Date: May 7, 2020

As COVID-19 continues to impact communities around the world, people and families everywhere are spending more time at home. In light of this, we’re launching a throwback Doodle series looking back at some of our popular interactive Google Doodle games!

Stay and play at home with today’s featured throwback: 

Our 2017 Doodle game celebrating the birth of Hip Hop!
 

Help stop the spread of COVID-19 by following these steps.  
 

Learn more here about the latest ways we’re responding, and how our products can help people stay connected during this time.

Location: Global

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Date: May 8, 2020

As COVID-19 continues to impact communities around the world, people and families everywhere are spending more time at home. In light of this, we’re launching a throwback Doodle series looking back at some of our popular interactive Google Doodle games!

Stay and play at home with today’s featured throwback: 

Our 2010 Doodle game celebrating PAC-MAN!
 

Help stop the spread of COVID-19 by following these steps.  
 

Learn more here about the latest ways we’re responding, and how our products can help people stay connected during this time.

Location: Global

Tags:

Date: May 9, 2020

Today’s Doodle celebrates British footballer and coach Frank Soo, a fearsome half-back and inside forward who broke racial barriers playing for the English national team during World War II. On this day in 1942, Soo made his international debut in a match against Wales, becoming the first person of non-European descent—and only ever of Asian heritage—to represent the country’s team at the highest level.

Frank Soo was born on March 8th, 1914 in Derbyshire, England and raised in Liverpool. He quickly earned a reputation as one of the best youth players in the city. At just 18 years old, he was scouted by Stoke City F.C., becoming the first professional player of Chinese ancestry in the English Football League.

Renowned for an artful playing style, dignified ball control, and precise passing, the charismatic Soo was named team captain at age 27 and rose to a level of national celebrity. In 1940, following the outbreak of World War II, Soo enlisted in the Royal Air Force and continued his legacy as the captain of its football team. Though England’s Football Association deemed its wartime matches unofficial, Soo went on to represent his country in nine such international fixtures by 1945.

Soo retired from playing in 1950 and moved on to a successful international coaching career that lasted over three decades. His legacy lives on in the increasingly diverse English team that plays today.

Thank you, Frank Soo, for showing the world the unifying power of sport. ​

Doodler Q&A with Matthew Cruickshank

Today’s Doodle was created by Doodler Matthew Cruickshank from North London.
Below, he shares some thoughts on the making of the Doodle:

 Q: What was your creative approach for this Doodle? Why did you choose this approach?

A: We found very old football trading cards online. This formed the basis of the Doodle, along with simple animation in order to celebrate Frank even more.

 Q: Did you draw inspiration from anything in particular for this Doodle?

A: I drew from my memories of being a very average footballer in England with a love of the game. But more importantly, I imagined how many barriers Frank broke down as a brilliant footballer in a minority. He took his talent to the pinnacle. 

 Q: What do you hope people will take away from this Doodle?

A: A love of football and a celebration of the diversity we have in the modern game today. Frank really played his part in achieving that.

Early concept sketches of the Doodle 

Location: United Kingdom

Tags: Animation, Sports, footballer, half-back, forward, soccer, football, English Football League

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Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson’s disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying K+ channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs) — not NMDA receptors. In so doing, amantadine enhances dendritic integration of excitatory synaptic potentials in SPNs and enhances — not antagonizes — the induction of long-term potentiation (LTP) at excitatory, axospinous synapses. Taken together, our studies suggest that the alleviation of LID in PD patients is mediated by diminishing the disparity in the excitability of direct- and indirect-pathway SPNs in the on state, rather than by disrupting LTP induction. This insight points to a pharmacological approach that could be used to effectively ameliorate LID and improve the quality of life for PD patients.

Although CEACAM1 (CC1) glycoprotein resides at the interface of immune liver injury and metabolic homeostasis, its role in orthotopic liver transplantation (OLT) remains elusive. We aimed to determine whether/how CEACAM1 signaling may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. In the mouse, donor liver CC1 null mutation augmented IRI-OLT (CC1-KO→WT) by enhancing ROS expression and HMGB1 translocation during cold storage, data supported by in vitro studies where hepatic flush from CC1-deficient livers enhanced macrophage activation in bone marrow–derived macrophage cultures. Although hepatic CC1 deficiency augmented cold stress–triggered ASK1/p-p38 upregulation, adjunctive ASK1 inhibition alleviated IRI and improved OLT survival by suppressing p-p38 upregulation, ROS induction, and HMGB1 translocation (CC1-KO→WT), whereas ASK1 silencing (siRNA) promoted cytoprotection in cold-stressed and damage-prone CC1-deficient hepatocyte cultures. Consistent with mouse data, CEACAM1 expression in 60 human donor liver biopsies correlated negatively with activation of the ASK1/p-p38 axis, whereas low CC1 levels associated with increased ROS and HMGB1 translocation, enhanced innate and adaptive immune responses, and inferior early OLT function. Notably, reduced donor liver CEACAM1 expression was identified as one of the independent predictors for early allograft dysfunction (EAD) in human OLT patients. Thus, as a checkpoint regulator of IR stress and sterile inflammation, CEACAM1 may be considered as a denominator of donor hepatic tissue quality, and a target for therapeutic modulation in OLT recipients.

Whether mutations in cancer driver genes directly affect cancer immune phenotype and T cell immunity remains a standing question. ARID1A is a core member of the polymorphic BRG/BRM-associated factor chromatin remodeling complex. ARID1A mutations occur in human cancers and drive cancer development. Here, we studied the molecular, cellular, and clinical impact of ARID1A aberrations on cancer immunity. We demonstrated that ARID1A aberrations resulted in limited chromatin accessibility to IFN-responsive genes, impaired IFN gene expression, anemic T cell tumor infiltration, poor tumor immunity, and shortened host survival in many human cancer histologies and in murine cancer models. Impaired IFN signaling was associated with poor immunotherapy response. Mechanistically, ARID1A interacted with EZH2 via its carboxyl terminal and antagonized EZH2-mediated IFN responsiveness. Thus, the interaction between ARID1A and EZH2 defines cancer IFN responsiveness and immune evasion. Our work indicates that cancer epigenetic driver mutations can shape cancer immune phenotype and immunotherapy.

The renaissance of complement diagnostics and therapeutics has introduced precision medicine into a widened field of complement-mediated diseases. In particular, complement-mediated diseases (or complementopathies) with ongoing or published clinical trials of complement inhibitors include paroxysmal nocturnal hemoglobinuria, cold agglutinin disease, hemolytic uremic syndrome, nephropathies, HELLP syndrome, transplant-associated thrombotic microangiopathy, antiphospholipid antibody syndrome, myasthenia gravis, and neuromyelitis optica. Recognizing that this field is rapidly expanding, we aim to provide a state-of-the-art review of (a) current understanding of complement biology for the clinician, (b) novel insights into complement with potential applicability to clinical practice, (c) complement in disease across various disciplines (hematology, nephrology, obstetrics, transplantation, rheumatology, and neurology), and (d) the potential future of precision medicine. Better understanding of complement diagnostics and therapeutics will not only facilitate physicians treating patients in clinical practice but also provide the basis for future research toward precision medicine in this field.

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.

Organ shortage continues to limit the lives of patients who require liver transplantation. While extending criteria for liver organs provides a needed resource, tissue damage from prolonged ischemic injury can result in early allograft dysfunction and consequent rejection. In this issue of the JCI, Nakamura et al. used a mouse transplantation model with prolonged ex vivo cold storage to explore liver graft protection. The authors found that liver grafts with absent carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) exhibited increased ischemia-reperfusion injury inflammation and decreased function in wild-type recipients. The authors went on to correlate CEACAM1 levels with postreperfusion damage in human liver transplant recipients. Notably, this study identified a potential biomarker for liver transplant donor graft quality.

Chronic pancreatitis (CP) is considered an irreversible fibroinflammatory pancreatic disease. Despite numerous animal model studies, questions remain about local immune characteristics in human CP. We profiled pancreatic immune cell characteristics in control organ donors and CP patients including those with hereditary and idiopathic CP undergoing total pancreatectomy with islet autotransplantation. Flow cytometric analysis revealed a significant increase in the frequency of CD68+ macrophages in idiopathic CP. In contrast, hereditary CP samples showed a significant increase in CD3+ T cell frequency, which prompted us to investigate the T cell receptor β (TCRβ) repertoire in the CP and control groups. TCRβ sequencing revealed a significant increase in TCRβ repertoire diversity and reduced clonality in both CP groups versus controls. Interestingly, we observed differences in Vβ-Jβ gene family usage between hereditary and idiopathic CP and a positive correlation of TCRβ rearrangements with disease severity scores. Immunophenotyping analyses in hereditary and idiopathic CP pancreases indicate differences in innate and adaptive immune responses, which highlights differences in immunopathogenic mechanisms of disease among subtypes of CP. TCR repertoire analysis further suggests a role for specific T cell responses in hereditary versus idiopathic CP pathogenesis, providing insights into immune responses associated with human CP.

Alterations in calcium signaling in pancreatic acinar cells can result in pancreatitis. Although pressure changes in the pancreas can elevate cytosolic calcium (Ca2+) levels, it is not known how transient pressure-activated elevations in calcium can cause prolonged calcium changes and consequent pancreatitis. In this issue of the JCI, Swain et al. describe roles for the mechanically activated plasma membrane calcium channels Piezo1 and transient receptor potential vanilloid subfamily 4 (TRPV4) in acinar cells. The authors used genetic deletion models and cell culture systems to investigate calcium signaling. Notably, activation of the Piezo1-dependent TRPV4 pathway was independent of the cholecystokinin (CCK) stimulation pathway. These results elegantly resolve an apparent discrepancy in calcium signaling and the pathogenesis of pancreatitis in pancreatic acinar cells.

Elevated pressure in the pancreatic gland is the central cause of pancreatitis following abdominal trauma, surgery, endoscopic retrograde cholangiopancreatography, and gallstones. In the pancreas, excessive intracellular calcium causes mitochondrial dysfunction, premature zymogen activation, and necrosis, ultimately leading to pancreatitis. Although stimulation of the mechanically activated, calcium-permeable ion channel Piezo1 in the pancreatic acinar cell is the initial step in pressure-induced pancreatitis, activation of Piezo1 produces only transient elevation in intracellular calcium that is insufficient to cause pancreatitis. Therefore, how pressure produces a prolonged calcium elevation necessary to induce pancreatitis is unknown. We demonstrate that Piezo1 activation in pancreatic acinar cells caused a prolonged elevation in intracellular calcium levels, mitochondrial depolarization, intracellular trypsin activation, and cell death. Notably, these effects were dependent on the degree and duration of force applied to the cell. Low or transient force was insufficient to activate these pathological changes, whereas higher and prolonged application of force triggered sustained elevation in intracellular calcium, leading to enzyme activation and cell death. All of these pathological events were rescued in acinar cells treated with a Piezo1 antagonist and in acinar cells from mice with genetic deletion of Piezo1. We discovered that Piezo1 stimulation triggered transient receptor potential vanilloid subfamily 4 (TRPV4) channel opening, which was responsible for the sustained elevation in intracellular calcium that caused intracellular organelle dysfunction. Moreover, TRPV4 gene–KO mice were protected from Piezo1 agonist– and pressure-induced pancreatitis. These studies unveil a calcium signaling pathway in which a Piezo1-induced TRPV4 channel opening causes pancreatitis.

Hepatocellular carcinoma (HCC) is clearly age-related and represents one of the deadliest cancer types worldwide. As a result of globally increasing risk factors including metabolic disorders, the incidence rates of HCC are still rising. However, the molecular hallmarks of HCC remain poorly understood. Neuropeptide Y (NPY) and NPY receptors represent a highly conserved, stress-activated system involved in diverse cancer-related hallmarks including aging and metabolic alterations, but its impact on liver cancer had been unclear. Here, we observed increased expression of NPY5 receptor (Y5R) in HCC, which correlated with tumor growth and survival. Furthermore, we found that its ligand NPY was secreted by peritumorous hepatocytes. Hepatocyte-derived NPY promoted HCC progression by Y5R activation. TGF-β1 was identified as a regulator of NPY in hepatocytes and induced Y5R in invasive cancer cells. Moreover, NPY conversion by dipeptidylpeptidase 4 (DPP4) augmented Y5R activation and function in liver cancer. The TGF-β/NPY/Y5R axis and DPP4 represent attractive therapeutic targets for controlling liver cancer progression.

BACKGROUND Neurofibroma/schwannoma hybrid nerve sheath tumors (N/S HNSTs) are neoplasms associated with larger nerves that occur sporadically and in the context of schwannomatosis or neurofibromatosis type 2 or 1. Clinical management of N/S HNSTs is challenging, especially for large tumors, and established systemic treatments are lacking.METHODS We used next-generation sequencing and array-based DNA methylation profiling to determine the clinically actionable genomic and epigenomic landscapes of N/S HNSTs.RESULTS Whole-exome sequencing within a precision oncology program identified an activating mutation (p.Asp769Tyr) in the catalytic domain of the ERBB2 receptor tyrosine kinase in a patient with schwannomatosis-associated N/S HNST, and targeted treatment with the small-molecule ERBB inhibitor lapatinib led to prolonged clinical benefit and a lasting radiographic and metabolic response. Analysis of a multicenter validation cohort revealed recurrent ERBB2 mutations (p.Leu755Ser, p.Asp769Tyr, p.Val777Leu) in N/S HNSTs occurring in patients who met diagnostic criteria for sporadic schwannomatosis (3 of 7 patients), but not in N/S HNSTs arising in the context of neurofibromatosis (6 patients) or outside a tumor syndrome (1 patient), and showed that ERBB2-mutant N/S HNSTs cluster in a distinct subgroup of peripheral nerve sheath tumors based on genome-wide DNA methylation patterns.CONCLUSION These findings uncover a key biological feature of N/S HNSTs that may have important diagnostic and therapeutic implications.FUNDING This work was supported by grant H021 from DKFZ-HIPO, the University Cancer Center Frankfurt, and the Frankfurt Research Funding Clinician Scientist Program.

An in-depth understanding of immune escape mechanisms in cancer is likely to lead to innovative advances in immunotherapeutic strategies. However, much remains unknown regarding these mechanisms and how they impact immunotherapy resistance. Using several preclinical tumor models as well as clinical specimens, we identified a mechanism whereby CD8+ T cell activation in response to programmed cell death 1 (PD-1) blockade induced a programmed death ligand 1/NOD-, LRR-, and pyrin domain–containing protein 3 (PD-L1/NLRP3) inflammasome signaling cascade that ultimately led to the recruitment of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) into tumor tissues, thereby dampening the resulting antitumor immune response. The genetic and pharmacologic inhibition of NLRP3 suppressed PMN-MDSC tumor infiltration and significantly augmented the efficacy of anti–PD-1 antibody immunotherapy. This pathway therefore represents a tumor-intrinsic mechanism of adaptive resistance to anti–PD-1 checkpoint inhibitor immunotherapy and is a promising target for future translational research.

Curing HIV infection will require the elimination of a reservoir of infected CD4+ T cells that persists despite HIV-specific cytotoxic T cell (CTL) responses. Although viral latency is a critical factor in this persistence, recent evidence also suggests a role for intrinsic resistance of reservoir-harboring cells to CTL killing. This resistance may have contributed to negative outcomes of clinical trials, where pharmacologic latency reversal has thus far failed to drive reductions in HIV reservoirs. Through transcriptional profiling, we herein identified overexpression of the prosurvival factor B cell lymphoma 2 (BCL-2) as a distinguishing feature of CD4+ T cells that survived CTL killing. We show that the inducible HIV reservoir was disproportionately present in BCL-2hi subsets in ex vivo CD4+ T cells. Treatment with the BCL-2 antagonist ABT-199 was not sufficient to drive reductions in ex vivo viral reservoirs when tested either alone or with a latency-reversing agent (LRA). However, the triple combination of strong LRAs, HIV-specific T cells, and a BCL-2 antagonist uniquely enabled the depletion of ex vivo viral reservoirs. Our results provide rationale for novel therapeutic approaches targeting HIV cure and, more generally, suggest consideration of BCL-2 antagonism as a means of enhancing CTL immunotherapy in other settings, such as cancer.

BACKGROUND The anti–programmed cell death 1 (anti–PD-1) antibody pembrolizumab is clinically active against non–small cell lung cancer (NSCLC). In addition to T cells, human natural killer (NK) cells, reported to have the potential to prolong the survival of patients with advanced NSCLC, also express PD-1. This study aimed to investigate the safety and efficacy of pembrolizumab plus allogeneic NK cells in patients with previously treated advanced NSCLC.METHODS In total, 109 enrolled patients with a programmed death ligand 1 (PD-L1) tumor proportion score (TPS) of 1% or higher were randomly allocated to group A (n = 55 patients given pembrolizumab plus NK cells) or group B (n = 54 patients given pembrolizumab alone). The patients received i.v. pembrolizumab (10 mg/kg) once every 3 weeks and continued treatment until the occurrence of tumor progression or unacceptable toxicity. The patients in group A continuously received 2 cycles of NK cell therapy as 1 course of treatment.RESULTS In our study, patients in group A had longer survival than did patients in group B (median overall survival [OS]: 15.5 months vs. 13.3 months; median progression-free survival [PFS]: 6.5 months vs. 4.3 months; P < 0.05). In group A patients with a TPS of 50% or higher, the median OS and PFS was significantly longer. Moreover, the patients in group A treated with multiple courses of NK cell infusion had better OS (18.5 months) than did those who received a single course of NK cell infusion (13.5 months).CONCLUSIONS Pembrolizumab plus NK cell therapy yielded improved survival benefits in patients with previously treated PD-L1+ advanced NSCLC.TRIAL REGISTRATION ClinicalTrials.gov NCT02843204.FUNDING This work was supported by grants from the National Natural Science Foundation of China (NSFC) – Guangdong Joint Foundation of China (no. U1601225); the NSFC (no. 81671965); the Guangdong Provincial Key Laboratory Construction Project of China (no. 2017B030314034); and the Key Scientific and Technological Program of Guangzhou City (no. 201607020016).

BACKGROUND Since December 2019, an outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, and is now becoming a global threat. We aimed to delineate and compare the immunological features of severe and moderate COVID-19.METHODS In this retrospective study, the clinical and immunological characteristics of 21 patients (17 male and 4 female) with COVID-19 were analyzed. These patients were classified as severe (11 cases) and moderate (10 cases) according to the guidelines released by the National Health Commission of China.RESULTS The median age of severe and moderate cases was 61.0 and 52.0 years, respectively. Common clinical manifestations included fever, cough, and fatigue. Compared with moderate cases, severe cases more frequently had dyspnea, lymphopenia, and hypoalbuminemia, with higher levels of alanine aminotransferase, lactate dehydrogenase, C-reactive protein, ferritin, and D-dimer as well as markedly higher levels of IL-2R, IL-6, IL-10, and TNF-α. Absolute numbers of T lymphocytes, CD4+ T cells, and CD8+ T cells decreased in nearly all the patients, and were markedly lower in severe cases (294.0, 177.5, and 89.0 × 106/L, respectively) than moderate cases (640.5, 381.5, and 254.0 × 106/L, respectively). The expression of IFN-γ by CD4+ T cells tended to be lower in severe cases (14.1%) than in moderate cases (22.8%).CONCLUSION The SARS-CoV-2 infection may affect primarily T lymphocytes, particularly CD4+ and CD8+ T cells, resulting in a decrease in numbers as well as IFN-γ production by CD4+ T cells. These potential immunological markers may be of importance because of their correlation with disease severity in COVID-19.TRIAL REGISTRATION This is a retrospective observational study without a trial registration number.FUNDING This work is funded by grants from Tongji Hospital for the Pilot Scheme Project, and partly supported by the Chinese National Thirteenth Five Years Project in Science and Technology for Infectious Disease (2017ZX10202201).

Treating neuropathic pain is challenging and novel non–opioid-based medicines are needed. Using unbiased receptomics, transcriptomic analyses, immunofluorescence, and in situ hybridization, we found that the expression of the orphan GPCR Gpr160 and GPR160 increased in the rodent dorsal horn of the spinal cord following traumatic nerve injury. Genetic and immunopharmacological approaches demonstrated that GPR160 inhibition in the spinal cord prevented and reversed neuropathic pain in male and female rodents without altering normal pain response. GPR160 inhibition in the spinal cord attenuated sensory processing in the thalamus, a key relay in the sensory discriminative pathways of pain. We also identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a GPR160 ligand. Inhibiting endogenous CARTp signaling in spinal cord attenuated neuropathic pain, whereas exogenous intrathecal CARTp evoked painful hypersensitivity through GPR160-dependent ERK and cAMP response element–binding protein (CREB). Our findings de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target, and provide insights into its signaling pathways. CARTp is involved in many diseases including depression and reward and addiction; de-orphanization of GPR160 is a major step forward understanding the role of CARTp signaling in health and disease.

IL-17–producing RORγt+ γδ T cells (γδT17 cells) are innate lymphocytes that participate in type 3 immune responses during infection and inflammation. Herein, we show that γδT17 cells rapidly proliferate within neonatal lymph nodes and gut, where, upon entry, they upregulate T-bet and coexpress IL-17, IL-22, and IFN-γ in a STAT3- and retinoic acid–dependent manner. Neonatal expansion was halted in mice conditionally deficient in STAT5, and its loss resulted in γδT17 cell depletion from all adult organs. Hyperactive STAT5 mutant mice showed that the STAT5A homolog had a dominant role over STAT5B in promoting γδT17 cell expansion and downregulating gut-associated T-bet. In contrast, STAT5B preferentially expanded IFN-γ–producing γδ populations, implying a previously unknown differential role of STAT5 gene products in lymphocyte lineage regulation. Importantly, mice lacking γδT17 cells as a result of STAT5 deficiency displayed a profound resistance to experimental autoimmune encephalomyelitis. Our data identify that the neonatal microenvironment in combination with STAT5 is critical for post-thymic γδT17 development and tissue-specific imprinting, which is essential for infection and autoimmunity.

BACKGROUND Preclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor.METHODS In this phase I trial, patients received either 1.5 × 106 MICs per kg BW on day –2 (n = 3, group A), or 1.5 × 108 MICs per kg BW on day –2 (n = 3, group B) or day –7 (n = 4, group C) before living donor kidney transplantation in addition to post-transplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360.RESULTS MIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 1010 donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19+CD24hiCD38hi transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature.CONCLUSION MIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs.TRIAL REGISTRATION EudraCT number: 2014-002086-30; ClinicalTrials.gov identifier: NCT02560220FUNDING Federal Ministry for Economic Affairs and Technology, Berlin, Germany, and TolerogenixX GmbH, Heidelberg, Germany.

Regenerative pain medicine, which seeks to harness the body’s own reparative capacity, is rapidly emerging as a field within pain medicine and orthopedics. It is increasingly appreciated that common analgesic mechanisms for these treatments depend on neuroimmune modulation. In this Review, we discuss recent progress in mechanistic understanding of nociceptive sensitization in chronic pain with a focus on neuroimmune modulation. We also examine the spectrum of regenerative outcomes, including preclinical and clinical outcomes. We further distinguish the analgesic mechanisms of regenerative therapies from those of cellular replacement, creating a conceptual and mechanistic framework to evaluate future research on regenerative medicine.

The mechanisms by which prostate cancer shifts from an indolent castration-sensitive phenotype to lethal castration-resistant prostate cancer (CRPC) are poorly understood. Identification of clinically relevant genetic alterations leading to CRPC may reveal potential vulnerabilities for cancer therapy. Here we find that CUB domain-containing protein 1 (CDCP1), a transmembrane protein that acts as a substrate for SRC family kinases (SFKs), is overexpressed in a subset of CRPC. Notably, CDCP1 cooperates with the loss of the tumor suppressor gene PTEN to promote the emergence of metastatic prostate cancer. Mechanistically, we find that androgens suppress CDCP1 expression and that androgen deprivation in combination with loss of PTEN promotes the upregulation of CDCP1 and the subsequent activation of the SRC/MAPK pathway. Moreover, we demonstrate that anti-CDCP1 immunoliposomes (anti–CDCP1 ILs) loaded with chemotherapy suppress prostate cancer growth when administered in combination with enzalutamide. Thus, our study identifies CDCP1 as a powerful driver of prostate cancer progression and uncovers different potential therapeutic strategies for the treatment of metastatic prostate tumors.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by loss of repression of the DUX4 gene; however, the DUX4 protein is rare and difficult to detect in human muscle biopsies, and pathological mechanisms are obscure. FSHD is also a chronic disease that progresses slowly over decades. We used the sporadic, low-level, muscle-specific expression of DUX4 enabled by the iDUX4pA-HSA mouse to develop a chronic long-term muscle disease model. After 6 months of extremely low sporadic DUX4 expression, dystrophic muscle presented hallmarks of FSHD histopathology, including muscle degeneration, capillary loss, fibrosis, and atrophy. We investigated the transcriptional profile of whole muscle as well as endothelial cells and fibroadiopogenic progenitors (FAPs). Strikingly, differential gene expression profiles of both whole muscle and, to a lesser extent, FAPs, showed significant overlap with transcriptional profiles of MRI-guided human FSHD muscle biopsies. These results demonstrate a pathophysiological similarity between disease in muscles of iDUX4pA-HSA mice and humans with FSHD, solidifying the value of chronic rare DUX4 expression in mice for modeling pathological mechanisms in FSHD and highlighting the importance FAPs in this disease.

Approximately half of the world’s population is infected with the stomach pathogen Helicobacter pylori. Infection with H. pylori is the main risk factor for distal gastric cancer. Bacterial virulence factors, such as the oncoprotein CagA, augment cancer risk. Yet despite high infection rates, only a fraction of H. pylori–infected individuals develop gastric cancer. This raises the question of defining the specific host and bacterial factors responsible for gastric tumorigenesis. To investigate the tumorigenic determinants, we analyzed gastric tissues from human subjects and animals infected with H. pylori bacteria harboring different CagA status. For laboratory studies, well-defined H. pylori strain B128 and its cancerogenic derivative strain 7.13, as well as various bacterial isogenic mutants were employed. We found that H. pylori compromises key tumor suppressor mechanisms: the host stress and apoptotic responses. Our studies showed that CagA induces phosphorylation of XIAP E3 ubiquitin ligase, which enhances ubiquitination and proteasomal degradation of the host proapoptotic factor Siva1. This process is mediated by the PI3K/Akt pathway. Inhibition of Siva1 by H. pylori increases survival of human cells with damaged DNA. It occurs in a strain-specific manner and is associated with the ability to induce gastric tumor.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A–dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non–muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C–null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A–dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

Seizures often herald the clinical appearance of gliomas or appear at later stages. Dissecting their precise evolution and cellular pathogenesis in brain malignancies could inform the development of staged therapies for these highly pharmaco-resistant epilepsies. Studies in immunodeficient xenograft models have identified local interneuron loss and excess glial glutamate release as chief contributors to network disinhibition, but how hyperexcitability in the peritumoral microenvironment evolves in an immunocompetent brain is unclear. We generated gliomas in WT mice via in utero deletion of key tumor suppressor genes and serially monitored cortical epileptogenesis during tumor infiltration with in vivo electrophysiology and GCAMP7 calcium imaging, revealing a reproducible progression from hyperexcitability to convulsive seizures. Long before seizures, coincident with loss of inhibitory cells and their protective scaffolding, gain of glial glutamate antiporter xCT expression, and reactive astrocytosis, we detected local Iba1+ microglial inflammation that intensified and later extended far beyond tumor boundaries. Hitherto unrecognized episodes of cortical spreading depolarization that arose frequently from the peritumoral region may provide a mechanism for transient neurological deficits. Early blockade of glial xCT activity inhibited later seizures, and genomic reduction of host brain excitability by deleting MapT suppressed molecular markers of epileptogenesis and seizures. Our studies confirmed xenograft tumor–driven pathobiology and revealed early and late components of tumor-related epileptogenesis in a genetically tractable, immunocompetent mouse model of glioma, allowing the complex dissection of tumor versus host pathogenic seizure mechanisms.

Immunosuppression continues to be a necessary component of transplantation, despite its association with a multitude of adverse effects. Numerous efforts have been made to circumvent the need for immunosuppression by using various techniques to achieve donor hyporesponsiveness. In this issue of the JCI, Morath et al. take this endeavor forward. Prior to transplantation, the researchers infused recipients with donor-modified immune cells and achieved immunologic hyporesponsiveness. This successful phase I trial also provides a possible avenue for achieving transplantation without the requisite immunosuppression.

BACKGROUND Glucose-6-phosphate dehydrogenase (G6PD) deficiency decreases the ability of red blood cells (RBCs) to withstand oxidative stress. Refrigerated storage of RBCs induces oxidative stress. We hypothesized that G6PD-deficient donor RBCs would have inferior storage quality for transfusion as compared with G6PD-normal RBCs.METHODS Male volunteers were screened for G6PD deficiency; 27 control and 10 G6PD-deficient volunteers each donated 1 RBC unit. After 42 days of refrigerated storage, autologous 51-chromium 24-hour posttransfusion RBC recovery (PTR) studies were performed. Metabolomics analyses of these RBC units were also performed.RESULTS The mean 24-hour PTR for G6PD-deficient subjects was 78.5% ± 8.4% (mean ± SD), which was significantly lower than that for G6PD-normal RBCs (85.3% ± 3.2%; P = 0.0009). None of the G6PD-normal volunteers (0/27) and 3 G6PD-deficient volunteers (3/10) had PTR results below 75%, a key FDA acceptability criterion for stored donor RBCs. As expected, fresh G6PD-deficient RBCs demonstrated defects in the oxidative phase of the pentose phosphate pathway. During refrigerated storage, G6PD-deficient RBCs demonstrated increased glycolysis, impaired glutathione homeostasis, and increased purine oxidation, as compared with G6PD-normal RBCs. In addition, there were significant correlations between PTR and specific metabolites in these pathways.CONCLUSION Based on current FDA criteria, RBCs from G6PD-deficient donors would not meet the requirements for storage quality. Metabolomics assessment identified markers of PTR and G6PD deficiency (e.g., pyruvate/lactate ratios), along with potential compensatory pathways that could be leveraged to ameliorate the metabolic needs of G6PD-deficient RBCs.TRIAL REGISTRATION ClinicalTrials.gov NCT04081272.FUNDING The Harold Amos Medical Faculty Development Program, Robert Wood Johnson Foundation grant 71590, the National Blood Foundation, NIH grant UL1 TR000040, the Webb-Waring Early Career Award 2017 by the Boettcher Foundation, and National Heart, Lung, and Blood Institute grants R01HL14644 and R01HL148151.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer, and there is great interest in applying inhibitors of HIF as anticancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors affect physiological responses to hypoxia at relevant doses. Here, we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumor growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatization and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerization with HIF-1β, did not respond to PT2385, indicating that these effects are on-target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerization with HIF-1β. Although PT2385 was well tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

BACKGROUND Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity.METHODS We treated 14 healthy women of diverse ethnicities (27.5 ± 1.1 years of age, BMI of 25.4 ± 1.2 kg/m2) with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for 4 weeks in an open-label study. The primary endpoint was the change in BAT metabolic activity as measured by [18F]-2-fluoro-d-2-deoxy-d-glucose (18F-FDG) PET/CT. Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by a frequently sampled intravenous glucose tolerance test.RESULTS Chronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers HDL and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has antidiabetic and antiinflammatory capabilities, increased with acute treatment and was 35% higher upon completion of the study. Finally, an intravenous glucose tolerance test revealed higher insulin sensitivity, glucose effectiveness, and insulin secretion.CONCLUSION These findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease.TRIAL REGISTRATION Clinicaltrials.gov NCT03049462.FUNDING This work was supported by grants from the Intramural Research Program of the NIDDK, NIH (DK075112, DK075116, DK071013, and DK071014).

The editors of JCI and JCI Insight are revisiting our editorial processes in light of the strain that the COVID-19 pandemic places on the worldwide scientific community. Here, we discuss adjustments to our decision framework in light of restrictions placed on laboratory working conditions for many of our authors.

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability–enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

Lipid-rich myelin forms electrically insulating, axon-wrapping multilayers that are essential for neural function, and mature myelin is traditionally considered metabolically inert. Surprisingly, we discovered that mature myelin lipids undergo rapid turnover, and quaking (Qki) is a major regulator of myelin lipid homeostasis. Oligodendrocyte-specific Qki depletion, without affecting oligodendrocyte survival, resulted in rapid demyelination, within 1 week, and gradually neurological deficits in adult mice. Myelin lipids, especially the monounsaturated fatty acids and very-long-chain fatty acids, were dramatically reduced by Qki depletion, whereas the major myelin proteins remained intact, and the demyelinating phenotypes of Qki-depleted mice were alleviated by a high-fat diet. Mechanistically, Qki serves as a coactivator of the PPARβ-RXRα complex, which controls the transcription of lipid-metabolism genes, particularly those involved in fatty acid desaturation and elongation. Treatment of Qki-depleted mice with PPARβ/RXR agonists significantly alleviated neurological disability and extended survival durations. Furthermore, a subset of lesions from patients with primary progressive multiple sclerosis were characterized by preferential reductions in myelin lipid contents, activities of various lipid metabolism pathways, and expression level of QKI-5 in human oligodendrocytes. Together, our results demonstrate that continuous lipid synthesis is indispensable for mature myelin maintenance and highlight an underappreciated role of lipid metabolism in demyelinating diseases.

BACKGROUND The live attenuated BPZE1 vaccine candidate induces protection against B. pertussis and prevents nasal colonization in animal models. Here we report on the responses in humans receiving a single intranasal administration of BPZE1.METHODS We performed multiple assays to dissect the immune responses induced in humans (n = 12) receiving BPZE1, with particular emphasis on the magnitude and characteristics of the antibody responses. Such responses were benchmarked to adolescents (n = 12) receiving the complete vaccination program of the currently used acellular pertussis vaccine (aPV). Using immunoproteomics analysis, potentially novel immunogenic B. pertussis antigens were identified.RESULTS All BPZE1 vaccinees showed robust B. pertussis–specific antibody responses with regard to significant increase in 1 or more of the following parameters: IgG, IgA, and memory B cells to B. pertussis antigens. BPZE1–specific T cells showed a Th1 phenotype, and the IgG exclusively consisted of IgG1 and IgG3. In contrast, all aPV vaccines showed a Th2-biased response. Immunoproteomics profiling revealed that BPZE1 elicited broader and different antibody specificities to B. pertussis antigens as compared with the aPV that primarily induced antibodies to the vaccine antigens. Moreover, BPZE1 was superior at inducing opsonizing antibodies that stimulated ROS production in neutrophils and enhanced bactericidal function, which was in line with the finding that antibodies against adenylate cyclase toxin were only elicited by BPZE1.CONCLUSION The breadth of the antibodies, the Th1-type cellular response, and killing mechanisms elicited by BPZE1 may hold prospects of improving vaccine efficacy and protection against B. pertussis transmission.TRIAL REGISTRATION ClinicalTrials.gov NCT02453048, NCT00870350.FUNDING ILiAD Biotechnologies, Swedish Research Council (Vetenskapsrådet), Swedish Heart-Lung Foundation.

BACKGROUND Beige adipose tissue is associated with improved glucose homeostasis in mice. Adipose tissue contains β3-adrenergic receptors (β3-ARs), and this study was intended to determine whether the treatment of obese, insulin-resistant humans with the β3-AR agonist mirabegron, which stimulates beige adipose formation in subcutaneous white adipose tissue (SC WAT), would induce other beneficial changes in fat and muscle and improve metabolic homeostasis.METHODS Before and after β3-AR agonist treatment, oral glucose tolerance tests and euglycemic clamps were performed, and histochemical analysis and gene expression profiling were performed on fat and muscle biopsies. PET-CT scans quantified brown adipose tissue volume and activity, and we conducted in vitro studies with primary cultures of differentiated human adipocytes and muscle.RESULTS The clinical effects of mirabegron treatment included improved oral glucose tolerance (P < 0.01), reduced hemoglobin A1c levels (P = 0.01), and improved insulin sensitivity (P = 0.03) and β cell function (P = 0.01). In SC WAT, mirabegron treatment stimulated lipolysis, reduced fibrotic gene expression, and increased alternatively activated macrophages. Subjects with the most SC WAT beiging showed the greatest improvement in β cell function. In skeletal muscle, mirabegron reduced triglycerides, increased the expression of PPARγ coactivator 1 α (PGC1A) (P < 0.05), and increased type I fibers (P < 0.01). Conditioned media from adipocytes treated with mirabegron stimulated muscle fiber PGC1A expression in vitro (P < 0.001).CONCLUSION Mirabegron treatment substantially improved multiple measures of glucose homeostasis in obese, insulin-resistant humans. Since β cells and skeletal muscle do not express β3-ARs, these data suggest that the beiging of SC WAT by mirabegron reduces adipose tissue dysfunction, which enhances muscle oxidative capacity and improves β cell function.TRIAL REGISTRATION Clinicaltrials.gov NCT02919176.FUNDING NIH: DK112282, P30GM127211, DK 71349, and Clinical and Translational science Awards (CTSA) grant UL1TR001998.

It’s no secret that I love Japan. I mean, I really, really love Japan. I felt more at home in Tokyo than I ever have anywhere else, and I think about going back all. the. time. I’m even thinking about it right now. You’re probably reading this right now because, at the very least, some tiny part of you is curious about whether you should do it. It might be the tiiiiiiiniest little part, but I’m sure it’s there. Maybe you don’t want to admit it because it seems pretty impossible, and yeah, I will admit that if you have a job that you don’t want to leave, strong family ties, kids, pets, or no money (among other things), it must seem like a distant what-if that will never happen. Here’s the thing. If you’re really, honestly interested, then make it happen. Because guess what? You freaking can make it happen, and don’t let anybody tell you no. If you’re coming up with a “But…” right now, I’ll stop you right there! “But I have kids/pets…” Figure out how to take them with you, because you can! “But I don’t want to leave my job…” Take a sabbatical for a year, look into transfers to a branch abroad, look for a better job in the same field in Japan, or look into whether this job is really worth giving up on this dream (maybe it isn’t). “But I can’t speak the language…” So? I moved to Japan and didn’t speak a word. Some people learn before they go, some people learn while they’re there (me), and some people never learn (I don’t recommend this). I could go on forever, but the whole world is at your fingers if you really want it! I seriously believe that. It’s not always easy, but if you want something badly enough, don’t you owe it to yourself to at least try? Anyways, let me give you the top reasons why I think that you should give living in Japan a try! 1. Living in a different culture opens your eyes. This especially is true if you immerse yourself in as much of the culture as you can. Make Japanese friends, learn about what people do on a daily basis and what they believe in. Try doing things in ways that are new to you. Try new foods! Mochi is the schiz, by the way! Once you’ve experienced doing new things, it will change how you do things even if you return back home. I will always have a no-shoes policy in my house (it’s so much cleaner!), I absolutely CRAVE a train system (if only!), and I have a newfound respect for walking and cycling. I never did this when I was little, but now, if I can, I walk! 2. You’ll have a fresh start. In your new home in Japan, you won’t have any of the drama that surrounded you in your old one. Thanks to the internet, we can still keep in touch with friends and family, but being a few thousand miles away from them will keep a lot of the drama to a minimum. Take a chance to stretch your wings and see what kind of person you are when you have the freedom to be you without their judgement. Trust me, it takes a weight off being in a new place where nobody knows who you used to be (or who they thought you used to be). Oh, and you know what? I bet that you will love yourself more than you ever did before. 3. Japan is a magical place! Seriously. Cherry blossoms, gorgeous temples and “castles” (I wouldn’t call them castles, but they’re called that nonetheless, and they’re really cool anyway), a rich history filled with Samurai and ninjas (who doesn’t love ninjas?), seasonal treats, and an entire culture that grew up reading manga. How does this not sound like an amazing place to live?!  And no offense to any other country, but Japanese trains come quickly, go almost everywhere, are extremely punctual, and pretty clean, which makes them (Tokyo especially) easily #1 in the world in public transportation. Now that sounds magical to me. 4. Universal Health Care. If you’re American like me, this will make a HUGE difference in your life. Trust me. If you come from pretty much any other 1st world nation, it probably won’t matter as much, though. But at least it’s good! 5. Japan is safer than where you came from. There’s no gun violence. There’s very low crime in general. You can walk in the dead of night in the seediest parts of town, as a woman, alone, and still feel perfectly safe from other people. From earthquakes is another matter, but you’ll get used to them really fast, and Japan is built to withstand all but the biggest. 6. Wa. There is a concept called wa in Japanese society, which essentially promotes practicing peace and harmony in your daily life. Wa is obvious in everything from traditional architecture and decor to the way that people act around each other– courteousness, quiet, and respect are what you expect most from your neighbors. You’re never going to wake up to your neighbors blaring music at 3am having a raucous party. Even drunken people wandering the street are more polite than not (although most of them just sort of stumble home or sit down where they are for the night– but remember, Japan is safe so they only thing they have to worry about is getting chilly). We could all use a little bit of harmony in our lives, and that’s something that Japan taught me to value. I’m surprised that yoga isn’t more popular, since they’re pretty in tune with each other. 7. All the new gadgets, and all of the old culture. Sure, Silicon Valley is where a lot of new apps are coming out, but if you want lots of little weird but useful gadgets to make your life easier (or more interesting), take a stroll through Akihabara. Plus, there are tons of cheap versions of what you’re used to, like large-capacity flash drives and SD cards. And I would be remiss in not mentioning the used electronics! Smartphones! Right next to small neighborhood temples, btw. It’s the only place to find Ayanami Rei in a kimono, wandering the street. The best of both worlds! 8. MANGA AND ANIME EVERYWHERE. This should be your main reason. This should be enough of a reason. Not only is it available everywhere, but events abound. If you wanted, you could go to an anime-related event every weekend of the year. Also, let’s not forget that it’s the only place to see all of the anime movies released in the theater, go to the official events (like Jump Festa, Comicket, World Cosplay Summit, and Anime Japan, among others), and see the musicals, seiyuu radio shows, and stage plays. If this isn’t reason enough, you’re probably in the wrong place. 9. It’s cheaper than you think. I lived in Tokyo, and then I moved back to the US, thinking that because I was living in a place often called “The Most Expensive City In The World,” it would be cheaper here. Nope.. Apartment rents, even in small cities, are at least the price that I was paying in Tokyo (~$600/mo). And try finding that in LA. So far I haven’t had any luck, and especially not in the areas that are actually sort-of-kind-of safe. Food is also about on-par with the US, especially domestic food. Considering that it’s an island, it’s actually really, really cheap. Food in Hawaii cost sometimes 3-4 times what I was able to get it for in Japan. Then, when you factor in healthcare, which is pretty cheap (what you pay for the insurance is based on your income, and then it covers 80% of all your bills — this is a simplification, but generally holds true), and transportation costs (you don’t need a car, therefore no gas, no insurance, no car maintenance fees), it’s downright cheap. Even living in Tokyo. 10. You will never run out of things to do. In nearly a decade, I never ran out of cool things to do. Can you say the same about the city that you live in now? Thought so. Ah man, I kinda feel ready to jump back on a plane and move across the ocean… three cats and all! Somebody hold me back… resistance is fading……………….  

☆ To see all of the parts in this series, click here ☆ I bet you thought I’d forgotten about this series, didn’t you! I never forget– sometimes I just lose steam, but I’m feeling good now, so lots of writing is coming! So, it was basically my 25th birthday. What did I write in my journal? It’s my 25th birthday in 3 days! :O So old!! I can’t believe that I ever wrote that… and also that it’s been more than ten years and I don’t feel any older. If anything, I feel younger! I have grown in a lot of ways, especially in the way that I relate to people around me and in how I take care of my health, and I guess I’ve matured in my general level of fangirlness…. No, wait. I’m still just as big a fangirl as ever, what am I saying? I’m still stalking the KuroKura tag on twitter every day, watching Lucifer mostly to see when he and Chloe are going to kiss or when he’s going to sleep with a guy again… but I digress! Let’s relive my fun first year in Japan instead! On my birthday, there happened to be a fanclub trip for Nagayama Takashi, the one and only actor whose fanclub I’ve ever been in. I went mostly because my friends A and M were going and to report on it for the internet at large, but it was really, really silly, and really, really fun! Well, silly is basically my #1 criteria for having fun, so that makes sense! We could all use more fun in our lives! The day started super early, and I was too excited to sleep, so I was dead tired. The event was scheduled to begin at 8:30 just outside of Tokyo station, where 288 girls (no boys, funny enough… actually, it’s not really funny) lined up in groups to get on one of the 7 waiting buses. Before that, we had to randomly draw for which tables we’d sit at, and were given this kind of horribly photoshopped and pixellated picture-card of Nagayan in front of Mt. Fuji that we were supposed to wear around our necks to identify ourselves as part of the tour (I wish that I still had it! How hilarious would that be!). On the back of the card were 4 empty squares and a little explanation that you were supposed to stamp them up along the way. Cool, sure! A, M, and I boarded bus 7 and ensconced ourselves in the back of the bus. It felt like everyone was staring at us, but well… we were the only non-japanese girls there. As the bus departed, a tour guide welcomed us over the PA explained how things were going to go. She said a little bit about the stamps, and then passed out fliers that we could use to order copies of the group pictures that we’d take later in the day with Nagayan (Wait, what did I do with that? I remember having a copy at some point). She also explained that our destination was going to be a mystery! Which, looking back, would have been a great setup for a horror movie! And theeeeeeen, we got the video. Oh, this video. Just watch for yourself (sorry for the bad quality, this was before smartphones, guys). There was a a short introduction from Nagayan, and then the song. And the dance. All of which he had created himself and wanted us to learn! The funny thing was that Nagayan is and was a serious actor. Outside of Tenimyu and Burimyu, his roles by and large were of and for adults. But that’s one of the things that makes Japan so great IMnot-so-humbleO– silliness is perfectly acceptable for adults! That given, I was still in the mindset of an American girl and in the beginning, my friends and I just sat there mouthing, “What the f—?” to each other. Sorry, Nagayan! It definitely grew on us, though! At about 10:15, we stopped at a rest stop for a potty break and a scavenger hunt! The tour guide told us to “get out and look for something special,” and that was it. It didn’t take long to find it, though, as by the time we’d wandered to the right location (the side of the building), there were quite a few girls lined up already! At the end of the line was Nagayan, sitting on the ground and wearing a green buddha mask. He didn’t say a word, so we followed the lead of the other girls, lined up, and said our prayers to Nagayan Buddha. For our piousness, we were allowed to choose a Chupa-Chups sucker to keep. It was so cute! Tell me if there is a single actor in the US who would do this? I doubt it! It’s certainly the kind of experience that you can only have in Japan XD Some girls looped around to get seconds, but ‘Buddha’ just pretended to be tired, slumping against the wall noiselessly. Btw, my sucker was a delicious strawberries and cream one!!! ♥ I ate it and tossed the wrapper…. which I wish that I’d kept for reasons that I’ll explain later! A and M on the bus next to me. We got back on the bus and left around 11:00. From there until the time that we arrived, there was no video, but this is where the IQ part of IQ TaiQ Barbequecame in! The tour guide administered us a timed IQ test on paper…… in Japanese! Er, yeah…. Well, I got one right at least!! Yay! Not bad for only being able to read a miniscule part of it. ^^;; I wrote “yomenai” (“I can’t read it” lit.-“It’s not readable”) in hiragana at the bottom, which later on I regretted doing! ^^;; The quiet scenery rolling by. After that, we had a bit of quiet time where I just watched the countryside and lakes go by and fell into a light sleep. At roughly 12:00, we pulled into our final mysterious mountain location! It looked a lot like any mountain location in eastern Washington or Arizona — a bit arid, but lots of trees and hills and of course a nice lake. We were lined up in lettered groups (There was from A to T, I believe. We were in group M) and we got to take the group pictures with Nagayan! It was fun– they had us sit/stand in 2 rows with Takashi in the very middle. Once we were already sitting down, he just kind of appeared and melded in with the group, and it took a while for some people to realize it, ourselves included haha! He asked us all to come closer (no problem!!) and the girls on his right completely freaked out, squealing ???? Japanese fangirls NEVER die. I love it! Right before we took the picture, Nagayan turned and asked if everyone was ready in Japanese, and then he turned to us and said loudly, “are you okay?” in English! Embvaaaarrrrasssing!! So, after that it was on to the BBQ! Outside of the BBQ, there were tables where we could turn in our order forms, as well as a special pocket mirror that you could buy with the Strawberry Meet logo on it. We entered the BBQ area, which was basically in back of a small resort, a BIG picnic-type area with numbered tables. There were 32 tables in all…. 32. Wow! On to the BBQ. To me, BBQ still meant hamburgers and hot dogs, chips and soda. To the Japanese, it meant that we cook our own yakiniku at the table! What a surprise for me at the time! My first time cooking yakiniku. Although I don’t eat meat anymore, I’d like to think that I’m a real pro at grilling eggplant now. ???? The three of us were at table 2 with 7 other girls. At first, it was a little awkward, and they didn’t really talk to us much, but they opened up when it turned out that we could understand a lot of what they were saying (A was especially awesome at Japanese), AND that one of them had done a homestay in America, so her English was pretty good. ^^ It turned out that not only was it my birthday, but it was also the birthday of another girl at our table!! Birthday twins!! So, the idea at this point was that Nagayan would randomly draw numbers and come around to visit and cook/eat with each of the tables. Ours came up surprisingly fast (I think we were the 7th table or so that he visited), and before we knew it, there he was, finding a space across the table from me (;_;) and helping us cook. Apparently we hadn’t been cooking fast enough for his liking, since we still had a LOT of meat and vegetables left, and so he started throwing all kinds of things on the grill. We told him that it was mine and the other girl’s birthday and he was like, ‘really?! Congratulations!!’ (He said it to me in English, but the other girl in Japanese) Then he told us that birthday people get priority, and put meat on both of our plates. Umm, yay. XD Soon after that, he reached over and gave me more and said “Present for me”….. Okay, that was the CUTEST thing I had probably ever seen in my life at the time! We tried to correct his English but I don’t think that he got it since he gave up really fast. He was REALLY cute, and even managed to ask us, “are you okay?” again for seemingly no reason. And then, sooner than we’d have liked, he was gone. ;__; We spent the rest of the BBQ listening to other tables talk to Nagayan and to the background music, which was of course our theme song. Some girls even stood up and started practicing the moves that had been on the video on the bus. Also randomly thrown in between the renditions were “Kokoro ga…” (his first single), and the other original song that he’d sang at his last fanclub event. After a few hours, we were told that it was time to get ready to leave, and to get back on the buses for the last part of our trip. Some girls practicing the dance… they were really good! This last part of our trip ended up being the craziest. They took us to a set of tennis courts (I’m sure this was a conscious choice) where they had us correct each other’s IQ tests. Oh man, this was HARD! But it was fun… if you’d gotten 9 or 10 right, you could get a stamp, although there were only a few girls that did. It seemed that, like me, that most people only had 1 or 2 right………. and they were native Japanese speakers that could read the test. I don’t feel so bad now. They also gave out stamps to people that had chosen strawberry suckers! This is where I wish that I had kept mine, though it didn’t really matter since I had chosen the wrong flavor. I was better suited to taking video at the time. Now, though, I think I would have just gone for it! After this, it was dance time! To my utter horror (I can’t dance ^^;;; except at Anime Expo but that’s another story), we had to get up in groups and perform the dance that we’d ‘learned.’ Nagayan led us each time, which was REALLY cute because he was REALLY into it, but it was still pretty nervewracking. It also took a LONG time to get through everyone. (almost 300 people doing it in about groups of 20). There was a lot of idle time. AND THEN, Nagayan had the best people get stamps and perform again for the little kids that had come up along the side...

☆ To see all of the parts in this series, click here ☆ Although it seemed as if my little fangirl heart couldn’t have taken any more, the day after the Nagayan Fanclub event, I went to see the Hyoutei myu for the first time. Just in case you’re reading this and going Huh? What’s a Hyoutei myu??, I can summarize by saying that it’s one in a series of musicals based on The Prince of Tennis. Yes, the manga that I ended up working on a few years later. ???? I was such a fangirl! Before I copy/paste my report on that musical, I want to say that the reason that I’m making this public again (they’re long gone now, and a lot of it was written privately) is because I want to show how much of a “silly,” squealing fangirl I was. I went from fan to insider purely because, later on, I took crazy-sounding chances and worked hard. And I sincerely believe that you, whomever you are, can do the same! By the way, all of this is eventually becoming fodder for an online comic that I’ve been formulating for a long time! No release date, though. I’m still working on Denkiki, this blog, my Youtube channel, and a few other things. And trying to put a new life together for myself. ???? In the meantime, let’s get to it! My friends and I ready for our dreams to come true, and our dreams were to see the Prince of Tennis musical! August 11, 2005 Tenimyu ~ Imperial Match Hyoutei!! This is the most detailed report on a musical that I’ve ever written. I’m going to do my best from now on to do the same each time!! (I did, for years) As Seigaku said, ♪♪DO YOUR BEST DO YOUR BEST!! DO YOUR BEST DO YOUR BEST!!♪♪ So far I’ve went to the opening performance of Hyoutei musical last night and to the second performance, which was tonight. ^^ I waited to write my report until seeing it a second time because I took very detailed notes the second time and added things and clarified things that the other people had seen afterwards! We had a tenimyu conference. =D (a tradition afterwards!!) It was a solid conclusion that cast improved a lot between the first and second performances. I can’t wait to see the last Tokyo performance, this Sunday. My bet is that they’re going to be VERY solid before then!! Oh wow. Deliciousness on a stick!! My overall feelings can be summed up in the chant that Hyoutei led, *clap clap* “Katsu no wa – Hyoutei! Makeru no Sei-gaku!!” 勝つのは氷帝！負けるの青学！！ (‘The winner will be Hyoutei! The loser will be Seigaku!’) Well, let’s start at what happened today before the musical!! Being hungry and having little other choice in the area for food, we went to the convenience store down the block from the musical… first off, I stopped and stared at this guy who was about to cross the road– who looked a lot like Ishibashi! But I couldn’t tell for sure because he’s changed his hair recently according to his picture set. He stared back at me, but it might have been because I was a tall red-headed foreigner wearing a Hyoutei jersey and a short black skirt.. ? LOL I don’t know if it was him, but I think that it was! He turned and looked back, and I think that he recognized me? I think.. maybe.. hmm..! He definitely recognized me yesterday, but that story is at the end of this report~ In the convenience store, while we are browsing the food sections, who do I notice has walked up alongside our aisle, but quite a few of the cast members! From what I remember, it was Yuu (Tezuka), Aiba (Fuji), Adachi (Kikumaru), Konishi (Kawamura), and Araki (Inui). Araki was wearing a cute hat so it took a minute to recognize him, and while most of the boys wandered along next to me without trouble in the aisle that I was in, Adachi stayed well away by the magazines. He was wearing a mask– which likely means that he has a cold!! Poor boy!! I only talked to Yuu– because I feel most comfortable talking to Yuu out of that group… ^^;; err, sorta. I (stupidly) shouted too loudly, “Yuu!!” when he came into my aisle, and of course he couldn’t ignore something like that. ^^;;; I feel a little bad, because I think that he gave me one of those, ‘why are you talking to me?’ looks. It’s not normal for a fan to actually talk to an actor here if they see them. I said, “hisashiburi!!’ (“It’s been a while!”) thinking that it’s been a while since I talked to him at all, to which he replied, “kino mita….” (“I saw you yesterday”)… Oh yeah…….. he’d obviously seen me at demachi yesterday (more later), and I felt kind of stupid and like I’d been really fannish, so I smiled and left him to do his things with the other boys. It wasn’t until later that I realized that I should have wished him good luck. Ah well, next time!! Itte yosh~! I probably acted way too familar, since we aren’t friends or anything, but it’s the way that I’ve always been with anyone that I’m fond of, friends or acquaintences. So it naturally carries off to them, without me even thinking of it. ^^; Ah well… in any case, I wish them all good luck for the future and rest of the performances!! On to the myu itself! (again! lol) Tenimyu ~ Imperial Match Hyoutei!! KENN special guest star. For both of these performances and for tomororw’s two shows also, KENN (Yuuta) is the special St. Rudolph guest star!! After that is Shiozawa for the next 3 days and Shinoda for the last day. I have tickets to shows with both of them (Thu and Sat for Shio, Sun of course for Shino), so I’m going to note the differences in separate posts. ^^ I can’t wait to see the differences!! ^____________________________^ This is my favorite~ I think that this is the best of all of the musicals. Hands down. Hyoutei was…. droolworthy. Hyoutei rocked the world, and they didn’t even show up for a little while. I’ll start at the beginning~ Tenimyu tenimyu tenimyu tenimyu tenimyu yaaay! <—— (me) First, as in all of the musicals, the curtain wa down andthere were suddenly the sounds of sneakers hitting the pavement. Cue the excitement level to rise and everyone to quiet down suddenly. The lights went down, and the curtain raised silently. A spotlight appeared off to the side, and onto the stage walks Sengoku (Wada) from the right, who does a short introduction. “Welcome,” etc. From the other side, lit with another spotlight, Yuuta (Kenn) made his entrance. They noticed each other and started to walk towards the center, but Yuuta suddenly stops as his cell phone goes off — with his ringer being Ore wa ore no namae de!! XDXDXD Wada starts to interrupt him, but his own phone goes off — with his ringer being Oretachi Jimi-su from bukimyu!! ROFLOL!! So hilarious!! They both managed to get off the phones shortly and walk over to each other, and began to argue — Sengoku asks Yuuta ‘So, who is the coolest player?’ Kenn begins right in saying that his buchou is the coolest, just look at him! And up above them on the screen appears a rather dorky shot of Akazawa and Kaneda from an earlier musical! Yuuta was embarrassed, saying, ‘ack!! I didn’t mean to put both of them up there!!’ Sengoku corrected him, saying, ‘My team is really cool’ — ‘Hey, who put that there?!’ Up on the screen had appeared a shot from Bukimyu of the Jimis mid-dance! Definitely NOT cool! XDXDXD In the end, they couldn’t decide who was the coolest, because music started up behind them…. Cue them to run off of stage and the second curtain to raise~ Starting with Fuji, all of Seigaku minus Ryoma took their places among the strong opening beats of the new Seigaku fight song, ♪Do Your Best! They did a long typical number for the kind of song- with lots of group singing and solos, the whole team going all out on their dancing just as they’d done for Bukimyu. Then they gathered in the center and Ryoma (Yanagi) was raised above! The crowd went noticeably more silent, this was the first time that Yanagi would be performing Ryoma on his own again!! How recovered WAS he? Yanagi didn’t move too much during this song, but he did do a bit of posing, and tried his very very best at singing. He still can’t sing well, but he’s obviously working on it. Hard. (So basically, he sounded terrible, but much better than he had at graduation myu!) Throughout the musical, Yanagi did very little dancing, and mostly posing. When he walked, he mostly walked without a limp, but there were a few times when it was noticeable, especially in the second performance. He could mostly manage it though, but he could not manage any kind of turns, as he stumbled slightly pretty much every time he tried. ;__;  Ganbarou Yanagi!! He’s improved a LOT!! Though there’s still a long road ahead, I think, he really seems to be trying hard. Everyone talked about it afterward and felt the same, very very respectful of what he must have to face and what he’s going through. What a strong boy~~ After Do Your Best, the lyrics consisting mostly of the team outlining their strengths and how they would try their hardest, Seigaku exited the stage and it all went dark. When the curtain raised again, the ichinen trio were sitting at desks on the left side of the stage, puzzling over the answers to their english test. Horio kept copying Kachiro, who shouted at him for it, and when they noticed us, they started in on another hilarious song, led by Horio, and beginning with english!! Horio belted out, “Hello, good day, how are you?” while Kachiro and Katsuo start chiming in. Kachiro seems to think that Horio is copying too much, so he stands up on the desk for his next refrain, and very very slashily (to me), Horio offers him a hand down. Then…….. they start a tap dance across stage that somehow incorporates the lyrics “A B C D”!! XDXD They have their usual display of awesome and hilarious dancing and suddenly rush off stage in the middle of the test when they realize that they’re about to miss the tennis team’s ranking tournament!! Most of Seigaku’s ranking matches are glossed over quickly, with the actors coming in and out quickly, pretending to hit a few balls or so. They give special attention to Ryoma vs. Oishi, after which Ryoma is congratulated for becoming a regular again. Next is the most important match, however. Inui vs. Momo and Inui vs. Tezuka. They do both matches at once, by having Inui stand on the right in the background, and Momo on the left in the background, with Tezuka in the front center (like a triangle). There is scary data-like music playing in the background (hard to describe.. it sounds like data). Inui is hitting balls and every other time that he hits a ball either Tezuka or Momo hits it. He finishes the Momo match first, and lo and behold ~ he beats Momo, who is no longer a regular because of it. Momo can’t believe it and stalks off stage. Then the Inu-Tez match is stopped, and the stage is resituated for a proper viewing of the rest of the match. The entire team is talking about how scary data tennis is, and I agree after seeing that. Inui is powerful in the musical! On to the real InuTez match. Inui tells Tezuka that he can break him with data tennis, because data doesn’t lie. In fact, after he counters Tezuka’s zero-shiki drop shot with data, he...

Being back in a cold and rainy climate reminds me of Tokyo. No, seriously. Washington has four seasons, just like Tokyo did, and just like Hawaii didn’t. I suppose that’s what has been making me feel really nostalgic these days. I’m in a place with the weather of Japan, but way less awesome. I have seriously owned around seventeen kajillion books about Japan in my lifetime, and I’ve given away, donated, or sold back almost the same amount. Some of them I bought, some of them were given to me, and I even found one or two. But the thing is that I have moved so many times that the only ones I’ve kept are those that I absolutely, positively, do not want to live without. (Well, maybe I could live without them, but then would I really be living?) Anyays! Right now, I only own three books in English about Japan, and these are them, and here is why I really like them:   1. The Otaku Encyclopedia: An Insider’s Guide to the Subculture of Cool Japan Disclaimer: A friend of mine wrote this, but that’s not why I’m recommending it.  Pat has written a bunch of books and papers, and they’re all great, but this is the one that I wish that every single otaku in the world could have. What is it? It’s seriously a dictionary, but not the kind of dictionary that we used when I was a little kid to look up stuff for our school essays. I never had a dictionary like this. You probably know what Hatsune Miku is, but do you know what a Heta-uma is? How about a kuchi-paku? Guess where you can find all of that information that you didn’t know that you needed to have? In this freaking book. I know a fair amount about Otaku culture. I lived and breathed it in Japan for almost a whole decade. But I didn’t know half of the stuff that Patrick wrote about in his book, and that’s why you need it. Plus, it’s got a lot of color, a cute mascot, and some really cool exclusive interviews. You can even learn about Tenimyu!   2. Tokyo on Foot So. I saw this book in the book store in Japan, even though it’s written in English. Maybe that’s because although there is a story in it, it’s mostly drawings and you don’t need to be able to read to get the gist of it. It was written/drawn by an artist that came to stay in Tokyo while his girlfriend was there for an internship. He spent almost every day of his six months there wandering the city with colored pencils and a pad of paper and drawing what he saw. Not only are his drawings aces, I absolutely love his little comments about places and people and things. Right after I bought this book (years ago), I was so enamored that I tried emulating his style with less than stellar results. Me and colored pencils don’t mix, which kind of makes this book even more cool (somehow)! Part of the reason that I really enjoyed this book was because it made me nostalgic for my own first days in Tokyo. I remembered thinking a lot of the same things. I just wish that I’d been good with colored pencils (and had enough confidence to write a book). You can read about my first year here on my blog, though! Honestly, I don’t think that this book is as much a must-have for otaku as the other two, but if art and impressions of Japan is your thing, I think you will love it as much as I do. I seriously only brought two English-language books back with me when I moved out of Japan, and this was one. The other was an ancient copy of The Mysterious Island that my father got when he was a kid and passed on to me.   3. Tokyo Geek’s Guide Aaaaalright. I was really, really skeptical about this one. I’ve seen a hundred other “guides to Tokyo” for otaku, but I didn’t keep any of them. This one, though? I am not only keeping it forever, I am going to give a copy to any of my friends traveling to Japan on their own to go otaku-shopping. Holy cow, I wish this book had existed when I first moved to Japan, because it covers things that it took me years of living there to find on my own! It’s a bona-fide travel guide, minus all of the generic stuff that you can find in a normal travel guide. It doesn’t focus on hotels or nice restaurants. Instead, it lists maid cafes, anime shops, and AWESOME stuff like Swallowtail (don’t know what that is? You need to get this book and find out because it is awesome!). The book is split into districts of Tokyo, and lists otaku-related info about each area along with detailed maps and how to get to all of these places. It’s kind of big and heavy for a travel guide, but it’s seriously the only one that I’m interested in having with me next time that I travel to Tokyo. There are places in it that I haven’t even been to. Oh, and bonus? There is a whole section in the latter part of the book talking about Geeky festivals like Comicket and JUMP Festa. I REALLY, REALLY WISH THAT THIS HAD BEEN AROUND WHEN I MOVED TO JAPAN. It’s 14 years too late for that, but not too late for my next trip, and not too late for yours! It’s also in full color. If you’re reading this, I think that you will probably want this book. GO BUY IT.   This has absolutely NOT been a paid advertisement. I am just a geeky girl honestly recommending things that she likes to you that she thinks you need. :3 See you again soon la la la!