Ten-years outcome analysis in patients with clinically localized prostate cancer treated by radical prostatectomy or external beam radiation therapy.

Purpose: Since there was no consensus on treatment options for localized prostate cancer, we performed a retrospective study to compare the long-term survival benefit of radiotherapy (RT) versus laparoscopic radical prostatectomy (LRP) in Taiwan. Methods: 218 patients with clinically localized prostate cancer treated between 2008 and 2017 (64 with LRP and 154 with RT) were enrolled in this study. The outcomes of RT and LRP were assessed after patients were stratified according to Gleason score, stage, and risk group. Crude survival, prostate cancer-specific survival, and metastasis-free survival were evaluated using the log-rank test. Results: The 5-year crude survival rate was 93.3% in the LRP group and 59.3% in the RT group. A significant survival benefit was found in the LRP group compared with the RT group (p = 0.004). Furthermore, significant differences were found in disease-specific survival (93.3% vs. 64.7%, p = 0.022) and metastasis-free survival (48% vs. 40.2%, p = 0.045) between the LRP and RT groups. Conclusions: Men with localized prostate cancer treated initially with LRP had a lower risk of prostate cancer-specific death and metastases compared with those treated with RT.

Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

Clinical Characteristics of Actinomyces viscosus Bacteremia.

Background and Objectives: Actinomyces species are part of the normal flora of humans and rarely cause disease. It is an uncommon cause of disease in humans. The clinical features of actinomycosis have been described, and various anatomical sites (such as face, bones and joints, respiratory tract, genitourinary tract, digestive tract, central nervous system, skin, and soft tissue structures) can be affected. It is not easy to identify actinomycosis because it sometimes mimics cancer due to under-recognition. As new diagnostic methods have been applied, Actinomyces can now more easily be identified at the species level. Recent studies have also highlighted differences among Actinomyces species. We report a case of Actinomyces viscosus bacteremia with cutaneous actinomycosis. Materials and Methods: A 66 years old male developed fever for a day with progressive right lower-leg erythematous swelling. Blood culture isolates yielded Actinomyces species, which was identified as Actinomyces viscosus by sequencing of the 16S rRNA gene. In addition, we searched for the term Actinomyces or actinomycosis cross-referenced with bacteremia or "blood culture" or "blood stream" from January 2010 to July 2020. The infectious diseases caused by species of A. viscosus from January 1977 to July 2020 were also reviewed. Results: The patient recovered well after intravenous ampicillin treatment. Poor oral hygiene was confirmed by dental examination. There were no disease relapses during the following period. Most cases of actinomycosis can be treated with penicillin. However, clinical alertness, risk factor evaluation, and identification of Actinomyces species can prevent inappropriate antibiotic or intervention. We also compiled a total of 18 cases of Actinomyces bacteremia after conducting an online database search. Conclusions: In summary, we describe a case of fever and progressive cellulitis. Actinomyces species was isolated from blood culture, which was further identified as Actinomyces viscosus by 16S rRNA sequencing. The cellulitis improved after pathogen-directed antibiotics. Evaluation of risk factors in patients with Actinomyces bacteremia and further identification of the Actinomyces species are recommended for successful treatment.

The Transition Zone Protein AHI1 Regulates Neuronal Ciliary Trafficking of MCHR1 and Its Downstream Signaling Pathway.

The Abelson-helper integration site 1 (AHI1) gene encodes for a ciliary transition zone localizing protein that when mutated causes the human ciliopathy, Joubert syndrome. We prepared and examined neuronal cultures derived from male and female embryonic Ahi1+/+ and Ahi1-/- mice (littermates) and found that the distribution of ciliary melanin-concentrating hormone receptor-1 (MchR1) was significantly reduced in Ahi1-/- neurons; however, the total and surface expression of MchR1 on Ahi1-/- neurons was similar to controls (Ahi1+/+). This indicates that a pathway for MchR1 trafficking to the surface plasma membrane is intact, but the process of targeting MchR1 into cilia is impaired in Ahi1-deficient mouse neurons, indicating a role for Ahi1 in localizing MchR1 to the cilium. Mouse Ahi1-/- neurons that fail to accumulate MchR1 in the ciliary membrane have significant decreases in two downstream MchR1 signaling pathways [cAMP and extracellular signal-regulated kinase (Erk)] on MCH stimulation. These results suggest that the ciliary localization of MchR1 is necessary and critical for MchR1 signaling, with Ahi1 participating in regulating MchR1 localization to cilia, and further supporting cilia as critical signaling centers in neurons.SIGNIFICANCE STATEMENT Our work here demonstrates that neuronal primary cilia are powerful and focused signaling centers for the G-protein-coupled receptor (GPCR), melanin-concentrating hormone receptor-1 (MCHR1), with a role for the ciliary transition zone protein, Abelson-helper integration site 1 (AHI1), in mediating ciliary trafficking of MCHR1. Moreover, our manuscript further expands the repertoire of cilia functions on neurons, a cell type that has not received significant attention in the cilia field. Lastly, our work demonstrates the significant influence of ciliary GPCR signaling in the overall signaling of neurons.

OTULIN limits cell death and inflammation by deubiquitinating LUBAC.

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.

Crystal Structure of Ripk4 Reveals Dimerization-Dependent Kinase Activity.

Receptor-interacting protein kinase 4 (RIPK4) is a highly conserved regulator of epidermal differentiation. Members of the RIPK family possess a common kinase domain as well as unique accessory domains that likely dictate subcellular localization and substrate preferences. Mutations in human RIPK4 manifest as Bartsocas-Papas syndrome (BPS), a genetic disorder characterized by severe craniofacial and limb abnormalities. We describe the structure of the murine Ripk4 (MmRipk4) kinase domain, in ATP- and inhibitor-bound forms. The crystallographic dimer of MmRipk4 is similar to those of RIPK2 and BRAF, and we show that the intact dimeric entity is required for MmRipk4 catalytic activity through a series of engineered mutations and cell-based assays. We also assess the impact of BPS mutations on protein structure and activity to elucidate the molecular origins of the disease.

The Joubert syndrome-associated missense mutation (V443D) in the Abelson-helper integration site 1 (AHI1) protein alters its localization and protein-protein interactions.

BACKGROUND: Missense mutations in AHI1 result in the neurodevelopmental ciliopathy called Joubert syndrome. RESULTS: Mutations in AHI1 decrease cilia formation, alter its localization and stability, and change its binding to HAP1 and NPHP1. CONCLUSION: Mutations in AHI1 affect ciliogenesis, AHI1 protein localization, and AHI1-protein interactions. SIGNIFICANCE: This study begins to describe how missense mutations in AHI1 can cause Joubert syndrome. Mutations in AHI1 cause Joubert syndrome (JBTS), a neurodevelopmental ciliopathy, characterized by midbrain-hindbrain malformations and motor/cognitive deficits. Here, we show that primary cilia (PC) formation is decreased in fibroblasts from individuals with JBTS and AHI1 mutations. Most missense mutations in AHI1, causing JBTS, occur in known protein domains, however, a common V443D mutation in AHI1 is found in a region with no known protein motifs. We show that cells transfected with AHI1-V443D, or a new JBTS-causing mutation, AHI1-R351L, have aberrant localization of AHI1 at the basal bodies of PC and at cell-cell junctions, likely through decreased binding of mutant AHI1 to NPHP1 (another JBTS-causing protein). The AHI1-V443D mutation causes decreased AHI1 stability because there is a 50% reduction in AHI1-V443D protein levels compared with wild type AHI1. Huntingtin-associated protein-1 (Hap1) is a regulatory protein that binds Ahi1, and Hap1 knock-out mice have been reported to have JBTS-like phenotypes, suggesting a role for Hap1 in ciliogenesis. Fibroblasts and neurons with Hap1 deficiency form PC with normal growth factor-induced ciliary signaling, indicating that the Hap1 JBTS phenotype is likely not through effects at PC. These results also suggest that the binding of Ahi1 and Hap1 may not be critical for ciliary function. However, we show that HAP1 has decreased binding to AHI1-V443D indicating that this altered binding could be responsible for the JBTS-like phenotype through an unknown pathway. Thus, these JBTS-associated missense mutations alter their subcellular distribution and protein interactions, compromising functions of AHI1 in cell polarity and cilium-mediated signaling, thereby contributing to JBTS.

Retinal degeneration and failure of photoreceptor outer segment formation in mice with targeted deletion of the Joubert syndrome gene, Ahi1.

Vertebrate photoreceptors have a modified cilium composed of a basal body, axoneme and outer segment. The outer segment includes stacked membrane discs, containing opsin and the signal transduction apparatus mediating phototransduction. In photoreceptors, two distinct classes of vesicles are trafficked. Synaptic vesicles are transported down the axon to the synapse, whereas opsin-containing vesicles are transported to the outer segment. The continuous replacement of the outer segments imposes a significant biosynthetic and trafficking burden on the photoreceptors. Here, we show that Ahi1, a gene that when mutated results in the neurodevelopmental disorder, Joubert syndrome (JBTS), is required for photoreceptor sensory cilia formation and the development of photoreceptor outer segments. In mice with a targeted deletion of Ahi1, photoreceptors undergo early degeneration. Whereas synaptic proteins are correctly trafficked, photoreceptor outer segment proteins fail to be transported appropriately or are significantly reduced in their expression levels (i.e., transducin and Rom1) in Ahi1(-/-) mice. We show that vesicular targeting defects in Ahi1(-/-) mice are cilium specific, and our evidence suggests that the defects are caused by a decrease in expression of the small GTPase Rab8a, a protein required for accurate polarized vesicular trafficking. Thus, our results suggest that Ahi1 plays a role in stabilizing the outer segment proteins, transducin and Rom1, and that Ahi1 is an important component of Rab8a-mediated vesicular trafficking in photoreceptors. The retinal degeneration observed in Ahi1(-/-) mice recapitulates aspects of the retinal phenotype observed in patients with JBTS and suggests the importance of Ahi1 in photoreceptor function.

Ahi1, whose human ortholog is mutated in Joubert syndrome, is required for Rab8a localization, ciliogenesis and vesicle trafficking.

The primary non-motile cilium, a membrane-ensheathed, microtubule-bundled organelle, extends from virtually all cells and is important for development. Normal functioning of the cilium requires proper axoneme assembly, membrane biogenesis and ciliary protein localization, in tight coordination with the intraflagellar transport system and vesicular trafficking. Disruptions at any level can induce severe alterations in cell function, giving rise to a myriad of human genetic diseases known as ciliopathies. Here we show that the Abelson helper integration site 1 (Ahi1) gene, whose human ortholog is mutated in Joubert syndrome, regulates cilium formation via its interaction with Rab8a, a small GTPase critical for polarized membrane trafficking. We find that the Ahi1 protein localizes to a single centriole, the mother centriole, which becomes the basal body of the primary cilium. In order to determine whether Ahi1 functions in ciliogenesis, loss of function analysis of Ahi1 was performed in cell culture models of ciliogenesis. Knockdown of Ahi1 expression by shRNAi in cells or targeted deletion of Ahi1 (Ahi1 knockout mouse) leads to impairments in ciliogenesis. In Ahi1-knockdown cells, Rab8a is destabilized and does not properly localize to the basal body. Since Rab8a is implicated in vesicular trafficking, we next examined this process in Ahi1-knockdown cells. Defects in the trafficking of endocytic vesicles from the plasma membrane to the Golgi and back to the plasma membrane were observed in Ahi1-knockdown cells. Overall, our data indicate that the distribution and functioning of Rab8a is regulated by Ahi1, not only affecting cilium formation, but also vesicle transport.

Species differences in the expression of Ahi1, a protein implicated in the neurodevelopmental disorder Joubert syndrome, with preferential accumulation to stigmoid bodies.

Joubert syndrome (JBTS) is an autosomal recessive disorder characterized by cerebellum and brainstem malformations. Individuals with JBTS have abnormal breathing and eye movements, ataxia, hypotonia, and cognitive difficulty, and they display mirror movements. Mutations in the Abelson-helper integration site-1 gene (AHI1) cause JBTS in humans, suggesting that AHI1 is required for hindbrain development; however AHI1 may also be required for neuronal function. Support for this idea comes from studies demonstrating that the AHI1 locus is associated with schizophrenia. To gain further insight into the function of AHI1 in both the developing and mature central nervous system, we determined the spatial and temporal expression patterns of the gene products of AHI1 orthologs throughout development, in human, mouse, and zebrafish. Murine Ahi1 was distributed throughout the cytoplasm, dendrites, and axons of neurons, but was absent in glial cells. Ahi1 expression in the mouse brain was observed as early as embryonic day 10.5 and persisted into adulthood, with peak expression during the first postnatal week. Murine Ahi1 was observed in neurons of the hindbrain, midbrain, and ventral forebrain. Generally, the AHI1/Ahi1/ahi1 orthologs had a conserved distribution pattern in human, mouse, and zebrafish, but mouse Ahi1 was not present in the developing and mature cerebellum. Ahi1 was also observed consistently in the stigmoid body, a poorly characterized cytoplasmic organelle found in neurons. Overall, these results suggest roles for AHI1 in neurodevelopmental processes that underlie most of the neuroanatomical defects in JBTS, and perhaps in neuronal functions that contribute to schizophrenia.