Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation.

Pyroptosis is an inflammatory form of programmed cell death following cellular damage or infection. It is a lytic process driven by gasdermin D-mediated cellular permeabilization and presumed osmotic forces thought to induce swelling and rupture. We found that pyroptotic cells do not spontaneously rupture in culture but lose mechanical resilience. As a result, cells were susceptible to rupture by extrinsic forces, such as shear stress or compression. Cell analyses revealed that all major cytoskeleton components were disrupted during pyroptosis and that sensitivity to rupture was calpain-dependent and linked with cleavage of vimentin and loss of intermediate filaments. Moreover, while release of lactate dehydrogenase (LDH), HMGB1, and IL-1ß occurred without rupture, rupture was required for release of large inflammatory stimuli-ASC specks, mitochondria, nuclei, and bacteria. Importantly, supernatants from ruptured cells were more immunostimulatory than those from nonruptured cells. These observations reveal undiscovered cellular events occurring during pyroptosis, define the mechanisms driving pyroptotic rupture, and highlight the immunologic importance of this event.

The SCF-Fbw7 ubiquitin ligase degrades MED13 and MED13L and regulates CDK8 module association with Mediator.

The Mediator complex is an essential transcription regulator that bridges transcription factors with RNA polymerase II. This interaction is controlled by dynamic interactions between Mediator and the CDK8 module, but the mechanisms governing CDK8 module-Mediator association remain poorly understood. We show that Fbw7, a tumor suppressor and ubiquitin ligase, binds to CDK8-Mediator and targets MED13/13L for degradation. MED13/13L physically link the CDK8 module to Mediator, and Fbw7 loss increases CDK8 module-Mediator association. Our work reveals a novel mechanism regulating CDK8 module-Mediator association and suggests an expanded role for Fbw7 in transcriptional control and an unanticipated relationship with the CDK8 oncogene.

Adult Acquired Flatfoot Deformity: Anatomy, Biomechanics, Staging, and Imaging Findings.

Adult acquired flatfoot deformity (AAFD) is a common disorder that typically affects middle-aged and elderly women, resulting in foot pain, malalignment, and loss of function. The disorder is initiated most commonly by degeneration of the posterior tibialis tendon (PTT), which normally functions to maintain the talonavicular joint at the apex of the three arches of the foot. PTT degeneration encompasses tenosynovitis, tendinosis, tendon elongation, and tendon tearing. The malaligned foot is initially flexible but becomes rigid and constant as the disorder progresses. Tendon dysfunction commonly leads to secondary damage of the spring ligament and talocalcaneal ligaments and may be associated with injury to the deltoid ligament, plantar fascia, and other soft-tissue structures. Failure of multiple stabilizers appears to be necessary for development of the characteristic planovalgus deformity of AAFD, with a depressed plantar-flexed talus bone, hindfoot and/or midfoot valgus, and an everted flattened forefoot. AAFD also leads to gait dysfunction as the foot is unable to change shape and function adequately to accommodate the various phases of gait, which require multiple rapid transitions in foot position and tone for effective ambulation. The four-tier staging system for AAFD emphasizes physical examination findings and metrics of foot malalignment. Mild disease is managed conservatively, but surgical procedures directed at the soft tissues and/or bones become necessary and progressively more invasive as the disease progresses. Although much has been written about the imaging findings of AAFD, this article emphasizes the anatomy and function of the foot's stabilizing structures to help the radiologist better understand this disabling disorder. Online supplemental material is available for this article. ©RSNA, 2019.

MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1ß Independently of Gasdermin-D.

Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1ß. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1ß released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1ß independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1ß independently of gasdermin-D.

p120-catenin is essential for terminal end bud function and mammary morphogenesis.

Although p120-catenin (p120) is crucial for E-cadherin function, ablation experiments in epithelial tissues from different organ systems reveal markedly different effects. Here, we examine for the first time the consequences of p120 knockout during mouse mammary gland development. An MMTV-Cre driver was used to target knockout to the epithelium at the onset of puberty. p120 ablation was detected in approximately one-quarter of the nascent epithelium at the forth week post-partum. However, p120 null cells were essentially nonadherent, excluded from the process of terminal end bud (TEB) morphogenesis and lost altogether by week six. This elimination process caused a delay in TEB outgrowth, after which the gland developed normally from cells that had retained p120. Mechanistic studies in vitro indicate that TEB dysfunction is likely to stem from striking E-cadherin loss, failure of cell-cell adhesion and near total exclusion from the collective migration process. Our findings reveal an essential role for p120 in mammary morphogenesis.

Assessing Cytoskeletal Destruction During Pyroptosis.

Pyroptosis is an inflammatory form of cell death driven by the activation of caspase-1 and/or caspase-11 which cleaves and activates the pore-forming and cell-permeabilizing protein gasdermin-D. Pyroptosis is characterized by cell swelling and release of inflammatory cytosolic content, which were thought to be driven by colloid-osmotic lysis. Instead, we previously demonstrated that in vitro, pyroptotic cells do not in fact lyse. We also demonstrated that calpain cleaves vimentin, leading to loss of intermediate filaments, which in turn makes cells fragile and susceptible to rupture by extrinsic pressure. However, if, as our observations suggest, cells do not swell due to osmotic forces, what then causes cell rupture? Interestingly, in addition to intermediate filament loss, we demonstrated that other cytoskeletal networks, such as microtubules, actin, and nuclear lamina, are similarly lost during pyroptosis; however, the mechanisms driving these cytoskeletal disruptions as well as their functional significance are unclear. To facilitate the study of these processes, we present here the immunocytochemical methods by which we detected and assayed cytoskeletal destruction during pyroptosis.

A C57BL/6 Mouse Model of SARS-CoV-2 Infection Recapitulates Age- and Sex-Based Differences in Human COVID-19 Disease and Recovery.

We present a comprehensive analysis of SARS-CoV-2 infection and recovery using wild type C57BL/6 mice and a mouse-adapted virus, and we demonstrate that this is an ideal model of infection and recovery that phenocopies acute human disease arising from the ancestral SARS-CoV-2. Disease severity and infection kinetics are age- and sex-dependent, as has been reported for humans, with older mice and males in particular exhibiting decreased viral clearance and increased mortality. We identified key parallels with human pathology, including intense virus positivity in bronchial epithelial cells, wide-spread alveolar involvement, recruitment of immune cells to the infected lungs, and acute bronchial epithelial cell death. Moreover, older animals experienced increased virus persistence, delayed dispersal of immune cells into lung parenchyma, and morphologic evidence of tissue damage and inflammation. Parallel analysis of SCID mice revealed that the adaptive immune response was not required for recovery from COVID disease symptoms nor early phase clearance of virus but was required for efficient clearance of virus at later stages of infection. Finally, transcriptional analyses indicated that induction and duration of key innate immune gene programs may explain differences in age-dependent disease severity. Importantly, these data demonstrate that SARS-CoV-2-mediated disease in C57BL/6 mice phenocopies human disease across ages and establishes a platform for future therapeutic and genetic screens for not just SARS-CoV-2 but also novel coronaviruses that have yet to emerge.

Blocked acinar development, E-cadherin reduction, and intraepithelial neoplasia upon ablation of p120-catenin in the mouse salivary gland.

p120 catenin is thought to be a key regulator of E-cadherin function and stability, but its role(s) in vivo is poorly understood. To examine these directly, we generated a conditional p120 knockout mouse and targeted p120 ablation to the embryonic salivary gland. Surprisingly, acinar differentiation is completely blocked, resulting in a gland composed entirely of ducts. Moreover, p120 ablation causes E-cadherin deficiency in vivo and severe defects in adhesion, cell polarity, and epithelial morphology. These changes closely phenocopy high-grade intraepithelial neoplasia, a condition that, in humans, typically progresses to invasive cancer. Tumor-like protrusions appear immediately after p120 ablation at e14 and expand into the lumen until shortly after birth, at which time the animals die with completely occluded glands. The data reveal an unexpected role for p120 in salivary acinar development and show that p120 ablation by itself induces effects consistent with a role in tumor progression.

LOX-1: A potential driver of cardiovascular risk in SLE patients.

Traditional cardiovascular disease (CVD) risk factors, such as hypertension, dyslipidemia and diabetes do not explain the increased CVD burden in systemic lupus erythematosus (SLE). The oxidized-LDL receptor, LOX-1, is an inflammation-induced receptor implicated in atherosclerotic plaque formation in acute coronary syndrome, and here we evaluated its role in SLE-associated CVD. SLE patients have increased sLOX-1 levels which were associated with elevated proinflammatory HDL, oxLDL and hsCRP. Interestingly, increased sLOX-1 levels were associated with patients with early disease onset, low disease activity, increased IL-8, and normal complement and hematological measures. LOX-1 was increased on patient-derived monocytes and low-density granulocytes, and activation with oxLDL and immune-complexes increased membrane LOX-1, TACE activity, sLOX-1 release, proinflammatory cytokine production by monocytes, and triggered the formation of neutrophil extracellular traps which can promote vascular injury. In conclusion, perturbations in the lipid content in SLE patients' blood activate LOX-1 and promote inflammatory responses. Increased sLOX-1 levels may be an indicator of high CVD risk, and blockade of LOX-1 may provide a therapeutic opportunity for ameliorating atherosclerosis in SLE patients.

A core function for p120-catenin in cadherin turnover.

p120-catenin stabilizes epithelial cadherin (E-cadherin) in SW48 cells, but the mechanism has not been established. Here, we show that p120 acts at the cell surface to control cadherin turnover, thereby regulating cadherin levels. p120 knockdown by siRNA expression resulted in dose-dependent elimination of epithelial, placental, neuronal, and vascular endothelial cadherins, and complete loss of cell-cell adhesion. ARVCF and delta-catenin were functionally redundant, suggesting that proper cadherin-dependent adhesion requires the presence of at least one p120 family member. The data reveal a core function of p120 in cadherin complexes, and strongly predict a dose-dependent loss of E-cadherin in tumors that partially or completely down-regulate p120.

A novel role for p120 catenin in E-cadherin function.

Indirect evidence suggests that p120-catenin (p120) can both positively and negatively affect cadherin adhesiveness. Here we show that the p120 gene is mutated in SW48 cells, and that the cadherin adhesion system is impaired as a direct consequence of p120 insufficiency. Restoring normal levels of p120 caused a striking reversion from poorly differentiated to cobblestone-like epithelial morphology, indicating a crucial role for p120 in reactivation of E-cadherin function. The rescue efficiency was enhanced by increased levels of p120, and reduced by the presence of the phosphorylation domain, a region previously postulated to confer negative regulation. Surprisingly, the rescue was associated with substantially increased levels of E-cadherin. E-cadherin mRNA levels were unaffected by p120 expression, but E-cadherin half-life was more than doubled. Direct p120-E-cadherin interaction was crucial, as p120 deletion analysis revealed a perfect correlation between E-cadherin binding and rescue of epithelial morphology. Interestingly, the epithelial morphology could also be rescued by forced expression of either WT E-cadherin or a p120-uncoupled mutant. Thus, the effects of uncoupling p120 from E-cadherin can be at least partially overcome by artificially maintaining high levels of cadherin expression. These data reveal a cooperative interaction between p120 and E-cadherin and a novel role for p120 that is likely indispensable in normal cells.

Interleukin-1ß Signaling in Dendritic Cells Induces Antiviral Interferon Responses.

Induction of interferon beta (IFN-ß), IFN-stimulated genes (ISGs), and inflammatory responses is critical for control of viral infection. We recently identified an essential linkage of stimulation of the inflammatory cytokine interleukin-1ß (IL-1ß) and induction of ISGs that function as host restriction pathways against the emerging flavivirus West Nile virus (WNV) in vivo Here we utilized ex vivo global transcriptome analysis of primary dendritic cells, known targets of WNV replication, to define gene signatures required for this IL-1ß-driven antiviral response. Dendritic cells that were deficient in IL-1 receptor signaling showed dysregulation of cell-intrinsic defense genes and loss of viral control during WNV infection. Surprisingly, we found that in wild-type cells, IL-1ß treatment, in the absence of infection, drove the transcription of IFN-ß and ISGs at late times following treatment. Expression of these antiviral innate immune genes was dependent on the transcription factor IFN regulatory factor 3 (IRF3) and appears to reflect a general shift in IL-1ß signaling from an early inflammatory response to a late IFN-mediated response. These data demonstrate that inflammatory and antiviral signals integrate to control viral infection in myeloid cells through a process of IL-1ß-to-IRF3 signaling crosstalk. Strategies to exploit these cytokines in the activation of host defense programs should be investigated as novel therapeutic approaches against individual pathogens.IMPORTANCE West Nile virus is an emerging mosquito-borne flavivirus that can result in serious illness, neuropathology, and death in infected individuals. Currently, there are no vaccines or therapies for human use against West Nile virus. Immune control of West Nile virus infection requires inflammatory and antiviral responses, though the effect that each arm of this response has on the other is unclear. The significance of our research is in defining how virus-induced inflammatory responses regulate critical antiviral immune programs for effective control of West Nile virus infection. These data identify essential mechanisms of immune control that can inform therapeutic efforts against West Nile virus, with potential efficacy against other neuroinvasive viruses.

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain.

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth.

Dramatic enhancement of the detection limits of bioassays via ultrafast deposition of polydopamine.

The ability to detect biomarkers with ultrahigh sensitivity radically transformed biology and disease diagnosis. However, owing to incompatibilities with infrastructure in current biological and medical laboratories, recent innovations in analytical technology have not received broad adoption. Here, we report a simple, universal 'add-on' technology (dubbed EASE) that can be directly plugged into the routine practices of current research and clinical laboratories and that converts the ordinary sensitivities of common bioassays to extraordinary ones. The assay relies on the bioconjugation capabilities and ultrafast and localized deposition of polydopamine at the target site, which permit a large number of reporter molecules to be captured and lead to detection-sensitivity enhancements exceeding 3 orders of magnitude. The application of EASE in the enzyme-linked-immunosorbent-assay-based detection of the HIV antigen in blood from patients leads to a sensitivity lower than 3 fg ml-1. We also show that EASE allows for the direct visualization, in tissues, of the Zika virus and of low-abundance biomarkers related to neurological diseases and cancer immunotherapy.

High-throughput genomic technology in research and clinical management of breast cancer. Plasma-based proteomics in early detection and therapy.

Protein-based breast cancer biomarkers are a promising resource for breast cancer detection at the earliest and most treatable stages of the disease. Plasma is well suited to proteomic-based methods of biomarker discovery because it is easily obtained, is routinely used in the diagnosis of many diseases, and has a rich proteome. However, due to the vast dynamic range in protein concentration and the often uncertain tissue and cellular origin of plasma proteins, proteomic analysis of plasma requires special consideration compared with tissue and cultured cells. This review briefly touches on the search for plasma-based protein biomarkers for the early detection and treatment of breast cancer.

In the superobese, weight loss and resolution of obesity comorbidities after biliopancreatic bypass and/or duodenal switch vary according to health insurance carrier: Medicaid vs Medicare vs Private insurance vs Self-Pay in 1681 Bariatric Outcomes Longitudinal Database patients.

BACKGROUND: Differences in Medicaid vs Medicare vs Private vs Self-Pay duodenal switch (DS) results are unknown. This study identified DS outcomes variations by health insurance. METHODS: Data from 1,681 DS patients were analyzed retrospectively: Medicaid (n = 138), Medicare (n = 313), Private insurance (n = 1,171), and Self-Pay (n = 59). General linear models included baseline and postoperative data and were modified for dichotomous variables. RESULTS: Hypertension, obstructive sleep apnea, abdominal hernia, diabetes, and 9 other hepatobiliary, and somatic conditions were lowest in Private (P < .05). Self-Pay cholelithiasis, gastroesophageal reflux disease, back and/or musculoskeletal pain, and 3 others were lowest; asthma, angina, congestive heart failure, alcohol use, liver disease, and 3 others were highest (P < .05). Medicare had highest abdominal hernia and musculoskeletal pain, pseudotumor cerebri; lowest asthma, and polycystic ovarian syndrome (P < .05). Medicaid hypertension, sleep apnea, cholelithiasis, gastroesophageal reflux disease, diabetes, back pain, and 5 others were highest (P < .05); dyslipidemia and alcohol use were lowest. CONCLUSIONS: Outcomes after DS vary by health insurance. These findings may facilitate management of DS patients.

Fetal brain lesions after subcutaneous inoculation of Zika virus in a pregnant nonhuman primate.

We describe the development of fetal brain lesions after Zika virus (ZIKV) inoculation in a pregnant pigtail macaque. Periventricular lesions developed within 10 d and evolved asymmetrically in the occipital-parietal lobes. Fetal autopsy revealed ZIKV in the brain and significant cerebral white matter hypoplasia, periventricular white matter gliosis, and axonal and ependymal injury. Our observation of ZIKV-associated fetal brain lesions in a nonhuman primate provides a model for therapeutic evaluation.

Semi-automated object tracking methods in biological imaging.

Time-lapse imaging is a rich data source offering potential kinetic information of cellular activity and behavior. Tracking and extracting measurements of objects from time-lapse datasets are challenges that result from the complexity and dynamics of each object's motion and intensity or the appearance of new objects in the field of view. A wide range of strategies for proper data sampling, object detection, image analysis, and post-analysis interpretation are available. Theory and methods for single-particle tracking, spot detection, and object linking are discussed in this unit, as well as examples with step-by-step procedures for utilizing semi-automated software and visualization tools for achieving tracking results and interpreting this output.

Preoperative simultaneous fractionated cisplatin and radiation therapy in the treatment of advanced operable stage III and IV squamous cell carcinoma of the head and neck.

BACKGROUND: In Stage III/IV head and neck squamous cell carcinoma of the head and neck, multidisciplinary treatment is not standardized. This study evaluated preoperative simultaneous radiation therapy and Cisplatin 20 mg/M(2)/4 days during weeks 1, 4, and 7 of irradiation (CTRT). METHODS: Records of 143 CTRT and 48 patients treated with other surgery/radiation/chemotherapy regimens (CONTROL) were reviewed. Chi-square, analysis of variance, and Kaplan-Meier statistical analysis were performed. RESULTS: CTRT improved outcomes in Grade 2 to 5 toxicity (76% CONTROL vs 45% CTRT, P < .0001), complete clinical response (68% CTRT vs 36% CONTROL, P < .003), histologic complete response (67% in CTRT vs 28% in CONTROL, P = .0002), recurrence (33% in CTRT vs 66% in CONTROL, P = .0007), and distant metastases (2% CTRT vs 37% CONTROL, P = .0003); Kaplan-Meier disease-free survival was 65% CTRT versus 34% CONTROL. CONCLUSIONS: CTRT increases complete clinical response, histologic complete response, organ preservation, and survival, with lower recurrence and reduced toxicity and rare recurrence. CTRT may be the first treatment for Stage III/IV head and neck squamous cell carcinoma of the head and neck.

Paget sarcoma of the pelvic bone with widespread metastatic disease on radiography, CT, MRI, and 18F-FDG PET/CT with pathologic correlation.

We report a case of Paget sarcoma of the left superior pubic ramus and disseminated metastatic disease in a 70-year-old man. Paget disease of the left hemipelvis with malignant degeneration in the region of the left superior pubic ramus was initially diagnosed on radiographs. Subsequent CT, MRI, PET/CT imaging, and CT-guided biopsy confirmed the diagnosis and showed extensive left-sided pelvic and abdominal lymphadenopathy with widespread metastatic disease to liver, spleen, and lungs.