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A 73-year-old woman in complete remission from localized small-cell lung cancer associated with Lambert-Eaton myasthenic syndrome (LEMS) 22 years earlier was referred to our hospital and diagnosed with non-small-cell lung cancer. After three courses of pembrolizumab, an immune checkpoint inhibitor, the patient complained of muscle weakness, fatigue, ptosis, and dysarthria. The anti-voltage-gated calcium channel antibody level was elevated, and waxing was observed on a high-frequency repetitive stimulation test using an electromyogram. We diagnosed her with recurrence of LEMS as an immune-related adverse event (irAE) induced by pembrolizumab. After intravenous immunoglobulin therapy, the patient's symptoms improved, and she was discharged.

Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in nonsmoking-related, non–small cell lung cancer (NSCLC). EGFR-mutant NSCLC has a noninflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune-checkpoint inhibitors, such as antiprogrammed cell death-1 (anti–PD-1), have weak antitumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by the sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, the simultaneous triple blockade had no such effect. The PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pretreatment with the EGFR-TKI, suggesting that the treatment schedule is crucial for the efficacy of the dual blockade therapy. Pretreatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing the expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a noninflamed TME.

Supplementary Data from CD8+ T-cell Responses Are Boosted by Dual PD-1/VEGFR2 Blockade after EGFR Inhibition in Egfr-Mutant Lung Cancer

Supplementary Figure from CD8+ T-cell Responses Are Boosted by Dual PD-1/VEGFR2 Blockade after EGFR Inhibition in Egfr-Mutant Lung Cancer

Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in nonsmoking-related, non–small cell lung cancer (NSCLC). EGFR-mutant NSCLC has a noninflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune-checkpoint inhibitors, such as antiprogrammed cell death-1 (anti–PD-1), have weak antitumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by the sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, the simultaneous triple blockade had no such effect. The PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pretreatment with the EGFR-TKI, suggesting that the treatment schedule is crucial for the efficacy of the dual blockade therapy. Pretreatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing the expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a noninflamed TME.

Supplementary Figure 3. Combined use of SHP099 with alectinib, crizotinib or osimertinib Cell viability after treatment with a single agent or a combination of agents relative to the control (set to 1). All drugs were loaded for 96 h. Error bars represent the standard error. *P < 0.01 (one-way ANOVA and the Bonferroni method). S, SHP099; A, alectinib; C, crizotinib; O, osimertinib.

Supplementary Figure 6. Body weight changes in a xenograft mouse model. Of the mice bearing H3122 tumors, those in the vehicle and SHP099 groups were euthanized on days 14 and 21 respectively, as they had reached the humanitarian endpoint due to tumor growth. Error bars represent the standard deviation.

Supplementary Figure 2. ROS1 TKI in combination with SHP099 (A) Immunoblots of the indicated proteins in HCC78 and ABC-20 cells treated for 24 h with DS-6051b (Ds, 100 nM) with or without SHP099 (SHP, 5 µM). Comb, combination. (B) RAS-GTP assay in H3122 cells treated with alectinib (Alec, 100 nM), ABC-20 cells treated with crizotinib (Cri, 1 µM) and HCC827 cells treated with osimertinib (Osi, 100 nM) for 24 h. All cell lines were also treated or not with SHP099 (SHP, 5 µM). Comb, combination. (C) Cell viability curves for cell lines treated with DS-6051b (DS) alone (circles), SHP099 (SHP) alone (triangles) or DC6051b and SHP099 (5 µM) (squares). All drugs were loaded for 96 h. Error bars represent the standard error.

Supplementary Figure 1. phospho-RTK array in PC-9 cells phospho-RTK array in PC-9 cells treated with 100 nM of the EGFR TKI erlotinib for up to 96 h. Multiple RTKs were gradually activated over time.

After molecular-targeted therapy, some cancer cells may remain that are resistant to therapies targeting oncogene alterations, such as those in the genes encoding the EGFR and anaplastic lymphoma kinase (ALK) as well as c-ros oncogene 1 (ROS1). The mechanisms underlying this type of resistance are unknown. In this article, we report the potential role of Src homology 2 domain–containing phosphatase 2 (SHP2) in the residual cells of ALK/ROS1/EGFR-altered non–small cell lung cancer (NSCLC). Molecular-targeted therapies failed to inhibit the ERK signaling pathway in the residual cells, whereas the SHP2 inhibitor SHP099 abolished their remaining ERK activity. SHP099 administered in combination with molecular-targeted therapy resulted in marked growth inhibition of cancer cells both in vitro and in vivo. Thus, treatment combining an SHP2 inhibitor and a tyrosine kinase inhibitor may be a promising therapeutic strategy for oncogene-driven NSCLC.

Supplementary Figure 7. SHP2 compared to selumetinib in a xenograft mouse model. (A) Immunoblots of the PC-9 xenograft tumors treated with the indicated agents for 7 days. (B) Body weight changes after combination therapy with osimertinib and SHP099 or osimertnib and selumetinib in PC-9 xenograft mice. Error bars represent the standard deviation. (C) Body weight changes after combination therapy with alectinib and SHP099 or alectinib and selumetinib in H3122 xenograft mice. Error bars represent the standard deviation. (D) Body weight changes after combination therapy with crizotinib and SHP099 or crizotinib and selumetinib in ABC-20 xenograft mice. Error bars represent the standard deviation. Osi, osimertinib; Alec, alectinib; Cri, crizotinib; Selu, selumetinib; SHP, SHP099.

Supplementary Figure 5. Combined use of RMC4550 with alectinib, crizotinib or osimertinib Immunoblots of the indicated proteins in H3122 and ABC-19 cells treated with alectinib (Alec, 100 nM), HCC78 and ABC-20 cells treated with crizotinib (Cri, 1 µM) and PC-9 and HCC827 cells treated with osimertinib (Osi, 100 nM). All cell lines were also treated with RMC-4550 (RMC, 5 µM). Comb, combination. (B) Cell viability curves plotted for the cell lines treated with the molecular-targeted drug alone (circles), RMC-4550 alone (triangles) or the molecular-targeted drug and RMC-4550 (5 µM) (squares). All drugs were loaded for 96 h. Error bars represent the standard error.

A 69-year-old man with refractory lung adenocarcinoma was treated with gemcitabine and vinorelbine. Dyspnea and hypertension developed after the 17th cycle of chemotherapy. Laboratory findings revealed intravascular hemolysis and renal dysfunction. Thrombotic microangiopathy (TMA) was confirmed by renal biopsy. Antihypertensive and steroid therapies were ineffective. After plasmapheresis, intravascular hemolysis and renal dysfunction gradually improved. However, the disease progressed, and he died 6 months after TMA diagnosis. Autopsy revealed similar pathological findings to those of the renal biopsy. It is important to discontinue gemcitabine at the onset of TMA and consider TMA when using gemcitabine for long periods.

Measuring the distribution, characteristics and dynamics of marine microscale plankton and other particulate matter is essential to understand the vertical flux of elements in the marine environment. Digital holographic microscopy is a powerful approach for measuring these and studying their 3-D trajectories in a relatively large observation volume. This article demonstrates a compact, in-line digital holographic microscope that allows large-volume and high-resolution recording of marine particles through combining a continuous wave laser and a short exposure CMOS camera with efficient global shutters. A resolution of better than 10 μm is demonstrated in air and the minimum distinguishable size of targets recorded in water is approximately 20 μm. The maximum volumetric throughput of the setup is 1904 mL/s. The microscope can take motion blur free holograms of particles moving at up to 490 mm/s in theory, and has been tested in the ∼200-mm/s flowing water. The orientation of the measured volume improves the ability of digital holography in profiling sinking rates and active vertical migration. The system was tested onboard a research vessel to record a range of live plankton and other particles. The motion of some samples, including the sinking motion and swimming motion, was analyzed using custom developed image processing software. The experimental results show that the combination of high resolution and a large volume over which motion of sparse-distribution particles can be tracked, can improve the ability to differentiate between different types of marine particle and identify behaviors of live plankton.

Hydrothermal vents and hydrocarbon seeps in the deep ocean are rare oases fueled by chemosynthesis. Biological communities inhabiting these ecosystems are often distributed in widely separated habitats, raising intriguing questions on how these organisms achieve connectivity and whether habitat types facilitate intraspecific divergence. The deep-sea patellogastropod limpet Bathyacmaea nipponica that colonizes both vents and seeps across ∼2,400 km in the Northwest Pacific is a feasible model to answer these questions. We analyzed 123 individuals from four vents and three seeps using a comprehensive method incorporating population genomics and physical ocean modeling. Genome survey sequencing and genotyping-by-sequencing resulted in 9,838 single-nucleotide polymorphisms for population genomic analyses. Genetic divergence and demographic analyses revealed four habitat-linked (i.e., three seep and one vent) genetic groups, with the vent genetic group established via the opportunistic invasion of a few limpet larvae from a nearby seep genetic group. TreeMix analysis uncovered three historical seep-to-vent migration events. ADMIXTURE and divMigrate analyses elucidated weak contemporary gene flow from a seep genetic group to the vent genetic group. Physical ocean modeling underlined the potential roles of seafloor topography and ocean currents in shaping the genetic connectivity, contemporary migration, and local hybridization of these deep-sea limpets. Our study highlighted the power of integrating genomic and oceanographic approaches in deciphering the demography and diversification of deep-sea organisms. Given the increasing anthropogenic activities (e.g., mining and gas hydrate extraction) affecting the deep ocean, our results have implications for the conservation of deep-sea biodiversity and establishment of marine protected areas.

After molecular-targeted therapy, some cancer cells may remain that are resistant to therapies targeting oncogene alterations, such as those in the genes encoding the EGFR and anaplastic lymphoma kinase (ALK) as well as c-ros oncogene 1 (ROS1). The mechanisms underlying this type of resistance are unknown. In this article, we report the potential role of Src homology 2 domain–containing phosphatase 2 (SHP2) in the residual cells of ALK/ROS1/EGFR-altered non–small cell lung cancer (NSCLC). Molecular-targeted therapies failed to inhibit the ERK signaling pathway in the residual cells, whereas the SHP2 inhibitor SHP099 abolished their remaining ERK activity. SHP099 administered in combination with molecular-targeted therapy resulted in marked growth inhibition of cancer cells both in vitro and in vivo. Thus, treatment combining an SHP2 inhibitor and a tyrosine kinase inhibitor may be a promising therapeutic strategy for oncogene-driven NSCLC.

We report the case of a 54-year-old man who was treated with nivolumab for recurrent squamous cell lung cancer. After 7 cycles of nivolumab treatment, the patient presented to our hospital with right eye vision loss. Gadolinium-enhanced magnetic resonance imaging of the brain showed enhancement around the optic nerve sheath. This finding and his symptoms led to the diagnosis of optic perineuritis (OPN). Steroid pulse therapy was administered twice although there was no remarkable improvement in his visual field defect. The relationship between OPN and nivolumab is unclear. However, immune-related adverse events caused by immune checkpoint inhibitors should be considered.

Objectives Pembrolizumab is a standard treatment for non-small cell lung cancer (NSCLC) with high-PD-L1 expression; however, its effect is dismal in patients with poor physical condition. Additionally, the effect of immunotherapy is generally limited in NSCLC harboring driver mutations such asEGFR, ALK, or RET gene aberrations. Results We report the beneficial effect of pembrolizumab in a patient with poor performance status and PD-L1+ lung adenocarcinoma with theCCDC6–RET fusion gene and co-occurring NF1/TP53 mutations, complicated by multiple cancer-associated thrombi and respiratory failure. Conclusions Further studies are warranted to establish the role of co-occurring NF1/TP53 mutations as a positive predictive biomarker for pembrolizumab in NSCLC harboring RET fusion genes.

This paper describes measurement methods of surface profiles that improve contact-type displacement sensor outputs by focusing on the contact point between the sphere tip of the sensor and the rough surface. We examined the geometry of a surface profile model and compared measurements using various methods with the measurement using a roughness meter. The spherical tip of the contact type displacement sensor touches the measurement surface and detects the displacement. The sphere tip radius of a typical contact-type displacement sensor ranges from 1–3 mm, causing the roughness curve to be “filtered” by the radius of the sphere. Three methods for estimating the valley portion of the surface profile are evaluated in this study: a) linear approximation of the concave portion of the surface profile, b) function approximation of the concave portion, and c) using the known nose radius of the machining tool. The following sphere tip radii were used to measure actual surface profiles: 0.25 mm, 0.5 mm, 1.0 mm and 1.5 mm. Given the conditions of the experimental model, we found that surface profiles with a roughness that approximates a predictable curve can be measured with a high degree of accuracy.

The distribution of species among spatially isolated habitat patches supports regional biodiversity and stability, so understanding the underlying processes and structure is a key target of conservation. Although multivariate statistics can infer the connectivity processes driving species distribution, such as dispersal and habitat suitability, they rarely explore structure. Methods from graph theory, applied to distribution data, give insights into both connectivity pathways and processes by intuitively formatting the data as a network of habitat patches. We apply these methods to empirical data from the hydrothermal vent habitats of the Northwest Pacific. Hydrothermal vents are ‘oases’ of biological productivity and endemicity on the seafloor that are imminently threatened by anthropogenic disturbances with unknown consequences to biodiversity. Here, we describe the structure of hydrothermal vent species assemblage networks, how local and regional parameters affect their structure, and the implications this has for conservation. Two complementary networks were formed from an extensive species assemblage dataset: a bipartite network of species nodes linked to vent site nodes at which they are present, and a similarity network of vent site nodes linked by weighted edges based on their pairwise assemblage similarity. Using these networks, we assessed the role of individual vent sites in linking their network and identified biogeographic sub-regions. The three sub-regions and two outlying sites are separated by their spatial arrangement and local environmental filters. Both networks detected vent sites that play a disproportionately important role in regional pathways, while the bipartite network also identified key vent sites maintaining the distinct species assemblages of their sub-regions. These regional connectivity pathways provide insights into historical colonisation routes, while sub-regional connectivity pathways are of value when selecting sites for conservation and/or estimating the multi-vent impacts from proposed deep-sea mining.

A new pardaliscid amphipod, Princaxelia marianaensissp. nov., is described from a single female captured at the Shinkai Seep Field, Mariana Trench, from a depth of 5,689–5,683 m. A key to species of Princaxelia is provided. This is the first species of Princaxelia to be described from the Mariana Trench, and the second report of this genus from this region.

Two species of dirivultid copepod (Siphonostomatoida), Stygiopontius senckenbergi Ivanenko & Ferrari, 2013 and Dirivultus kaiko sp. nov., were discovered from hydrothermal vent fields in the Okinawa Trough, the western North Pacific. Since S. senckenbergi was originally described based on two adult males from the New Ireland Fore-Arc system, Papua New Guinea, the discovery here represents the second record. This species was attached on ventral setae covered with filamentous bacteria of the deep-sea squat lobster Shinkaia crosnieri (Decapoda: Munidopsidae). The female of S. senckenbergi is described for the first time as well as the nauplius I and the copepodid IV. Sexual dimorphism is shown on several appendages (i.e. antennule, leg 2 and leg 5). The nauplius I shows typical lethithotrophic characteristics. Dirivultus kaiko sp. nov., which was found on the tentacular crown of the siboglinid tubeworm Lamellibrachia columna, differs from its two congeners by the antennule, maxilliped and leg 5 of both sexes. These are the second and third dirivultid species to be reported from Japan.

Objectives Immune checkpoint inhibitors offer longer survival than chemotherapy in several clinical trials for advanced non-small cell lung cancer. In subset analyses of clinical trials, immune checkpoint inhibitors extended survival in patients aged ≥65 years, but the effects in patients aged ≥75 years are controversial. We performed multicenter, collaborative and retrospective analyses of immune checkpoint inhibitor efficacy and safety in non-small cell lung cancer patients aged ≥75 years. Methods We retrospectively studied 434 advanced non-small cell lung cancer patients who received immune checkpoint inhibitors from December 2015 to December 2017, and retrospectively applied the Geriatric (G) 8 screening tool with medical records. Results Of the 434 patients who received immune checkpoint inhibitors, 100 were aged ≥75 years. Five patients with performance status 3 were omitted from the final analysis. Immune checkpoint inhibitors were given as a first-line treatment to 20 patients. The objective response rates, median progression-free survival rates and median survival times were 35.0%, 6.1 months and 10.7 months for first-line treatment, and 20.0%, 2.9 months and 14.7 months for second- or later-line treatments, respectively. The median modified G8 score was 11.0. The median survival time was longer in the high modified G8 (≥12.0) group than in the low modified G8 (≤11.0) group (18.7 vs. 8.7 months; P = 0.02). Likewise, the median survival time was 15.5 months (performance status 0–1) vs. 3.2 months (performance status 2) (P Conclusions In this study, immune checkpoint inhibitors were effective and tolerable for patients aged ≥75 years. The modified G8 screening tool and performance status were associated with the outcome of older non-small cell lung cancer patients treated with immune checkpoint inhibitors.

Genetic connectivity provides a basis for evaluating the resilience of animal communities by elucidating gene flow and serves as a proxy for realized dispersal through planktonic larval dispersal, settlement, and reproductive success. Gandalfus yunohana (Takeda, Hashimoto & Ohta, 2000) is a brachyuran crab endemic to deep-sea hydrothermal vents in the northwestern Pacific. Although an iconic and often dominant species, the connectivity among its populations is yet to be examined. We obtained barcoding sequences of the mitochondrial COI gene of G. yunohana from four vent fields including two on the Izu Arc, one on the northern Mariana Arc, and one in the Okinawa Trough. Genetic diversity of populations on the Izu and northern Mariana arcs were similar and shared the dominant haplotypes, showing no genetic subdivision regardless of the habitat depth. The Okinawa Trough population, for which only one specimen was available, was not genetically different from specimens from the Izu/Mariana arc populations. Estimation of the number of immigrants among populations suggests that the migration from north to south is higher than in the reverse direction. Our results resonate with previous laboratory-culture experiments, suggesting a high dispersal capability for G. yunohana. Visual observations and sampling, however, suggest that G. yunohana is exceedingly rare in the Okinawa Trough. This perhaps results from a lack of sufficient larval supply from the Izu-Bonin-Mariana Arc vents, and there may not be a reproductive population in the Okinawa Trough.

Stygiopontius copepods (Dirivultidae, Siphonostomatoida, Crustacea) are among the most successful meiobenthic organisms at deep-sea hydrothermal vents. Most of their ecology is not yet known, including the spatiotemporal differences in their sex ratios and their controlling factors. We investigated spatial variation in the sex ratio of adult Stygiopontius senokuchiae and its association with environmental parameters, including food quality at hydrothermal vent chimney structures in the calderas of three neighboring sea knolls (Bayonnaise Knoll, Myojin Knoll, and Myojin-sho Caldera) in the western North Pacific Ocean. Their sex ratio was significantly biased to males from 1:1, which was different from some of the other Stygiopontius species. The ratios did not show a significant correlation with the density of total adults. While previous studies have shown that the abundance of S. senokuchiae is positively associated with δ13C values of detritus on active chimneys, multivariate analyses in this study did not detect any significant association between their sex ratio and any investigated parameters including δ13C. These findings suggest that neither population density nor chemoautotrophic food availability drives the sexual difference in the spatial distribution around vents among the adults of the species.

The Scaly-foot Snail, Chrysomallon squamiferum, presents a combination of biomineralised features, reminiscent of enigmatic early fossil taxa with complex shells and sclerites such as sachtids, but in a recently-diverged living species which even has iron-infused hard parts. Thus the Scaly-foot Snail is an ideal model to study the genomic mechanisms underlying the evolutionary diversification of biomineralised armour. Here, we present a high-quality whole-genome assembly and tissue-specific transcriptomic data, and show that scale and shell formation in the Scaly-foot Snail employ independent subsets of 25 highly-expressed transcription factors. Comparisons with other lophotrochozoan genomes imply that this biomineralisation toolkit is ancient, though expression patterns differ across major lineages. We suggest that the ability of lophotrochozoan lineages to generate a wide range of hard parts, exemplified by the remarkable morphological disparity in Mollusca, draws on a capacity for dynamic modification of the expression and positioning of toolkit elements across the genome., The Scaly-foot Snail, Chrysomallon squamiferum, is a model for understanding the evolution of biomineralised armour. Here, the authors present a chromosome-level reference genome assembly and tissue-specific transcriptomic data for this enigmatic organism.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated as INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription dataset of INp10 from biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help better understand ecological strategies of this possibly widespread marine-specific methanotrophic clade. Importance The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus spp. suggests that the hydrothermal field is a promising locale to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus.

Newborn screening of inborn errors of metabolism using tandem mass spectrometry has become a public health strategy in many developed countries. Retrospective analyses using stored dried blood specimens have been limited, mainly due to a lack of biochemical information on the long-term stability of acylcarnitines and amino acids in stored specimens. We studied the characteristic profiles of the stability of amino acid, free carnitine, and acyl carnitines in dried blood specimens stored in a refrigerator after newborn screening.Dried blood specimens from 198 healthy newborns, which had been stored in a refrigerator at 5 °C after newborn screening, were prospectively subjected to tandem mass spectrometry analyses after 1, 3, 6 months, 1 and 2 years of storage. We also retrospectively re-analyzed the stored samples from 90 newborns, which had been analyzed and stored at 5 °C for 4 years.We found that proline (Pro) and tyrosine (Tyr) were stable for 2 years, and that alanine (Ala), arginine (Arg), and phenylalanine (Phe) decayed with linear regression. The C0 increased during the time-course of 2 years, whereas most acylcarnitines gradually decayed and some showed a linear correlation. The retrospective analysis of samples stored for 4 years revealed that Ala, Phe, Pro and Tyr were almost stable, leucine (Leu), valine (Val) decayed with linear regression, C0 increased, and C10, C12, C14, C14:1, C16, C18, C18:1 decreased, while maintaining a linear correlation.These data suggested that some metabolic parameters from refrigerator-stored dried blood specimens were applicable for the detection of inborn errors of metabolism.

Ocean bottom electromagnetometers (OBEMs) installed on the seafloor around Nishinoshima Island (Bonin Islands) were missing after a December volcanic eruption. In February 2021, one was found on a beach on Iriomote Island (Ryukyu Islands), implying that it drifted westward for 1,700 km. The reason(s) for the disappearance of the OBEMs and the path followed by the recovered OBEM while drifting are important information for future ocean bottom observations and seafloor volcanology in general. We conducted particle drifting simulations with and without horizonal eddy diffusion to estimate the possible drift path and duration of the recovered OBEM. Our simulations show that particles transported from Nishinoshima have a 7-10 % probability of arriving at Iriomote Island, which is thus not a rare occurrence. Transport durations in our simulations varied widely between 140 and 602 days depending on the drift paths. The most likely drift duration in our simulation was 150 – 180 days, with or without eddy diffusion, corresponding to the release from the seafloor of the OBEM between 22 August and 21 September 2020. These dates follow shortly after intensifying eruptions at Nishinoshima, which may have affected the seafloor around the island. A similar drift duration and path was reported for pumices that erupted from Fukutoku-Oka-no-Ba submarine volcano (northern Bonin Islands) during 18-21 January 1986 and arrived in the Ryukyu Islands in late May 1986. Such drifting simulations may prove useful for identifying the sources of drift pumices, and thus otherwise undetectable eruptions. Finally, the Fukutoku-Oka-no-Ba submarine volcano erupted on 13 August 2021, producing abundant pumice rafts that, based on our results, will likely arrive in the Ryukyu Islands in the coming months.

Variations in offspring production according to feeding strategies or food supply have been recognized in many animals from various ecosystems. Despite an unusual trophic structure based on non-photosynthetic primary production, these relationships remain largely under-studied in chemosynthetic ecosystems. Here, we useRimicarisshrimps from deep-sea hydrothermal vents as a study case to explore relations between reproduction, diets and food supply in these environments. For that, we compared reproductive outputs of three congeneric shrimps differing by their diets. They inhabit vents located under oligotrophic waters of tropical gyres with opposed latitudes, allowing us to also examine the prevalence of phylogenetic vs environmental drivers in their reproductive rhythms. For this we used both our original data and a compilation of published observations on the presence of ovigerous females covering various seasons over the past 35 years. We report distinct egg production trends betweenRimicarisspecies relying solely on chemosymbiosis –R. exoculataandR. kairei– and those relying on mixotrophy –R. chacei– whereR. chaceiproduces broods with higher numbers of smaller eggs. Besides, our data and historical records suggest a reproductive period with substantial proportions of brooding females mostly between January and early April for all examined species whatever the region. Intriguingly, this periodicity does not correspond to seasonal variations in surface production with presence of brooding females during either boreal winter or austral summer. These observations contrast with the long-standing paradigm in deep-sea species for which periodic reproductive patterns have always been attributed to seasonal variations of photosynthetic production sinking from surface. Our results suggest the presence of intrinsic basis for biological rhythms in the deep sea, and bring to light the importance of having year-round observations in order to understand life history of vent animals.

Increasing research has been conducted about patients’ preferences for their role in treatment decision making. Nowadays, a patient-centred approach, so-called ‘shared decision making,’ emphasises ...

Introduction The highly selective ALK receptor tyrosine kinase (ALK) inhibitor alectinib is standard therapy for ALK-positive lung cancers; however, some tumors quickly develop resistance. Here, we investigated the mechanism associated with rapid acquisition of resistance using clinical samples. Methods Autopsied samples were obtained from lung, liver, and renal tumors from a 51-year-old male patient with advanced ALK-positive lung cancer who had acquired resistance to alectinib in only 3 months. We established an alectinib-resistant cell line (ABC-14) from pleural effusion and an alectinib/crizotinib-resistant cell line (ABC-17) and patient-derived xenograft (PDX) model from liver tumors. Additionally, we performed next-generation sequencing, direct DNA sequencing, and quantitative real-time reverse transcription polymerase chain reaction. Results ABC-14 cells harbored no ALK mutations and were sensitive to crizotinib while also exhibiting MNNG HOS transforming gene (MET) gene amplification and amphiregulin overexpression. Additionally, combined treatment with crizotinib/erlotinib inhibited cell growth. ABC-17 and PDX tumors harbored ALK G1202R, and PDX tumors metastasized to multiple organs invivo, whereas the third-generation ALK-inhibitor, lorlatinib, diminished tumor growth invitro and invivo. Next-generation sequencing indicated high tumor mutation burden and heterogeneous tumor evolution. The autopsied lung tumors harbored ALK G1202R (c. 3604 G>A) and the right renal metastasis harbored ALK G1202R (c. 3604 G>C); the mutation thus comprised different codon changes. Conclusions High tumor mutation burden and heterogeneous tumor evolution might be responsible for rapid acquisition of alectinib resistance. Timely lorlatinib administration or combined therapy with an ALK inhibitor and other receptor tyrosine-kinase inhibitors might constitute a potent strategy.

Anti-programmed cell death 1 (PD-1) antibodies have poor efficacy in epidermal growth factor receptor (EGFR)-mutated lung cancer. We herein report a 72-year-old man with programmed cell death-ligand 1 (PD-L1)-negative lung adenocarcinoma harboring an EGFR mutation that responded to nivolumab for more than 2 years. A pathological examination revealed infiltration of CD8-positive lymphocytes and macrophages expressing CD68, CD206, and PD-L1 into the PD-L1-negative tumor; CD206 expression is a marker of immunosuppressive tumor-associated macrophages (TAMs). The presence of PD-L1-positive TAMs in the tumor environment might be a predictor of a positive response to anti-PD-1 antibodies.

This paper describes an evaluation method of shot peened surface using image processing. Shot peening is a process that applies compressive residual stress to the product surface, and its evaluation is performed visually by an expert. If visual inspection can be replaced with image processing, the inspection of the entire product will be easier. Therefore, first reference samples that experts evaluate are prepared, next these samples are evaluated by image processing. relationship between expert evaluations and image processing evaluations are compared and the estimation function is defined using gausian distribution. Unknown processed surfaces are evaluated as a classification problem. For image processing, after binarization and labeling, the number of labels and the area ratio of binarization are used.

Osimertinib, a third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is the standard treatment for patients with lung cancer harboring EGFR T790M; however, acquired resistance is inevitable due to genetic and epigenetic changes in cancer cells. In addition, a recent randomized clinical trial revealed that the combination of osimertinib and bevacizumab failed to exhibit superior progression-free survival compared with osimertinib alone. The present study aimed to investigate the effect of triple therapy with osimertinib, bevacizumab and cetuximab in xenograft tumors with different initial tumor volumes (conventional model, 200 mm3 and large model, 500 mm3). The results demonstrated that osimertinib significantly inhibited tumor growth in both the conventional and large models; however, maximum tumor regression was attenuated in the large model in which hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-α (TGF-α) expression levels increased. Although the combination of osimertinib and bevacizumab exerted a greater inhibitory effect on tumor growth compared with osimertinib in the conventional model, the effect of this combination therapy was attenuated in the large model. TGF-α attenuated sensitivity to osimertinib in vitro; however, this negative effect was counteracted by the combination of osimertinib and cetuximab, but not osimertinib and bevacizumab. In the large xenograft tumor model, the triple therapy induced the greatest inhibitory effect on tumor growth compared with osimertinib alone and its combination with bevacizumab. Clinical trials of the triple therapy are required for patients with lung cancer with EGFR mutations and HIF-1α/TGF-α.