodes proximal to Epilog sensors for all seizure events. Epileptologists accurately identified seizures in 71% of Epilog recordings and 84% of single-channel wired recordings and were 92% accurate identifying seizures with Epilog when those seizures ended in a clinical convulsion compared to those that did not (>55%). Conclusions: Epileptologists are able to visualize seizure activity on single-channels of EEG at locations where Epilog sensors are easily placed on the scalp below hairline. Manual review of seizure annotations can be done quickly and accurately (>70% TP and >98% PPV) on single-channel EEG data. Reviewing single-channel EEG is more accurate than what has been reported in the literature on self-reporting seizures in seizure diaries, the current standard of care for seizure counting outside of the EMU. Significance: Wearable EEG will be important for seizure monitoring outside of the hospital. Epileptologists can accurately identify seizures in single-channel EEG, better than patient self-reporting in diaries based on the literature. Automated or semi-automated seizure detection on single channels of EEG could be used in the future to objectively count seizures to complement the standard of care outside of the EMU without the overt burden upon epileptologist review.

Summary: Background: COH04S1, a synthetic attenuated modified vaccinia virus Ankara vector co-expressing SARS-CoV-2 spike and nucleocapsid antigens, was tested for safety and immunogenicity in healthy adults. Methods: This combined open-label and randomised, phase 1 trial was done at the City of Hope Comprehensive Cancer Center (Duarte, CA, USA). We included participants aged 18–54 years with a negative SARS-CoV-2 antibody and PCR test, normal haematology and chemistry panels, a normal electrocardiogram and troponin concentration, negative pregnancy test if female, body-mass index of 30 kg/m2 or less, and no modified vaccinia virus Ankara or poxvirus vaccine in the past 12 months. In the open-label cohort, 1·0 × 107 plaque-forming units (PFU; low dose), 1·0 × 108 PFU (medium dose), and 2·5 × 108 PFU (high dose) of COH04S1 were administered by intramuscular injection on day 0 and 28 to sentinel participants using a queue-based statistical design to limit risk. In a randomised dose expansion cohort, additional participants were randomly assigned (3:3:1), using block size of seven, to receive two placebo vaccines (placebo group), one low-dose COH04S1 and one placebo vaccine (low-dose COH04S1 plus placebo group), or two low-dose COH04S1 vaccines (low-dose COH04S1 group). The primary outcome was safety and tolerability, with secondary objectives assessing vaccine-specific immunogenicity. The primary immunological outcome was a four times increase (seroconversion) from baseline in spike-specific or nucleocapsid-specific IgG titres within 28 days of the last injection, and seroconversion rates were compared with participants who received placebo using Fisher's exact test. Additional secondary outcomes included assessment of viral neutralisation and cellular responses. This trial is registered with ClinicalTrials.gov, NCT046339466. Findings: Between Dec 13, 2020, and May 24, 2021, 56 participants initiated vaccination. On day 0 and 28, 17 participants received low-dose COH04S1, eight received medium-dose COH04S1, nine received high-dose COH04S1, five received placebo, 13 received low-dose COH04S1 followed by placebo, and four discontinued early. Grade 3 fever was observed in one participant who received low-dose COH04S1 and placebo, and grade 2 anxiety or fatigue was seen in one participant who received medium-dose COH04S1. No severe adverse events were reported. Seroconversion was observed in all 34 participants for spike protein and 32 (94%) for nucleocapsid protein (p

Background: Innominate vein stenosis and venous tortuosity are common findings during cardiac implantable electronic device upgrades or replacements and present a challenge to the implanting physician. Various techniques have been described to facilitate lead placement, including serial dilation, balloon venoplasty, and percutaneous access medial to the stenosis, each with its own benefits and risks. Objective: The purpose of this study was to assess the feasibility, safety, and efficacy of the wire countertraction (“body flossing”) technique to facilitate sheath placement through tortuous and stenotic vessels. Methods: Patients undergoing cardiac implantable electronic device procedures requiring the body flossing technique due to inability to place vascular sheaths over the wire through stenoses or tortuosity were retrospectively analyzed. Clinical characteristics, procedural equipment, and outcomes were analyzed. Results: Simultaneous countertraction was successful in all attempted cases, including 8 patients with stenoses and 2 with tortuosity. In 2 of the stenosis cases, venoplasty had previously failed. No complications occurred. Conclusion: Simultaneous countertraction (body flossing) is an effective tool to overcome venous stenosis and tortuosity that are amenable to wire advancement but not to vascular sheaths. It seems to be a safe and effective alternative to other techniques used in these scenarios.

Right ventricular infarction can precipitate severe right-to-left shunting and refractory hypoxia from a previously dormant patent foramen ovale. Right ventricle mechanical circulatory support and patent foramen ovale closure can play a crucial role in the treatment of hypoxia and right ventricular recovery. We report a case of successful percutaneous patent foramen ovale closure on right ventricle mechanical circulatory support in a patient with right ventricular shock. (Level of Difficulty: Intermediate.)

Summary: Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder, in which an abnormal and toxic protein called progerin, accumulates in cell nuclei, leading to major cellular defects. Among them, chromatin remodeling drives gene expression changes, including miRNA dysregulation. In our study, we evaluated miRNA expression profiles in HGPS and control fibroblasts. We identified an enrichment of overexpressed miRNAs belonging to the 14q32.2-14q32.3 miRNA cluster. Using 3D FISH, we demonstrated that overexpression of these miRNAs is associated with chromatin remodeling at this specific locus in HGPS fibroblasts. We then focused on miR-376b-3p and miR-376a-3p, both overexpressed in HGPS fibroblasts. We demonstrated that their induced overexpression in control fibroblasts decreases cell proliferation and increases senescence, whereas their inhibition in HGPS fibroblasts rescues proliferation defects and senescence and decreases progerin accumulation. By targeting these major processes linked to premature aging, these two miRNAs may play a pivotal role in the pathophysiology of HGPS.

Inverted urothelial papilloma (IUP) is an unusual tumor that typically presents as a non-invasive, endophytic urothelial neoplasm. Though fairly well described in the bladder, IUP of the upper urinary tract is fairly rare, with only 68 cases documented in English language literature. Our patient presented with an IUP of the distal left ureter and was treated with a distal ureterectomy and reimplant of the ureter with a psoas hitch.

Ureteral fibroepithelial polyps (UFP) are relatively rare, benign tumors. A total of 236 total cases were documented between 1932 and 2013. Notably, imaging studies, including computerized tomography (CT) and magnetic resonance imaging (MRI) are often negative. This report details a case of a patient with a UFP who presented with hematuria. CT suggested a possible 1.8 cm mass, but subsequent MRI was negative. Cystoscopy showed a polyp that prolapsed out of the left ureter and into the bladder with peristalsis. The patient subsequently underwent retrograde ureteroscopy and holmium laser excision of the polyp.

Metastasis of malignant melanoma to the bladder is rarely seen clinically with only 29 cases reported in English-language literature. However, autopsy series of melanoma patients have shown that metastasis to the bladder could be as high as 37%, implying that numerous cases remain undiagnosed. This report details a case of malignant melanoma metastasis to the bladder. Patient presented with severe anemia and history of gross hematuria. Computerized tomography (CT) revealed a 2.7 cm mass in the bladder. Cystoscopy confirmed presence of a mass. Patient underwent cystoscopy and transurethral resection of the tumor, revealed to be melanoma on pathology.

Purpose: Single agent PD-1 inhibitors have yielded durable responses in a minority of gastroesophageal cancers. Radiation therapy has been recognized to promote antitumor immune responses and may synergize with anti-PD-1 agents. We sought to evaluate if combining palliative radiation therapy with pembrolizumab can augment antitumor immune responses in gastroesophageal cancer. Methods and Materials: Patients had metastatic gastroesophageal cancer with indication for palliative radiation therapy with ≥2 disease sites outside of the radiation field assessable for abscopal response and biopsies for laboratory correlative analyses. Palliative radiation was delivered to a dose of 30 Gy over 10 fractions. Pembrolizumab, 200 mg, was administered concurrently intravenously every 3 weeks until disease progression, unacceptable toxicity, or study withdrawal, for up to 2 years. Endpoints included PD-L1 expression in pre- and posttreatment biopsies and abscopal objective response rate per Response Evaluation Criteria in Solid Tumors. Results: Of 14 enrolled patients, the objective response rate was 28.6% (95% confidence interval, 8.4%-58.1%), and the median duration of response was not reached (95% confidence interval, 6.9-NR months). Overall, 2 patients had treatment-related grade 3 to 4 adverse events with no grade 5 events. One patient discontinued therapy due to grade 4 colitis. We did not observe an association between radiation and abscopal changes in PD-L1 expression via assessment of an analogous PD-L1 Combined Positive Score, Tumor Proportion Score, Mononuclear Immune Cell Density Score, or proportion of PD-L1-expressing immune cells between pre- and posttreatment tumor biopsies. Conclusions: Combining palliative radiation therapy and pembrolizumab provided promising durable responses in this patient population but we were unable to definitively distinguish abscopal biologic changes. Biomarker analyses beyond PD-L1 expression are needed to better understand putative mechanisms and identify patients who will benefit from this approach.

Objectives: The objective of this study was to compare safety and efficacy of patent foramen ovale (PFO) closure compared with medical therapy in patients with cryptogenic stroke (CS). Background: The role of PFO closure in preventing recurrent stroke in patients with prior CS has been controversial. Methods: We searched PubMed, EMBASE, the Cochrane Central Register of Controlled trials, and the clinical trial registry maintained at clinicaltrials.gov for randomized control trials that compared device closure with medical management and reported on subsequent stroke and adverse events. Event rates were compared using a forest plot of relative risk using a random-effects model assuming interstudy heterogeneity. Results: A total of 6 studies (n = 3747) were included in the final analysis. Mean follow-up ranged from 2 to 5.9 years. Pooled analysis revealed that device closure compared to medical management was associated with a significant reduction in stroke (risk ratio [RR] = 0.41, 95% CI = 0.20–0.83, I2 = 51%, P = 0.01). There was, however, a significant increase in atrial fibrillation with device therapy (RR = 5.29, 95% CI = 2.32–12.06, I2 = 38%, P

Rational: Phosphaturic mesenchymal tumors (PMTs) are rare tumors often causing tumor-induced osteomalacia (TIO), with surgical resection being the first line in the treatment of TIO. There are very few reports of PMTs located in the brain. The authors present one such case, it's management and outcome as well as a review of the literature. Patient concerns: The authors report of a 58 year old male with a background of osteoporosis, bone pain, muscle weakness and gait disturbances for the past several years. He presented to our institution with newly developed absence spells. He underwent imaging which revealed a brain tumor. Interventions: The patient underwent surgery for what was suspected as a meningioma. A gross total resection was done. The pathological diagnosis was that of phosphaturic mesenchymal tumor. Outcomes: Removal of the tumor helped in alleviating the patient's back symptoms immediately after surgery. The patient was discharged home and was followed up by an endocrinologist, with improvement of his bone pain, his muscle weakness and his gait disturbances. Lessons: PMT with involvement of the brain can be treated surgically. Removal of the tumor will alleviate symptoms in patients harboring this rare brain tumor. Keywords: Tumor induced osteomalacia, Phosphaturic mesenchymal tumor, Oncogenic osteomalacia

miR-224 has recently emerged as a driver oncomiR in sporadic colorectal carcinogenesis, but its pathogenetic role is still controversial. A large phenotypical and molecularly characterized series of preinvasive and invasive colorectal lesions was investigated for miR-224 expression by qRT-PCR and in situ hybridization. The caspase-3 and caspase-7 status was also assessed and correlated to miR-224 dysregulation. miR-224 was significantly upregulated during the adenoma-carcinoma sequence and in the context of inflammatory bowel disease dysplastic lesions, whereas its expression was significantly downregulated among BRAF-mutated tumors and in the presence of a DNA mismatch repair deficiency. miR-224 targets caspase-3 and caspase-7 in colorectal cancer, and this inverse relation was already evident from the earliest phases of transformation in intestinal mucosa. The miR-224/caspases axis may represent an interesting field of study for innovative biomarkers/therapeutics for BRAF-mutated/DNA mismatch repair-deficient tumors.

Background & Aims: Pancreatitis is a major cause of morbidity and mortality and is a risk factor for pancreatic tumorigenesis. Upon tissue damage, an inflammatory response, made up largely of macrophages, provides multiple growth factors that promote repair. Here, we examine the molecular pathways initiated by macrophages to promote pancreas recovery from pancreatitis. Methods: To induce organ damage, mice were subjected to cerulein-induced experimental pancreatitis and analyzed at various times of recovery. CD11b-DTR mice were used to deplete myeloid cells. Hbegff/f;LysM-Cre mice were used to ablate myeloid cell–derived heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). To ablate EGFR specifically during recovery, pancreatitis was induced in Egfrf/f;Ptf1aFlpO/+;FSF-Rosa26CAG-CreERT2 mice followed by tamoxifen treatment. Results: Macrophages infiltrating the pancreas in experimental pancreatitis make high levels of HB-EGF. Both depletion of myeloid cells and ablation of myeloid cell HB-EGF delayed recovery from experimental pancreatitis, resulting from a decrease in cell proliferation and an increase in apoptosis. Mechanistically, ablation of myeloid cell HB-EGF impaired epithelial cell DNA repair, ultimately leading to cell death. Soluble HB-EGF induced EGFR nuclear translocation and methylation of histone H4, facilitating resolution of DNA damage in pancreatic acinar cells in vitro. Consistent with its role as the primary receptor of HB-EGF, in vivo ablation of EGFR from pancreatic epithelium during recovery from pancreatitis resulted in accumulation of DNA damage. Conclusions: By using novel conditional knockout mouse models, we determined that HB-EGF derived exclusively from myeloid cells induces epithelial cell proliferation and EGFR-dependent DNA repair, facilitating pancreas healing after injury. Keywords: DNA Damage, EGFR, Inflammation, Macrophages

Background: We conducted a first-in-human dose-escalation study with the oral FASN inhibitor TVB-2640 to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D), as monotherapy and with a taxane. Methods: This completed open-label outpatient study was conducted at 11 sites in the United States and United Kingdom. Patients with previously-treated advanced metastatic solid tumors and adequate performance status and organ function were eligible. TVB-2640 was administered orally daily until PD. Dose escalation initially followed an accelerated titration design that switched to a standard 3 + 3 design after Grade 2 toxicity occurred. Disease-specific cohorts were enrolled at the MTD. Statistical analyses were primarily descriptive. Safety analyses were performed on patients who received at least 1 dose of study drug. (Clinicaltrials.gov identifier NCT02223247) Findings: The study was conducted from 21 November 2013 to 07 February 2017. Overall, 136 patients received TVB-2640, 76 as monotherapy (weight-based doses of 60 mg/m2 to 240 mg/m2 and flat doses of 200 and 250 mg) and 60 in combination, (weight-based doses of 60 mg/m2 to 100 mg/m2 and flat dose of 200 mg) (55 paclitaxel, 5 docetaxel). DLTs with TVB-2640 were reversible skin and ocular effects. The MTD/RP2D was 100 mg/m2. The most common TEAEs (n,%) with TVB-2640 monotherapy were alopecia (46; 61%), PPE syndrome (35; 46%), fatigue (28; 37%), decreased appetite (20; 26%), and dry skin (17; 22%), and with TVB-2640+paclitaxel were fatigue (29 ; 53%), alopecia (25; 46%), PPE syndrome (25; 46%), nausea (22; 40%), and peripheral neuropathy (20; 36%). One fatal case of drug-related pneumonitis occurred with TVB-2640+paclitaxel; no other treatment-related deaths occurred. Target engagement (FASN inhibition) and inhibition of lipogenesis were demonstrated with TVB-2640. The disease control rate (DCR) with TVB-2640 monotherapy was 42%; no patient treated with monotherapy had a complete or partial response (CR or PR). In combination with paclitaxel, the PR rate was 11% and the DCR was 70%. Responses were seen across multiple tumor types, including in patients with KRASMUT NSCLC, ovarian, and breast cancer. Interpretation: TVB-2640 demonstrated potent FASN inhibition and a predictable and manageable safety profile, primarily characterized by non-serious, reversible adverse events affecting skin and eyes. Further investigation of TVB-2640 in patients with solid tumors, particularly in KRASMUT lung, ovarian, and breast cancer, is warranted. Funding: This trial was funded by 3-V Biosciences, Inc. (now known as Sagimet Biosciences Inc.).

Mouse models have made innumerable contributions to understanding the genetic basis of neurological disease and pathogenic mechanisms and to therapy development. Here we consider the current state of mouse genetic models of Developmental and Epileptic Encephalopathy (DEE), representing a set of rare but devastating and largely intractable childhood epilepsies. By examining the range of mouse lines available in this rapidly moving field and by detailing both expected and unusual features in representative examples, we highlight lessons learned in an effort to maximize the full potential of this powerful resource for preclinical studies.

Despite impressive global school enrollment, especially at the primary school level, there are still some corners of the world where equal access to education remains far from reach. The current study sought to expand on previous research by further examining child, parent, and household characteristics that are associated with children's schooling status (CSS), namely enrollment, dropout and non-enrollment. The study utilized a cross-sectional 2014 dataset of the Uwezo Uganda National Learning Assessment survey with a sample size of n = 28,147. The survey recorded an 87.94% child enrollment rate, 2.91% of the sample dropped out of school, and 9.16% reported non-enrollment. Two multiple multinomial logistic models were employed, one with only main effects and one with an interaction term between a mother's education level and the child's gender. Based on the effect sizes and ranked in descending order of magnitude, household head's primary level of education and mother's level of education, preschool attendance, household head's secondary or higher level of education, presence of one or no biological parent/s, disability, household wealth, the child's age and the number of children in the household significantly predicted never enrolling in school. Similarly, ranked in descending order of magnitude, a mother's primary level of education, the presence of one or no biological parent/s, disability, age, the child's gender, household wealth, and preschool attendance significantly predicted dropping out of school. Finally, the interaction model indicated that girls whose mother had a primary education were less likely to either drop out or never enroll in school compared to those with a mother who did not complete formal education.

Mutations in the X-linked gene IQSEC2 are associated with multiple cases of epilepsy, epileptic encephalopathy, intellectual disability and autism spectrum disorder, the mechanistic understanding and successful treatment of which remain a significant challenge in IQSEC2 and related neurodevelopmental genetic diseases. To investigate disease etiology, we studied behaviors and synaptic function in IQSEC2 deficient mice. Hemizygous Iqsec2 null males exhibit growth deficits, hyperambulation and hyperanxiety phenotypes. Adult hemizygotes experience lethal spontaneous seizures, but paradoxically have a significantly increased threshold to electrically induced limbic seizures and relative resistance to chemically induced seizures. Although there are no gross defects in brain morphology, hemizygotes exhibit stark hippocampal reactive astrogliosis. Electrophysiological recordings of hippocampal neurons reveal increased excitatory drive specifically onto interneurons, and significant alterations in intrinsic electrical properties specific to the interneuron population. As they age, hemizygotes also develop an increased abundance of parvalbumin-positive interneurons in the hippocampus, neurons in which IQSEC2 is expressed in addition to the excitatory neurons. These findings point to a novel role of IQSEC2 in hippocampal interneuron synaptic function and development with implications for a class of intractable neurodevelopmental diseases.

ARFGEF1 encodes a guanine exchange factor involved in intracellular vesicle trafficking, and is a candidate gene for childhood genetic epilepsies. To model ARFGEF1 haploinsufficiency observed in a recent Lennox Gastaut Syndrome patient, we studied a frameshift mutation (Arfgef1fs) in mice. Arfgef1fs/+ pups exhibit signs of developmental delay, and Arfgef1fs/+ adults have a significantly decreased threshold to induced seizures but do not experience spontaneous seizures. Histologically, the Arfgef1fs/+ brain exhibits a disruption in the apical lining of the dentate gyrus and altered spine morphology of deep layer neurons. In primary hippocampal neuron culture, dendritic surface and synaptic but not total GABAA receptors (GABAAR) are reduced in Arfgef1fs/+ neurons with an accompanying decrease in the number of GABAAR-containing recycling endosomes in cell body. Arfgef1fs/+ neurons also display differences in the relative ratio of Arf6+:Rab11+:TrfR+ recycling endosomes. Although the GABAAR-containing early endosomes in Arfgef1fs/+ neurons are comparable to wildtype, Arfgef1fs/+ neurons show an increase in the number of GABAAR-containing lysosomes in dendrite and cell body. Together, the altered endosome composition and decreased neuronal surface GABAAR results suggests a mechanism whereby impaired neuronal inhibition leads to seizure susceptibility.

Summary: Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. Here, we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. We explored how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases. : Preger-Ben Noon et al. find that shavenbaby gene enhancers contain regulatory information for driving several expression patterns (i.e., enhancers are pleiotropic) and that, in some cases, the transcription factor binding sites that activate these enhancers are reused during development. Keywords: enhancer, pleitropy, gene regulation, shavenbaby, Drosophila

Summary: We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course. : A/E pathogens intimately adhere to the gut mucosa. Mullineaux-Sanders et al. demonstrate that inducing specific dysbiosis at the peak of murine infection with Citrobacter rodentium prevents mucosal colonization. This occurs via a mechanism independent of virulence gene expression modulation, indicating that enteric pathogens may rely on commensals for effective infection. Keywords: C. rodentium, enteric infection, antibiotics, microbiota, colonization resistance, AMR

This is the first prospective study of the effects of human gut microbiota and metabolites on immune checkpoint inhibitor (ICT) response in metastatic melanoma patients. Whereas many melanoma patients exhibit profound response to ICT, there are fewer options for patients failing ICT—particularly with BRAF-wild-type disease. In preclinical studies, specific gut microbiota promotes regression of melanoma in mice. We therefore conducted a study of the effects of pretreatment gut microbiota and metabolites on ICT Response Evaluation Criteria in Solid Tumors response in 39 metastatic melanoma patients treated with ipilimumab, nivolumab, ipilimumab plus nivolumab (IN), or pembrolizumab (P). IN yielded 67% responses and 8% stable disease; P achieved 23% responses and 23% stable disease. ICT responders for all types of therapies were enriched for Bacteroides caccae. Among IN responders, the gut microbiome was enriched for Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis. Among P responders, the microbiome was enriched for Dorea formicogenerans. Unbiased shotgun metabolomics revealed high levels of anacardic acid in ICT responders. Based on these pilot studies, both additional confirmatory clinical studies and preclinical testing of these bacterial species and metabolites are warranted to confirm their ICT enhancing activity.

This is the first prospective study of a combination therapy involving a cardenolide and a MEK inhibitor for metastatic melanoma. Whereas BRAF mutant melanomas can exhibit profound responses to treatment with BRAF and MEK inhibitors, there are fewer options for BRAF wild-type melanomas. In preclinical studies, we discovered that cardenolides synergize with MEK inhibitor to promote the regression of patient-derived xenografts irrespective of BRAF mutation status. We therefore conducted a phase 1B study of digoxin 0.25 mg and trametinib 2 mg given orally once daily in 20 patients with advanced, refractory, BRAF wild-type melanomas. The most common adverse events were rash, diarrhea, nausea, and fatigue. The response rate was 4/20 or 20% with response durations of 2, 4, 6, and 8 months. The disease control rate (including partial responses and stable disease) was 13/20 or 65% of patients, including 5/6 or 83% of patients with NRAS mutant melanomas and 8/14 or 57% of NRAS wild-type melanomas. Patients with stable disease had disease control for 2, 2, 2, 4, 5, 6, 7, 10, and 10 months. Xenografts from four patients recapitulated the treatment responses observed in patients. Based on these pilot results, an expansion arm of digoxin plus MEK inhibitor is warranted for NRAS mutant metastatic melanoma patients who are refractory or intolerant of immunotherapy. Key points: Digoxin plus trametinib is well tolerated and achieves a high rate of disease control in BRAF wild-type metastatic melanoma patients.

Despite the growing interest in Mg and its alloys, their use has been largely limited due to their high reactivity in aqueous environments. Improving the understanding of the basic principles of Mg corrosion represents the first step to explain and, eventually, improve the corrosion behaviour of Mg alloys. Herein an original mechanistic surface kinetic DFT model that clarifies the mechanism of anomalous HE on anodically polarised Mg is presented. In accordance with several experimental observations, this model describes anomalous HE proceeding at the regions dominated by anodic dissolution via the reaction of an Mg*H intermediate with water. The Mg*H intermediates undergo oxidation upon anodic polarisation, resulting in hydrogen evolution and Mg dissolution. Furthermore, it is revealed that increasing rates of an electrochemical cathodic reaction are possible within a dissolving anode. Keywords: Magnesium, Hydrogen evolution, Dissolution, Density functional theory DFT, Negative difference effect NDE

Dynamin 1 is a large neuron-specific GTPase involved in the endocytosis and recycling of pre-synaptic membranes and synaptic vesicles. Mutations in the gene encoding dynamin 1 (DNM1) underlie two epileptic encephalopathy syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Mice homozygous for the Dnm1 “fitful” mutation, a non-synonymous coding variant in an alternatively spliced exon of Dnm1 (exon 10a; isoform designation: Dnm1aFtfl) have an epileptic encephalopathy-like disorder including lethal early onset seizures, locomotor and neurosensory deficits. Although fitful heterozygotes have milder recurrent seizures later in life, suggesting an additive or semi-dominant mechanism, the molecular etiology must also consider the fact that Dnm1aFtfl exerts a dominant negative effect on endocytosis in vitro. Another complication is that the fitful mutation induces alterations in the relative abundance of Dnm1 splice variants; mutants have a downregulation of Dnm1a and an upregulation of Dnm1b, changes which may contribute to the epileptic pathology. To examine whether Dnm1a loss of function, Dnm1aFtfl dominance or compensation by Dnm1b is the most critical for severe seizures, we studied alternate isoform-specific mutant mice. Mice lacking Dnm1 exon 10a or Dnm1 exon 10b have neither spontaneous seizures nor other overt abnormalities, suggesting that in normal conditions the major role of each isoform is redundant. However, in the presence of Dnm1aFtfl only exon 10a deleted mice experience severe seizures. These results reveal functional differences between Dnm1a and Dnm1b isoforms in the presence of a challenge, i.e. toxic Dnm1Ftfl, while reinforcing its effect explicitly in this model of severe pediatric epilepsy.

Purpose: Capacity to monitor non-communicable diseases (NCDs) at state or local levels is limited. Emerging approaches include using biomeasures and electronic health record (EHR) data. In 2004, New York City (NYC) performed a population-based health study on adult residents using biomeasures (NYC Health and Nutrition Examination Study, or NYC HANES), modeled after NHANES. A second NYC HANES was launched in 2013 to examine change over time, evaluate municipal policies, and validate a proposed EHR-based surveillance system. We describe the rationale and methods of NYC HANES 2013–2014. Methods: NYC HANES was a population-based, cross-sectional survey of NYC adults using three-stage cluster sampling. Between August 2013 and June 2014, selected participants completed a health interview and physical exam (blood pressure, body mass index, and waist circumference). Fasting biomeasures included diabetes, lipid profiles, kidney function, environmental biomarkers, and select infectious diseases. Results: Of the 3065 households approached, 2742 were eligible and 1827 were successfully screened (67%). A total of 1524 of eligible participants completed the survey (54%), for an overall response rate of 36%. Conclusion: Completing a second NYC HANES a decade after the first study affords an opportunity to understand changes in prevalence, awareness and control of NCDs and evaluate municipal efforts to manage them.

In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8aV929F). Here, we confirmed the prior suggestion that Scn8aV929F exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8aV929F is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2V752F as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2V752F functional analysis revealed an overall gain-of-function and for NaV1.6V929F revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2V752F due to “functional silencing” of NaV1.6V929F suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6V929F loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as absence epilepsy.

In this study, we attempt to target the mitogen-activated protein kinase (MAPK) pathway in acute myeloid leukemia (AML) cells using a recombinant anthrax lethal toxin (LeTx). LeTx consists of protective antigen (PrAg) and lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves MAPK kinases, inhibiting the MAPK pathway. We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic responses to LeTx. Cytotoxicity of LeTx was mimicked by the specific mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126, indicating that LeTx-induced cell death is mediated through the MEK1/2-extracellular signal-regulated kinase (ERK1/2) branch of the MAPK pathway. The four LeTx-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase inhibitor LY294002. Co-treatment of AML cells with both LeTx and LY294002 did not lead to increased sensitivity, showing a lack of additive/synergistic effects when both pathways are inhibited. Flow cytometry analysis of MAPK pathway activation revealed the presence of phospho-ERK1/2 only in LeTx-sensitive cells. Staining for Annexin V/propidium iodide and active caspases showed an increase in double-positive cells and the absence of caspase activation following treatment, indicating that LeTx-induced cell death is caspase-independent and nonapoptotic. We have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the MAPK pathway. Furthermore, we have demonstrated that LeTx-induced cytotoxicity in AML cells is nonapoptotic and dependent on phospho-ERK1/2 levels.

Mutations in the neuronal voltage-gated sodium channel genes SCN1A and SCN2A are associated with inherited epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (severe myoclonic epilepsy of infancy). The clinical presentation and severity of these epilepsies vary widely, even in people with the same mutation, suggesting the action of environmental or genetic modifiers. To gain support for the hypothesis that genetic modifiers can influence clinical presentation in patients with SCN1A-derived GEFS+, we used mouse models to study the effect of combining the human GEFS+ mutation SCN1A-R1648H with SCN2A, KCNQ2, and SCN8A mutations. Knock-in mice heterozygous for the R1648H mutation (Scn1aRH/+) have decreased thresholds to induced seizures and infrequent spontaneous seizures, whereas homozygotes display spontaneous seizures and premature lethality. Scn2aQ54 transgenic mice have a mutation in Scn2a that results in spontaneous, adult-onset partial motor seizures, and mice carrying the Kcnq2-V182M mutation exhibit increased susceptibility to induced seizures, and rare spontaneous seizures as adults. Combining the Scn1a-R1648H allele with either Scn2aQ54 or Kcnq2V182M/+ results in early-onset, generalized tonic–clonic seizures and juvenile lethality in double heterozygous mice. In contrast, Scn8a mutants exhibit increased resistance to induced seizures. Combining the Scn1a-R1648H and Scn8a-med-jo alleles restores normal thresholds to flurothyl-induced seizures in Scn1aRH/+ heterozygotes and improved survival of Scn1aRH/RH homozygotes. Our results demonstrate that variants in Scn2a, Kcnq2, and Scn8a can dramatically influence the phenotype of mice carrying the Scn1a-R1648H mutation and suggest that ion channel variants may contribute to the clinical variation seen in patients with monogenic epilepsy.