Postoperative Prognosis According to Pathologic Categorization of Desmoplastic Reaction in Patients with Extrahepatic Cholangiocarcinoma.

BACKGROUND: Recent studies have demonstrated the importance of desmoplastic reaction (DR) in predicting postoperative prognosis for patients with colorectal carcinoma. However, the impact of DR on the prognosis of extrahepatic cholangiocarcinomas (EHCCs) is not established. This study aimed to clarify the associations of pathologic DR categories with clinicopathologic factors and postoperative prognosis of perihilar cholangiocarcinoma (PHCC) and distal cholangiocarcinoma (DCC). METHODS: A pathologic review of 174 patients with PHCC and 109 patients with DCC who underwent surgical resection was performed. The patients were classified into three DR categories (immature, intermediate, and mature) based on the histologic features within the fibrotic stroma in the invasive front. The association between DR categories and the distribution of fibroblasts with anti-α-smooth muscle actin (SMA) expression, seeming to be tumor-promoting cancer-associated fibroblasts (CAFs), was evaluated in 191 tissue microarray specimens of EHCCs. RESULTS: Intermediate/immature DR categories were significantly associated with a more invasive nature, including higher pT and pN stages and more tumor buds than the mature category in both PHCC and DCC. The DR categories could stratify overall survival (OS) and relapse-free survival (RFS) in both PHCC and DCC patients. In the multivariate analysis, the DR category was an independent prognostic factor for OS and RFS in both PHCC and DCC (p < 0.001). The mature and immature DR categories were significantly associated respectively with the confined and pervasive distribution of fibroblasts with α-SMA expression. CONCLUSION: In patients with EHCCs, DR categorization was an independent prognostic factor reflecting the distribution of tumor-promoting CAFs in the invasive front.

Predictors of Long-Term Survival in Pancreatic Ductal Adenocarcinoma after Pancreatectomy: TP53 and SMAD4 Mutation Scoring in Combination with CA19-9.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a fatal cancer for which even unfavorable clinicopathological factors occasionally fail to preclude long-term survival. We sought to establish a scoring system that utilizes measurable pre-intervention factors for predicting survival following surgical resection. METHODS: We retrospectively analyzed 34 patients who died from short-term recurrences and 32 long-term survivors among 310 consecutively resected patients with PDA. A logistic regression model was used to define factors related to clinical parameters, molecular profiles of 18 pancreatic cancer-associated genes, and aberrant expression of major tumor suppressors. RESULTS: Carbohydrate antigen 19-9 (CA19-9) had the best ability to classify patients with short-term recurrence and long-term survivors [odds ratio 21.04, 95% confidence interval (CI) 4.612-96.019], followed by SMAD4 and TP53 mutation scoring (odds ratio 41.322, 95% CI 3.156-541.035). Missense TP53 mutations were strongly associated with the nuclear expression of p53, whereas truncating mutations were associated with the absence of nuclear p53. The former subset was associated with a worse prognosis. The combination of aberrant SMAD4 and mutation types of TP53 exhibited a better resolution for distinguishing patients with short-term recurrences from long-term survivors (compared with the assessment of the number of mutated KRAS, CDKN2A, TP53, and SMAD4 genes). Calibration of mutation scores combined with CA19-9 in a logistic regression model setting demonstrated a practical effect in classifying long survivors and patients with early recurrence (c-statistic = 0.876). CONCLUSIONS: Genetic information, i.e., TP53 mutation types and SMAD4 abnormalities, combined with CA19-9, will be a valuable tool for improving surgical strategies for pancreatic cancer.

Prognostic relevance of tertiary lymphoid organs following neoadjuvant chemoradiotherapy in pancreatic ductal adenocarcinoma.

The efficacy of preoperative neoadjuvant chemoradiotherapy (NAC) in cases of pancreatic cancer with extremely poor prognoses has been reported. In this study, we aimed to identify novel biomarkers that reflect prognoses following chemoradiotherapy using tertiary lymphoid organs (TLO) expressed in the tumor microenvironment. Resected tumor specimens were obtained from 140 pancreatic cancer patients. We retrospectively investigated the clinical relevance of TLO by categorizing patients into those who underwent upfront surgery (surgery first [SF]) and those who received NAC. The immunological elements within TLO were analyzed by immunohistochemistry (IHC). In the IHC analysis, the proportions of CD8+ T lymphocytes, PNAd+ high endothelial venules, CD163+ macrophages and Ki-67+ cells within the TLO were higher in the NAC group than in the SF group. In contrast, the proportion of programmed cell death-1+ immunosuppressive lymphocytes within TLO was lower in the NAC group than in the SF group. The NAC group demonstrated favorable prognoses compared with the SF group. In the multivariate analysis, the TLO/tumor ratio was determined as an independent predictive prognostic factor. In conclusion, the administration of preoperative chemoradiotherapy may influence the immunological elements in the tumor microenvironment and result in favorable prognoses in pancreatic ductal adenocarcinoma patients.

Impact of tumour budding grade in 310 patients who underwent surgical resection for extrahepatic cholangiocarcinoma.

AIMS: Tumour budding is a risk factor for poor prognosis in various cancers. Tumour buds may present an epithelial-mesenchymal transition (EMT) morphological phenotype. This study aimed to elucidate the prognostic impact of tumour budding grade and its association with clinicopathological and EMT-related features in perihilar cholangiocarcinoma (PHCC) or distal cholangiocarcinoma (DCC). METHODS AND RESULTS: Subjects included 195 PHCC and 115 DCC patients. The numbers of tumour buds in different patients were stratified for postoperative survival using the recursive partitioning technique. Consequently, the numbers of tumour buds in PHCC patients were classified into three grades; namely, low (0-4 buds); intermediate (5-11 buds); and high (≥12 buds); those of DCC patients were classified into two grades; namely, low (0-4 buds) and high (≥5 buds). In both PHCC and DCC patients, high tumour budding grade was associated with poor histological differentiation, higher pT factor, presence of lymphatic, venous, perineural invasion and regional lymph node metastasis. In PHCC patients, residual invasive tumour in the resected margin was also associated with high tumour budding grade. For both PHCC and DCC patients, high tumour budding grade was an independent adverse prognostic factor in multivariate analysis (P < 0001 and P = 0.046, respectively). Immunohistochemical examination revealed that the number of tumour buds increased in patients with tumours showing a mesenchymal profile (negative for E-cadherin and positive for vimentin). CONCLUSIONS: Higher tumour budding grade is associated with invasive clinicopathological features, adverse postoperative prognosis and EMT status in extrahepatic cholangiocarcinoma.

Male scent-marking pheromone of Bombus ardens ardens (Hymenoptera; Apidae) attracts both conspecific queens and males.

To explore the role of the volatiles emitted from male labial gland (LG) of the bumblebee Bombus ardens ardens, we investigated the responses of virgin queens and males to volatiles using a gas chromatography-electroantennographic detector (GC-EAD) system and Y-tube olfactometer. GC-EAD analysis revealed that citronellol, the main compound detected in the male LG, caused clear electrophysiological responses in the antennae of B. a. ardens virgin queens and males although two minor compounds elicited antennal responses when applied in a high concentration. Behavioral tests using a Y-tube olfactometer showed that queens and males were significantly attracted to both LG extracts and citronellol more than to the solvent alone. This is the first study to demonstrate that citronellol as a major compound of male scent-marking pheromone in B. a. ardens functions as a sex attractant for queens. The results also suggest that this compound has another function as a trail marker used by males.

Expression analysis of Egr-1 ortholog in metamorphic brain of honeybee (Apis mellifera L.): Possible evolutionary conservation of roles of Egr in eye development in vertebrates and insects.

Specific genes quickly transcribed after extracellular stimuli without de novo protein synthesis are known as immediate early genes (IEGs) and are thought to contribute to learning and memory processes in the mature nervous system of vertebrates. A recent study revealed that the homolog of Early growth response protein-1 (Egr-1), which is one of the best-characterized vertebrate IEGs, shared similar properties as a neural activity-dependent gene in the adult brain of insects. With regard to the roles of vertebrate Egr-1 in neural development, the contribution to the development and growth of visual systems has been reported. However, in insects, the expression dynamics of the Egr-1 homologous gene during neural development remains poorly understood. Our expression analysis demonstrated that AmEgr, a honeybee homolog of Egr-1, was transiently upregulated in the developing brain during the early to mid pupal stages. In situ hybridization and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry revealed that AmEgr was mainly expressed in post-mitotic cells in optic lobes, the primary visual center of the insect brain. These findings suggest the evolutionarily conserved role of Egr homologs in the development of visual systems in vertebrates and insects.

Expression profile of the sex determination gene doublesex in a gynandromorph of bumblebee, Bombus ignitus.

Gynandromorphy that has both male and female features is known in many insect orders, including Hymenoptera. In most cases, however, only external morphology and behavioral aspects have been studied. We found a gynandromorph of bumblebee, Bombus ignitus, that showed almost bilateral distribution of external sexual traits, with male characters observed on the left side and female characters on the right side. This individual never exhibited sexual behavior toward new queens. The dissection of the head part showed that it had bilaterally dimorphic labial glands, only the left of which was well developed and synthesized male-specific pheromone components. In contrast, the gynandromorph possessed an ovipositor and a pair of ovaries in the abdominal part, suggesting that it had a uniformly female reproductive system. Furthermore, we characterized several internal organs of the gynandromorph by a molecular biological approach. The expression analyses of a sex determination gene, doublesex, in the brain, the fat bodies, the hindgut, and the ovaries of the gynandromorph revealed a male-type expression pattern exclusively in the left brain hemisphere and consistent female-type expression in other tissues. These findings clearly indicate the sexual discordance between external traits and internal organs in the gynandromorph. The results of genetic analyses using microsatellite markers suggested that this individual consisted of both genetically male- and female-type tissues.

Cross-mating between the alien bumblebee Bombus terrestris and two native Japanese bumblebees, B. hypocrita sapporensis and B. cryptarum florilegus, in the Nemuro Peninsula, Japan.

The rapid naturalization of Bombus terrestris across the Nemuro Peninsula has led to a decline in two closely related native Japanese species, namely Bombus hypocrita sapporensis and Bombus cryptarum florilegus, both belonging to the common subgenus Bombus. Although it is widely believed that cross-mating of native and non-native species is influenced by the common male sex pheromone in this region, no study has been conducted to substantiate this claim. Thus, we investigated the cross-activities of male sex pheromones between native and non-native bumblebees, as well as the frequencies of cross-mating, using chemical and DNA assays. Our gas chromatography-electroantennographic detector analyses and behavioral tests revealed the presence of sex pheromonal cross-activities between B. terrestris and the two Japanese bumblebees species. Furthermore, DNA analyses revealed the occurrence of cross-mating between native and non-native species in the Nemuro Peninsula. Overall, these results indicate the immediate need for conservation measures to safeguard Japanese bumblebee populations in the Nemuro Peninsula.

Protective Effects of Imeglimin and Metformin Combination Therapy on ß-Cells in db/db Male Mice.

Imeglimin and metformin act in metabolic organs, including ß-cells, via different mechanisms. In the present study, we investigated the impacts of imeglimin, metformin, or their combination (Imeg + Met) on ß-cells, the liver, and adipose tissues in db/db mice. Imeglimin, metformin, or Imeg + Met treatment had no significant effects on glucose tolerance, insulin sensitivity, respiratory exchange ratio, or locomotor activity in db/db mice. The responsiveness of insulin secretion to glucose was recovered by Imeg + Met treatment. Furthermore, Imeg + Met treatment increased ß-cell mass by enhancing ß-cell proliferation and ameliorating ß-cell apoptosis in db/db mice. Hepatic steatosis, the morphology of adipocytes, adiposity assessed by computed tomography, and the expression of genes related to glucose or lipid metabolism and inflammation in the liver and fat tissues showed no notable differences in db/db mice. Global gene expression analysis of isolated islets indicated that the genes related to regulation of cell population proliferation and negative regulation of cell death were enriched by Imeg + Met treatment in db/db islets. In vitro culture experiments confirmed the protective effects of Imeg + Met against ß-cell apoptosis. The expression of Snai1, Tnfrsf18, Pdcd1, Mmp9, Ccr7, Egr3, and Cxcl12, some of which have been linked to apoptosis, in db/db islets was attenuated by Imeg + Met. Treatment of a ß-cell line with Imeg + Met prevented apoptosis induced by hydrogen peroxide or palmitate. Thus, the combination of imeglimin and metformin is beneficial for the maintenance of ß-cell mass in db/db mice, probably through direct action on ß-cells, suggesting a potential strategy for protecting ß-cells in the treatment of type 2 diabetes.

CCR7 Mediates Cell Invasion and Migration in Extrahepatic Cholangiocarcinoma by Inducing Epithelial-Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC.

Positive geographic correlation between soldiers' weapon size and defensive prowess in a eusocial aphid, Ceratovacuna japonica.

Some aphid species produce a soldier caste with enlarged forelegs and horns (weapons). It has been hypothesised that the evolution of morphological specialization by soldiers in social aphids is accelerated by high predation pressure, but this possibility has not been tested. Here, we investigated the relationship between local predator abundance and soldiers' weapon size and aggressiveness in a prey-predator system comprising a eusocial aphid, Ceratovacuna japonica, and its predators (larvae of the butterfly Taraka hamada and of the moth Atkinsonia ignipicta) in two populations with different predator abundances. We found that the soldiers in the predator-abundant population had larger weapons and were more aggressive than those in the population with lower predator abundance. Furthermore, the soldiers' defensive prowess (evaluated as the survival of aphids in the presence of predators) was greater in the predator-abundant population. These results provide the first evidence that a population of eusocial aphids experiencing high predation pressure has soldiers with pronounced defensive traits and defensive prowess.

Description of a new species of Tardigrada Hypsibius nivalis sp. nov. and new phylogenetic line in Hypsibiidae from snow ecosystem in Japan.

Snow ecosystems are an important component of polar and mountainous regions, influencing water regime, biogeochemical cycles and supporting snow specific taxa. Although snow is considered to be one of the most unique, and at the same time a disappearing habitat, knowledge of its taxonomic diversity is still limited. It is true especially for micrometazoans appearing in snow algae blooming areas. In this study, we used morphological and molecular approaches to identify two tardigrade species found in green snow patches of Mt. Gassan in Japan. By morphology, light (PCM) and scanning electron microscopy (SEM), and morphometry we described Hypsibius nivalis sp. nov. which differs from other similar species by granular, polygonal sculpture on the dorsal cuticle and by the presence of cuticular bars next to the internal claws. Additionally, phylogenetic multilocus (COI, 18S rRNA, 28S rRNA) analysis of the second taxon, Hypsibius sp. identified by morphology as convergens-pallidus group, showed its affinity to the Hypsibiidae family and it is placed as a sister clade to all species in the Hypsibiinae subfamily. Our study shows that microinvertebrates associated with snow are poorly known and the assumption that snow might be inhabited by snow-requiring tardigrade taxa cannot be ruled out. Furthermore, our study contributes to the understanding subfamily Hypsibiinae showing that on its own the morphology of specimens belonging to convergens-pallidus group is insufficient in establishing a true systematic position of specimens.

Comparison of Echocardiographic Changes Between Surgery and Medication Treatment in Patients With Primary Aldosteronism.

Background Primary aldosteronism can cause cardiac dysfunction, including left ventricular hypertrophy, left ventricular diastolic dysfunction, and left atrial enlargement. A few studies have compared the cardioprotective effects between surgery and medication for primary aldosteronism, although most have not adjusted for baseline disease status. In this study, we investigated the difference in cardiovascular outcomes between surgery and medication treatment for primary aldosteronism after adjusting for baseline clinical characteristics, including aldosterone level and pretreatment echocardiographic information. Methods and Results We retrospectively analyzed 220 patients diagnosed with primary aldosteronism who underwent adrenalectomy (n=144) or medication treatment (n=76) between 2009 and 2019. Echocardiographic changes were evaluated pretreatment and 1 year posttreatment. The surgery group had lower potassium, lower plasma renin activity, and higher plasma aldosterone concentration than the medication group, indicating a severe primary aldosteronism phenotype in the former. The decrease in left ventricular mass index after treatment was significantly greater in the surgery group than in the medication group (P=0.047). However, this relationship was not noted after multivariable regression analysis (standard ß=-0.08, P=0.17). Additionally, decreased parameter values related to left ventricular diastolic dysfunction and left atrial enlargement were not different between the groups. Pretreatment echocardiographic values were most associated with changes in all echocardiographic parameters. The findings were consistent in the propensity score-matched analysis. Conclusions This study's findings suggest that there is no difference in cardioprotective efficacy between surgical and medication treatment under similar disease severity; however, it should be considered that several study participants with severe hyperaldosteronism were managed surgically.

Genes associated with hot defensive bee ball in the Japanese honeybee, Apis cerana japonica.

BACKGROUND: The Japanese honeybee, Apis cerana japonica, shows a specific defensive behavior, known as a "hot defensive bee ball," used against the giant hornet, Vespa mandarinia. Hundreds of honeybee workers surround a hornet and make a "bee ball" during this behavior. They maintain the ball for around 30 min, and its core temperature can reach 46. Although various studies have been conducted on the characteristics of this behavior, its molecular mechanism has yet to be elucidated. Here, we performed a comprehensive transcriptomic analysis to detect candidate genes related to balling behavior. RESULTS: The expression levels of differentially expressed genes (DEGs) in the brain, flight muscle, and fat body were evaluated during ball formation and incubation at 46 °C. The DEGs detected during ball formation, but not in response to heat, were considered important for ball formation. The expression of genes related to rhodopsin signaling were increased in all tissues during ball formation. DEGs detected in one or two tissues during ball formation were also identified. CONCLUSIONS: Given that rhodopsin is involved in temperature sensing in Drosophila, the rhodopsin-related DEGs in A. cerana japonica may be involved in temperature sensing specifically during ball formation.

Cryoconite - From minerals and organic matter to bioengineered sediments on glacier's surfaces.

Cryoconite is a mixture of mineral and organic material covering glacial ice, playing important roles in biogeochemical cycles and lowering the albedo of a glacier surface. Understanding the differences in structure of cryoconite across the globe can be important in recognizing past and future changes in supraglacial environments and ice-organisms-minerals interactions. Despite the worldwide distribution and over a century of studies, the basic characteristics of cryoconite, including its forms and geochemistry, remain poorly studied. The major purpose of our study is the presentation and description of morphological diversity, chemical and photoautotrophs composition, and organic matter content of cryoconite sampled from 33 polar and mountain glaciers around the globe. Observations revealed that cryoconite is represented by various morphologies including loose and granular forms. Granular cryoconite includes smooth, rounded, or irregularly shaped forms; with some having their surfaces covered by cyanobacteria filaments. The occurrence of granules increased with the organic matter content in cryoconite. Moreover, a major driver of cryoconite colouring was the concentration of organic matter and its interplay with minerals. The structure of cyanobacteria and algae communities in cryoconite differs between glaciers, but representatives of cyanobacteria families Pseudanabaenaceae and Phormidiaceae, and algae families Mesotaeniaceae and Ulotrichaceae were the most common. The most of detected cyanobacterial taxa are known to produce polymeric substances (EPS) that may cement granules. Organic matter content in cryoconite varied between glaciers, ranging from 1% to 38%. The geochemistry of all the investigated samples reflected local sediment sources, except of highly concentrated Pb and Hg in cryoconite collected from European glaciers near industrialized regions, corroborating cryoconite as element-specific collector and potential environmental indicator of anthropogenic activity. Our work supports a notion that cryoconite may be more than just simple sediment and instead exhibits complex structure with relevance for biodiversity and the functioning of glacial ecosystems.

Lymph node ratio is a powerful prognostic index in patients with stage III distal rectal cancer: a Japanese multicenter study.

PURPOSE: The present study aims to define the prognostic impact of the lymph node ratio (LNR) in patients with stage III distal rectal cancer. METHODS: We analyzed data from 501 patients who underwent curative resection (total mesorectal excision, TME) for stage III distal rectal cancer at 12 institutions between 1991 and 1998. Patients were divided into four groups according to quartiles based on LNR. RESULTS: Among the 501 patients, 381 underwent TME with pelvic sidewall dissection (PSD). The median numbers of lymph nodes retrieved with and without PSD were 45 and 17, respectively (P < 0.0001). Forty-nine patients with lymph node retrieved less than 12 were excluded from further analyses. Among various clinicopathological parameters, univariate analysis identified age (P = 0.0059), histological grade (P < 0.0001), depth of tumor invasion (P = 0.0003), and number of positive nodes (P < 0.0001) and LNR (P < 0.0001) as prognostic factors. The Cox proportional hazards model revealed that age (P = 0.014), histological grade (P < 0.0001), depth of tumor invasion (P = 0.0002), and LNR (group 3, P = 0.0012; group 4, P < 0.0001) were independent prognostic factors. When the American Joint Committee on Cancer (AJCC) seventh staging system was added as a covariate, both AJCC stage (P < 0.0001) and LNR (P < 0.0001) were independent prognostic factors. CONCLUSIONS: Adding the LNR concept to the AJCC cancer staging system will improve accuracy in evaluating the nodal status of distal rectal cancer.

Snow algae blooms are beneficial for microinvertebrates assemblages (Tardigrada and Rotifera) on seasonal snow patches in Japan.

Although studies on snow algae and macroinvertebrates have been frequently conducted on snow patches, only few surveys have been focused on microinvertebrates which reach high biomass and play various trophic roles in other cold habitats. The aims of this study were (1) to search for microinvertebrates in seasonal surface snow patches located on the slope of Mt. Gassan, in northern Japan, and (2) to identify factors determining their distribution associated with snow algal blooms of various colorations (orange, green, and golden-brown) collected from the same sampling site over two seasons (2018, 2019). Microscopic observation revealed presence of two major groups of microinvertebrates: Tardigrada and Rotifera. They were concentrated in green snow colored by blooms of Chloromonas sp. in comparison to orange or golden-brown snow and only a few were found in white snow. Mean body length of tardigrades increased throughout the melt season, their intestine content was green and they laid eggs on colored snow. These results suggest that tardigrades preferentially grew and reproduced on green snow patches. Population densities of tardigrades, rotifers and concentration of chlorophyll a were significantly correlated. Our study indicates that green snow patches in temperate mountainous forests constitute important and unique low-temperature ecosystems for microinvertebrates. Snow covered by algae is an unrecognized novel habitats for tardigrades and rotifers.