TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity.

To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of "at the ready" antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in the heart and lungs, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in cotranscribed clusters and examples in which TDMD underlies "arm switching," a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8-null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

Tau seeding without tauopathy.

Neurodegenerative tauopathies such as Alzheimer's disease (AD) are caused by brain accumulation of tau assemblies. Evidence suggests tau functions as a prion, and cells and animals can efficiently propagate unique, transmissible tau pathologies. This suggests a dedicated cellular replication machinery, potentially reflecting a normal physiologic function for tau seeds. Consequently, we hypothesized that healthy control brains would contain seeding activity. We have recently developed a novel monoclonal antibody (MD3.1) specific for tau seeds. We used this antibody to immunopurify tau from the parietal and cerebellar cortices of 19 healthy subjects without any neuropathology, ranging 19 to 65 years. We detected seeding in lysates from the parietal cortex, but not in the cerebellum. We also detected no seeding in brain homogenates from wildtype or human tau knockin mice, suggesting that cellular/genetic context dictates development of seed-competent tau. Seeding did not correlate with subject age or brain tau levels. We confirmed our essential findings using an orthogonal assay, real-time quaking-induced conversion, which amplifies tau seeds in vitro. Dot blot analyses revealed no AT8 immunoreactivity above background levels in parietal and cerebellar extracts and ∼1/100 of that present in AD. Based on binding to a panel of antibodies, the conformational characteristics of control seeds differed from AD, suggesting a unique underlying assembly, or structural ensemble. Tau's ability to adopt self-replicating conformations under nonpathogenic conditions may reflect a normal function that goes awry in disease states.

Fic-mediated AMPylation tempers the unfolded protein response during physiological stress.

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

Deep Learning-Based H-Score Quantification of Immunohistochemistry-Stained Images.

Immunohistochemistry (IHC) is a well-established and commonly used staining method for clinical diagnosis and biomedical research. In most IHC images, the target protein is conjugated with a specific antibody and stained using diaminobenzidine (DAB), resulting in a brown coloration, whereas hematoxylin serves as a blue counterstain for cell nuclei. The protein expression level is quantified through the H-score, calculated from DAB staining intensity within the target cell region. Traditionally, this process requires evaluation by 2 expert pathologists, which is both time consuming and subjective. To enhance the efficiency and accuracy of this process, we have developed an automatic algorithm for quantifying the H-score of IHC images. To characterize protein expression in specific cell regions, a deep learning model for region recognition was trained based on hematoxylin staining only, achieving pixel accuracy for each class ranging from 0.92 to 0.99. Within the desired area, the algorithm categorizes DAB intensity of each pixel as negative, weak, moderate, or strong staining and calculates the final H-score based on the percentage of each intensity category. Overall, this algorithm takes an IHC image as input and directly outputs the H-score within a few seconds, significantly enhancing the speed of IHC image analysis. This automated tool provides H-score quantification with precision and consistency comparable to experienced pathologists but at a significantly reduced cost during IHC diagnostic workups. It holds significant potential to advance biomedical research reliant on IHC staining for protein expression quantification.

Myofiber-type-dependent 'boulder' or 'multitudinous pebble' formations across distinct amylopectinoses.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen's spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes' functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous ('pebbles') or giant and single ('boulders'), and that this is primarily determined by the myofiber types in which they form, 'pebbles' in glycolytic and 'boulders' in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

Novel multi-spectral short-wave infrared imaging for assessment of human burn wound depth.

Burn depth determination is critical for patient care but is currently lacking accuracy. Recent animal studies showed that Short Wave Infrared (SWIR) imaging can distinguish between superficial and deep burns. This is a first human study correlating reflectance of multiple SWIR bands using a SWIR assessment tool (SWAT) with burn depth classifications by surgeons and histology. Burns and adjacent normal skin in 11 patients with thermal injuries were imaged with visual and narrow bands centred at 1200, 1650, 1940 and 2250 nm and biopsies were taken from select areas. Reflectance intensities for each band in 273 regions of interest (ROI) were divided by the normal skin reflectance and combined into three Reflectance Indices (RIs). In addition, burns in ROIs and biopsies were classified by five surgeons and three pathologists, respectively, as superficial partial, deep partial, or full thickness. Results show that for burn depth increase classified by the surgeons, reflectance increased at 1200 and 2250, decreased at 1940, and didn't change at 1650 nm. In contrast, all three RIs increase with burn depth and predict the deep and full depths ROIs representing operable regions (Area Under Curve >0.6507, p < 0.0001). Pathologists' classification matched surgeons' classification of burn category only in eight of 21 biopsies (38.1%), but reflectance at all bands and one RI for all deep partial and full thickness biopsies were larger than in non-biopsy normal and superficial partial thickness ROIs (p < 0.0118). In conclusion, multi-spectral imaging with a new SWAT is a promising approach for evaluation of burn wound depth.

Fli1 Promotes Vascular Morphogenesis by Regulating Endothelial Potential of Multipotent Myogenic Progenitors.

[Figure: see text].

Development of a Chronic Canine Ovariohysterectomy Model to Evaluate Vaginal Cuff Healing Using Two Closure Systems.

Background and Purpose. This study established a suitable animal model of ovariohysterectomy; characterized the course and pattern of vaginal healing after ovariohysterectomy; and compared healing obtained after closure of the vaginal cuff with a novel cuff-closure device (Zip-stitch® clips) and VICRYL® sutures. Research Design and Study Sample. This prospective, randomized, controlled, blinded animal study was conducted in 27 mongrel hounds according to an IACUC-approved protocol. Each animal underwent ovariohysterectomy followed by vaginal cuff closure with Zip-stitch or VICRYL. At two or six weeks, animals were sacrificed for gross and histological analysis. Data Collection. The primary endpoint was the difference in the fraction of vaginal cuff healed six weeks after application of the closure device. Secondary endpoints included histopathologic cellular and tissue responses, including inflammation, necrosis, infection, and vascular and muscle changes. Results. In the test group, there were two distinct locations where fibrotic or granular tissue fusion between the anterior and posterior vaginal walls was observed: in tissue "captured" by a clip or in tissue around the clip. The fraction of the vaginal cuff healed was similar in animals treated with Zip-stitch clips and those treated with sutures at six weeks (68±10% vs 67±18%; P=.148, test for non-inferiority) after surgery. The test article performed similarly or better than the control article in terms of the intensity or extent of the secondary endpoints. Conclusions. Subject to further confirmation, this study supports Zip-stitch clips as a method to maintain immediate post-operative approximation of the vaginal cuff leading to healing but did not achieve statistical significance in its primary endpoint.

Conjunctival changes following Muller's muscle conjunctival resection.

PURPOSE: To analyze the conjunctival changes, especially goblet cell populations, following Muller's muscle conjunctival resection (MMCR) by histologically evaluating pre and post-MMCR specimens. METHODS: This is a retrospective analysis of conjunctival samples sent for histologic evaluation from two patient populations: those who had previously undergone a MMCR and required repeat surgery and controls who underwent a MMCR surgery in a previously unoperated eyelid. Specimens underwent hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining to accentuate goblet cell identification and were evaluated by two ocular pathologists to quantify goblet cell populations and note other anatomical changes. Statistical analysis of goblet cell populations was then performed. RESULTS: Four samples were identified for each group: (1) post-MMCR and (2) control. The mean age was 67 years in the post-MMCR group and 66 years in the control group. The mean goblet cell population was 7 ± 5 cells/mm in the post-MMCR conjunctiva and was 39 ± 16 cells/mm in the control group, which was statistically significant (p = 0.01). Samples from both groups demonstrated scarring and inflammatory cell infiltrate. CONCLUSIONS: While there was a relative loss of goblet cell populations in the conjunctiva overlying the region of surgery following MMCR, the lack of dry eye symptoms or changes in tear production reported in prior studies suggests that there may be enough goblet cell population reserve in the remaining accessory lacrimal glands and in the unaltered conjunctiva to provide sufficient lubrication and ocular protection.

Epigenetic Reader BRD4 (Bromodomain-Containing Protein 4) Governs Nucleus-Encoded Mitochondrial Transcriptome to Regulate Cardiac Function.

BACKGROUND: BET (bromodomain and extraterminal) epigenetic reader proteins, in particular BRD4 (bromodomain-containing protein 4), have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small-molecule BET protein inhibitors such as JQ1 have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and to unveil potential pharmacological side effects. METHODS: By engineering a cardiomyocyte-specific BRD4 knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analyses to evaluate BRD4 function in developing and mature hearts. RESULTS: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, estrogen-related receptor α, a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. CONCLUSIONS: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function.

Genome-wide analyses as part of the international FTLD-TDP whole-genome sequencing consortium reveals novel disease risk factors and increases support for immune dysfunction in FTLD.

Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) represents the most common pathological subtype of FTLD. We established the international FTLD-TDP whole-genome sequencing consortium to thoroughly characterize the known genetic causes of FTLD-TDP and identify novel genetic risk factors. Through the study of 1131 unrelated Caucasian patients, we estimated that C9orf72 repeat expansions and GRN loss-of-function mutations account for 25.5% and 13.9% of FTLD-TDP patients, respectively. Mutations in TBK1 (1.5%) and other known FTLD genes (1.4%) were rare, and the disease in 57.7% of FTLD-TDP patients was unexplained by the known FTLD genes. To unravel the contribution of common genetic factors to the FTLD-TDP etiology in these patients, we conducted a two-stage association study comprising the analysis of whole-genome sequencing data from 517 FTLD-TDP patients and 838 controls, followed by targeted genotyping of the most associated genomic loci in 119 additional FTLD-TDP patients and 1653 controls. We identified three genome-wide significant FTLD-TDP risk loci: one new locus at chromosome 7q36 within the DPP6 gene led by rs118113626 (p value = 4.82e - 08, OR = 2.12), and two known loci: UNC13A, led by rs1297319 (p value = 1.27e - 08, OR = 1.50) and HLA-DQA2 led by rs17219281 (p value = 3.22e - 08, OR = 1.98). While HLA represents a locus previously implicated in clinical FTLD and related neurodegenerative disorders, the association signal in our study is independent from previously reported associations. Through inspection of our whole-genome sequence data for genes with an excess of rare loss-of-function variants in FTLD-TDP patients (n ≥ 3) as compared to controls (n = 0), we further discovered a possible role for genes functioning within the TBK1-related immune pathway (e.g., DHX58, TRIM21, IRF7) in the genetic etiology of FTLD-TDP. Together, our study based on the largest cohort of unrelated FTLD-TDP patients assembled to date provides a comprehensive view of the genetic landscape of FTLD-TDP, nominates novel FTLD-TDP risk loci, and strongly implicates the immune pathway in FTLD-TDP pathogenesis.

Foreign-Body Granulomata Caused by Injected Permanent Filler Masquerading as Cutaneous Sarcoidosis.

Foreign-body granuloma formation following filler injections is most commonly seen with permanent fillers; these reactions can occur years following the injections and often require either an intralesional steroid injection or surgical excision. The authors present a case of a 75-year-old woman with a history of systemic sarcoidosis previously treated with numerous immunosuppressive medications who was examined for bilateral infraorbital nodules and swelling that were unresponsive to treatment. She underwent a bilateral anterior orbitotomy through a transconjunctival approach with mass excision. The histologic analysis was consistent with foreign-body granulomata juxtaposed to implantable material, specifically ArteFill, which was injected many years prior. There were no separate noncaseating granulomas to suggest sarcoidosis as the underlying etiology. It is important to consider prior filler injections in patients with sarcoidosis who present with subcutaneous nodules as this changes management and may prevent the need for more aggressive immunosuppressive treatment.

A rare case of atypical pleomorphic adenoma arising from periocular ectopic lacrimal gland.

PURPOSE: To describe features of atypical pleomorphic adenoma, a rare clinical entity, particularly when found in ectopic periocular lacrimal gland tissue. METHODS: Case report of biopsy-confirmed periocular atypical pleomorphic adenoma. RESULTS: A 35-year-old female presented with a unique orbital lesion found to be ectopic lacrimal gland demonstrating atypical pleomorphic adenoma on formal histopathologic review. Pleomorphic adenoma is pathologically characterized as an epithelial lesion intermixed with mesenchymal elements. It is further classified as atypical with the presence of features such as hypercellularity, regions of necrosis or hyalinization, cellular dysplasia, capsular violation, and malignant characteristics without frank local extension or distant metastases. CONCLUSIONS: Due to its rarity, the natural history and prognosis of atypical pleomorphic adenoma is unclear. Physicians need to recognize this entity, and complete surgical excision with strict follow-up regimens are likely warranted.

Unusual Location for a Periorbital Dermoid Cyst.

Dermoid cysts in the orbit classically present in children as a mass in the superotemporal or superonasal orbit along the zygomatico-frontal or fronto-ethmoidal suture lines. The presence of a dermoid cyst in the superficial eyelid, not associated with the tarsus, has only been reported once previously. The authors present a case of a 60-year-old man with a painless right lower eyelid mass inferonasally that was completely excised and found to be a dermoid cyst. The presence of a dermoid cyst involving the superficial lower eyelid is very rare. Furthermore, dermoid cysts in adults typically present in the setting of trauma, which was absent in this case. Therefore, it is important to consider dermoid cysts in the differential diagnosis of eyelid lesions in adults.

Amyloid fibril polymorphism in the heart of an ATTR amyloidosis patient with polyneuropathy attributed to the V122Δ variant.

ATTR amyloidosis is a phenotypically heterogeneous disease characterized by the pathological deposition of transthyretin in the form of amyloid fibrils into various organs. ATTR amyloidosis may stem from mutations in variant (ATTRv) amyloidosis, or aging in wild-type (ATTRwt) amyloidosis. ATTRwt generally manifests as a cardiomyopathy phenotype, whereas ATTRv may present as polyneuropathy, cardiomyopathy, or mixed, in combination with many other symptoms deriving from secondary organ involvement. Over 130 different mutational variants of transthyretin have been identified, many of them being linked to specific disease symptoms. Yet, the role of these mutations in the differential disease manifestation remains elusive. Using cryo-electron microscopy, here we structurally characterized fibrils from the heart of an ATTRv patient carrying the V122Δ mutation, predominantly associated with polyneuropathy. Our results show that these fibrils are polymorphic, presenting as both single and double filaments. Our study alludes to a structural connection contributing to phenotypic variation in ATTR amyloidosis, as polymorphism in ATTR fibrils may manifest in patients with predominantly polyneuropathic phenotypes.

Multi-organ structural homogeneity of amyloid fibrils in ATTRv-T60A amyloidosis patients, revealed by Cryo-EM.

ATTR amyloidosis is a degenerative disorder characterized by the systemic deposition of the protein transthyretin. These amyloid aggregates of transthyretin (ATTR) can deposit in different parts of the body causing diverse clinical manifestations. Our laboratory aims to investigate a potential relationship between the different genotypes, organ of deposition, clinical phenotypes, and the structure of ATTR fibrils. Using cryo-electron microscopy, we have recently described how the neuropathic related mutations ATTRv-I84S and ATTRv-V122∆ can drive structural polymorphism in ex vivo fibrils. Here we question whether the mutation ATTRv-T60A, that commonly triggers cardiac and neuropathic symptoms, has a similar effect. To address this question, we extracted and determined the structure of ATTR-T60A fibrils from multiple organs (heart, thyroid, kidney, and liver) from the same patient and from the heart of two additional patients. We have found a consistent conformation among all the fibril structures, acquiring the "closed-gate morphology" previously found in ATTRwt and others ATTRv related to cardiac or mixed manifestations. The closed-gate morphology is composed by two segments of the protein that interact together forming a polar channel, where the residues glycine 57 to isoleucine 68 act as a gate of the polar cavity. Our study indicates that ATTR-T60A fibrils present in peripheral organs adopt the same structural conformation in all patients, regardless of the organ of deposition.

Cancer Cachexia in STK11/LKB1 -mutated NSCLC is Dependent on Tumor-secreted GDF15.

Cachexia is a wasting syndrome comprised of adipose, muscle, and weight loss observed in cancer patients. Tumor loss-of-function mutations in STK11/LKB1 , a regulator of the energy sensor AMP-activated protein kinase, induce cancer cachexia (CC) in preclinical models and are associated with cancer-related weight loss in NSCLC patients. Here we characterized the relevance of the NSCLC-associated cachexia factor growth differentiation factor 15 (GDF15) in several patient-derived and genetically engineered STK11/LKB1 -mutant NSCLC cachexia lines. Both tumor mRNA expression and serum concentrations of tumor-derived GDF15 were significantly elevated in multiple mice transplanted with patient-derived STK11/LKB1 -mutated NSCLC lines. GDF15 neutralizing antibody administered to mice transplanted with patient- or mouse-derived STK11/LKB1 -mutated NSCLC lines suppressed cachexia-associated adipose loss, muscle atrophy, and changes in body weight. The silencing of GDF15 in multiple human NSCLC lines was also sufficient to eliminate in vivo circulating GDF15 levels and abrogate cachexia induction, suggesting that tumor and not host tissues represent a key source of GDF15 production in these cancer models. Finally, reconstitution of wild-type STK11/LKB1 in a human STK11/LKB1 loss-of-function NSCLC line that normally induces cachexia in vivo correlated with the absence of tumor-secreted GDF15 and rescue from the cachexia phenotype. The current data provide evidence for tumor-secreted GDF15 as a conduit and a therapeutic target through which NSCLCs with STK11/LKB1 loss-of-function mutations promote cachexia-associated wasting.

Dysregulation of FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis.

Bi-functional enzyme FicD regulates the endoplasmic reticulum chaperone BiP using AMPylation and deAMPylation during ER homeostasis and stress, respectively. Human FicD with an arginine-to-serine mutation disrupts FicD deAMPylation activity resulting in severe neonatal diabetes. We generated the FicD R371S mutation in mice to create a pre-clinical murine model for neonatal diabetes. We observed elevated BiP AMPylation levels across multiple tissues and signature markers for diabetes including glucose intolerance and reduced serum insulin levels. While the pancreas of FicD R371S mice appeared normal at birth, adult FicD R371S mice displayed disturbed pancreatic islet organization that progressed with age. FicD R371S mice provide a preclinical mouse model for the study of UPR associated diabetes and demonstrate the essentiality of FicD for tissue resilience.