Tissue Imprints Research Articles

Coinfection with Dolphin Morbillivirus (DMV) and Gammaherpesvirus in a Spinner Dolphin (Stenella longirostris) Stranded in Sri Lanka.

Following the X-Press Pearl maritime disaster off the coast of Sri Lanka, a stranded spinner dolphin (Stenella longirostris) was recovered, and the cause of death was investigated. Post-mortem examinations revealed evidence of by-catch, but a natural coinfection with dolphin morbillivirus (DMV) and gammaherpesvirus was detected by further analyses, marking the first documented case of a dual viral infection in this species within the region. Molecular diagnostics, including PCR and sequencing, were performed on tissue imprints collected on FTA® cards, confirming the presence of DMV in the prescapular lymph node and gammaherpesvirus in the lesions in the oral cavity. The concurrent detection of DMV and gammaherpesvirus raises significant concerns regarding the potential impacts of environmental stressors, such as chemical pollutants from the X-Press Pearl maritime disaster, on exacerbating susceptibility to viral infections in marine mammals. These findings highlight the need for ongoing surveillance of cetacean populations in the Indian Ocean to better understand pathogen circulation and health and conservation implications of anthropogenic activities on the marine ecosystem.

THE FIRST CENOZOIC OCTOPOD: A LOWER EOCENE RECORD FROM BOLCA, NORTHEASTERN ITALY

To date, soft-tissue remains of extinct incirrate octopods have been described exclusively from UpperCretaceous deposits. Here, three specimens of an incirrate octopodid with well-preserved soft tissue imprints are described for the first time from Paleogene strata. This material originates from the upper Ypresian (lower Eocene) fish-bearing levels of the Bolca Konservat-Lagerstätte in the Pesciara of Bolca in north-eastern Italy. Previously, these specimens had tentatively been interpreted as teuthids. Based on a detailed study of their anatomical structure, a new genus and species of octopodid, Bolcaoctopus pesciaraensis, belonging to the extant family Octopodidae, are here erected. Bolcaoctopus gen. nov. may be distinguished from the Late Cretaceous genus Styletoctopus by the presence of long and thin arms and an elongated body shape with a narrowed apical end and covered by longitudinal wrinkles. Although finds of fossil octopuses in the Bolca Konservat-Lagerstätte remain extremely rare, it is worth noting that Cenozoic marine vertebrate localities constitute an important potential source of additional finds of coleoid cephalopods, the study of which may shed light on a poorly understood period in the evolutionary history of these molluscs.

Isoporous Membrane Mediated Imprinting Mass Spectrometry Imaging for Spatially-Resolved Metabolomics and Rapid Histopathological Diagnosis.

Surface-assisted laser desorption/ionization (SALDI) mass spectrometry imaging (MSI) holds great value in spatial metabolomics and tumor diagnosis. Tissue imprinting on the SALDI target can avoid laser-induced tissue ablation and simplifies the sample preparation. However, the tissue imprinting process always causes lateral diffusion of biomolecules, thereby losing the fidelity of metabolite distribution on tissue. Herein, a membrane-mediated imprinting mass spectrometry imaging (MMI-MSI) strategy is proposed using isoporous nuclepore track-etched membrane as a mediating imprinting layer to selectively transport metabolites through uniform and vertical pores onto silicon nanowires (SiNWs) array. Compared with conventional direct imprinting technique, MMI-MSI can not only exclude the adsorption of large biomolecules but also avoid the lateral diffusion of metabolites. The whole time for MMI-based sample preparation can be reduced to 2min, and the lipid peak number can increase from 46 to 113 in kidney tissue detection. Meanwhile, higher resolution of MSI can be achieved due to the confinement effect of the pore channel in the diffusion of metabolites. Based on MMI-MSI, the tumor margins of liver cancer can be clearly discriminated and their different subtypes can be precisely classified. This work demonstrates MMI-MSI is a rapid, highly sensitive, robust and high-resolution technique for spatially-resolved metabolomics and pathological diagnosis.

Group 1 ILCs: Heterogeneity, plasticity, and transcriptional regulation.

Group 1 innate lymphoid cells (ILCs), comprising ILC1s and natural killer cells (NK cells), belong to a large family of developmentally related innate lymphoid cells that lack rearranged antigen-specific receptors. NK cells and ILC1s both require the transcription factor T-bet for lineage commitment but additionally rely on Eomes and Hobit, respectively, for their development and effector maturation programs. Both ILC1s and NK cells are essential for rapid responses against infections and mediate cancer immunity through production of effector cytokines and cytotoxicity mediators. ILC1s are enriched in tissues and hence generally considered tissue resident cells whereas NK cells are often considered circulatory. Despite being deemed different cell types, ILC1s and NK cells share many common features both phenotypically and functionally. Recent studies employing single cell RNA sequencing (scRNA-seq) technology have exposed previously unappreciated heterogeneity in group 1 ILCs and further broaden our understanding of these cells. Findings from these studies imply that ILC1s in different tissues and organs share a common signature but exhibit some unique characteristics, possibly stemming from tissue imprinting. Also, data from recent fate mapping studies employing Hobit, RORγt, and polychromic reporter mice have greatly advanced our understanding of the developmental and effector maturation programs of these cells. In this review, we aim to outline the fundamental traits of mouse group 1 ILCs and explore recent discoveries related to their developmental programs, phenotypic heterogeneity, plasticity, and transcriptional regulation.

Noninvasive Detection of Skin Cancer by Imprint Desorption Electrospray Ionization Mass Spectrometry Imaging.

We report a technique for the noninvasive detection of skin cancer by imprint desorption electrospray ionization mass spectrometry imaging (DESI-MSI) using a transfer agent that is pressed against the tissue of interest. By noninvasively pressing a tape strip against human skin, metabolites, fatty acids, and lipids on the skin surface are transferred to the tape with little spatial distortion. Running DESI-MSI on the tape strip provides chemical images of the molecules on the skin surface, which are valuable for distinguishing cancer from healthy skin. Chemical components of the tissue imprint on the tape strip and the original basal cell carcinoma (BCC) section from the mass spectra show high consistency. By comparing MS images (about 150-μm resolution) of same molecules from the tape strip and from the BCC section, we confirm that chemical patterns are successfully transferred to the tape stripe. We also used the technique to distinguish cherry angiomas from normal human skin by comparing the molecular patterns from a tape strip. These results demonstrate the potential of the imprint DESI-MSI technique for the noninvasive detection of skin cancers as well as other skin diseases before and during clinical surgery.

Comparison between Sampling Techniques for Virological Molecular Analyses: Dolphin Morbillivirus and Herpesvirus Detection from FTA® Card and Frozen Tissue.

Stranded animals offer valuable information on marine mammal physiology and pathology; however, the decomposition state of the carcasses and lack of a rigorous cold chain for sample preservation can sometimes discourage diagnostic analyses based on nucleic acid detection. The present paper aims at evaluating the reliability of FTA® card tissue imprints as an alternative matrix to frozen tissues for virological analyses based on biomolecular methods. Given the contribution of Cetacean morbillivirus (CeMV) to strandings and the increase of herpesvirus detection in cetaceans, these two pathogens were selected as representative of RNA and DNA viruses. Dolphin morbillivirus (DMV) and herpesvirus presence was investigated in parallel on tissue imprints on FTA® cards and frozen tissues collected during necropsy of dolphins stranded in Italy. Samples were analysed by nested RT-PCR for DMV and nested-PCR for herpesvirus. Only one animal was positive for herpesvirus, hampering further considerations on this virus. DMV was detected in all animals, both in FTA® card imprints and tissue samples, with differences possibly related to the decomposition condition category of the carcasses. Tissue sampling on FTA® cards seems a promising alternative to frozen tissues for biomolecular analyses, especially when ensuring adequate storage and shipment conditions for frozen tissues is difficult.

Retinoic acid drives intestine-specific adaptation of effector ILC2s originating from distant sites.

Adaptation of immune cells to tissue-specific microenvironments is a crucial process in homeostasis and inflammation. Here, we show that murine effector type 2 innate lymphoid cells (ILC2s) from various organs are equally effective in repopulating ILC2 niches in other anatomical locations where they adapt tissue-specific phenotypes of target organs. Single-cell transcriptomics of ILC2 populations revealed upregulation of retinoic acid (RA) signaling in ILC2s during adaptation to the small intestinal microenvironment, and RA signaling mediated reprogramming of kidney effector ILC2s toward the small intestinal phenotype in vitro and in vivo. Inhibition of intestinal ILC2 adaptation by blocking RA signaling impaired worm expulsion during Strongyloides ratti infection, indicating functional importance of ILC2 tissue imprinting. In conclusion, this study highlights that effector ILC2s retain the ability to adapt to changing tissue-specific microenvironments, enabling them to exert tissue-specific functions, such as promoting control of intestinal helminth infections.

Anti-cancer activity of Chaga mushroom (Inonotus obliquus) against dog bladder cancer organoids.

Despite its disadvantages, chemotherapy is still commonly used for the treatment of bladder cancer (BC). Developing natural supplements that can target cancer stem cells (CSCs) which cause drug resistance and distant metastasis is necessary. Chaga mushrooms are popular to have several health-promoting and anti-cancer potentials. Organoid culture can recapitulate tumor heterogeneity, epithelial environment, and genetic and molecular imprints of the original tissues. In the previous study, we generated dog bladder cancer organoids (DBCO) as a novel experimental model of muscle-invasive BCO. Therefore, the present study aimed to examine the anti-tumor potentials of Chaga mushroom extract (Chaga) against DBCO. Four strains of DBCO were used in the present study. Treatment with Chaga inhibited the cell viability of DBCO in a concentration-dependent way. Treatment of DBCO with Chaga has significantly arrested its cell cycle and induced apoptosis. Expression of bladder CSC markers, CD44, C-MYC, SOX2, and YAP1, declined in the Chaga-treated DBCO. Also, Chaga inhibited the phosphorylation of ERK in DBCO. Expression of downstream signals of ERK, C-MYC, and Cyclins (Cyclin-A2, Cyclin-D1, Cyclin-E1, and CDK4) was also inhibited by Chaga in DBCO. Interestingly, the combinational treatment of DBCO with Chaga and anti-cancer drugs, vinblastine, mitoxantrone, or carboplatin, showed a potentiating activity. In vivo, Chaga administration decreased tumor growth and weight of DBCO-derived xenograft in mice with the induction of necrotic lesions. In conclusion, Chaga diminished the cell viability of DBCO by inhibiting proliferation-related signals and stemness conditions as well as by arresting the cell cycle. Collectively, these data suggest the value of Chaga as a promising natural supplement that could potentiate the effect of adjuvant chemotherapy, lower its adverse effects, and thus, limit the recurrence and metastasis of BC.

The regulation mechanism of hematopoiesis during ischemic and hemolytic anemia in grass carp(Ctenopharyngodon idella)

Grass carp (C. idella) is an important farmed freshwater fish in China. In recent years, grass carp hemorrhagic diseases caused by viruses, bacteria, and parasites have hindered the aquaculture industry; however, there are few studies on the hematopoiesis of grass carp. It is reported that erythropoiesis is a complex biological process, which requires hematopoietic-stimulating factors, inhibitors, the hematopoietic microenvironment, and stromal cells to interact for fine regulation. In this study, ischemic anemia (a blood loss model) and hemolytic anemia (a hemolysis model) were established to stimulate hematopoiesis of grass carp. The hematopoietic organs of grass carp were studied using tissue imprinting, hematoxylin-eosin (H&E) staining, western blot (WB), and immunofluorescence assay (IFA). Hematopoietic-stimulating factors and iron metabolism-related factors were detected by quantitative real-time PCR (qRT-PCR). The results of tissue imprinting showed that there were proerythrocytes in the head kidney, spleen, and middle kidney. In the model of ischemic anemia, a hematopoietic island could be found in head kidney and spleen at 24 h. The hematopoietic island appeared in the middle kidney on the 7th day. The results of H&E staining showed that when the peripheral blood decreased sharply, the spleen compensated for the peripheral blood and decreased the melanin macrophage center in head kidney, spleen, and middle kidney. The results of WB, IFA, and qRT-PCR showed that the hematopoietic pathway and iron metabolism were activated in both models. In sum, the head kidney, spleen, and middle kidney of grass carp are its main hematopoietic organs, and the head kidney is the most important. The spleen is an important blood bank of grass carp and hematopoiesis compensation occurs in the spleen and mesonephros during severe ischemia.

IDENTIFICATION AND RETROSPECTIVE EVALUATION OF A FILARIOID NEMATODE SPECIES IN MANAGED GRASSHOPPER SPARROWS (AMMODRAMUS SAVANNARUM).

The grasshopper sparrow (Ammodramus savannarum) is a species of ground-dwelling passerine bird with 12 different subspecies. The Florida subspecies (Ammodramus savannarum floridanus) is classified as federally endangered, with the most common threats including habitat loss, nest predation, and floods. A managed breeding program was established at White Oak Conservation (Yulee, FL) in 2015 with eastern grasshopper sparrows (Ammodramus savannarum pratensis) as a model for breeding Florida grasshopper sparrows as part of an assurance colony. A filarioid parasite species (Aproctella sp.) was characterized by PCR after identification by blood films and postmortem examinations of both subspecies housed at White Oak Conservation. This Aproctella species was distinct from others with available sequence. Records from 157 eastern and Florida grasshopper sparrows were reviewed, and correlations between presence of filariasis and subspecies, sex, body condition score, and presence of systemic isosporosis, squamous metaplasia, coelomitis, airsacculitis, or a combination of conditions were investigated. Twenty-nine (18.5%) birds (13 of 71 Florida grasshopper sparrows; 16 of 86 eastern grasshopper sparrows) were positive for filariasis by blood film review, grossly or by tissue imprint at postmortem examination, or histologically. Filariasis was significantly correlated with systemic isosporosis, coelomitis, and airsacculitis; was not correlated with subspecies, sex, or squamous metaplasia; and had a questionable correlation with body condition score. This report provides evidence that this Aproctella species has potential to contribute to morbidity and mortality in the grasshopper sparrow. This information will be helpful for implementing effective measures against suspected vectors and for the development of best practice strategies for the health management of the species in breeding programs.

Porous Graphitic Carbon-Based Imprint Mass Spectrometry Imaging with an Ambient Liquid Extraction Technique for Enhancing Coverage of Glycerolipids and Sphingolipids in Brain Tissue.

Localization of lipidomes and tracking their spatial changes by mass spectrometry imaging (MSI) is critical for the mechanism studies on living process, disease, and therapeutic treatment. However, due to the strong ion suppression in complex biotissue, the imaging coverage for lipids with low polarity or low abundances, such as glycerolipids and sphingolipids, is usually limited. To address this issue, we utilized a porous graphitic carbon (PGC) material to imprint brain tissue sections for selective enrichment of neutral lipids with polar phospholipids removed. Then, the tissue imprint was scanned for desorption by the ambient liquid extraction MSI system. It was found that on the PGC surface, hydrophobic interaction dominates in protic solvents, and polar interaction dominates in aprotic solvents. Accordingly, methanol was selected as the spray solvent for tissue imprinting, and 75% acetonitrile-methanol was selected as the desorption solvent for the ambient liquid extraction MSI system. The results showed that glycerides had high recoveries after the imprinting-desorption process (recovery ∼ 70%) with phospholipids eliminated (recovery < 7%). To increase the transferring efficiencies of lipids from tissue onto PGC, electrospray was used for solvent application during imprinting, and the signals of diglycerides (DGs) in the imprint MSI of brain tissue increased by 2-3 times as compared to those via air spray. Finally, the new imprint MSI approach was applied to the imaging of the rat cerebellum and was compared with direct tissue MSI. The results showed that with imprint MSI, the coverage of DGs, sphingomyelins (SMs), and ceramides was enhanced by 4-5-fold (32 vs 6, 4 vs 1, and 5 vs 0). The ion images showed that with imprint MSI, higher signal intensities and clearer spatial distribution of DGs and SMs were obtained without interference from phosphatidylcholine signals compared with tissue MSI. This new method provides a complementary approach for traditional MSI to address the issues in imaging poorly ionizable or low-abundance lipids.

Parenteral vaccination strategies target protective immunity to the small and large intestine mucosa

Abstract Prophylactic vaccines are most commonly given by a parenteral route. However, this strategy often confers poor protection against GI pathogens because antigens are primarily presented in peripheral lymph nodes, which do not provide the prerequisite ‘tissue imprinting signals’ to target memory T and B cells to the gut. Here, we describe a platform that enables parenteral vaccines to target protection to the GI tract. This was achieved by subcutaneous (s.c.) delivery of compounds known to imprint gut tropism onto lymphocytes. Am80, a retinoic acid receptor agonist, targeted immunity to the small intestine through up-regulation of gut-homing receptors α4β7 and CCR9, and promotion of IgA class-switching. S.c. injection of OVA + CpG combined with repeated injections of Am80 generated significantly higher and durable fecal anti-OVA IgA titers, compared to OVA + CpG without Am80. Vaccination with Am80 also increased antigen-specific CD4 and CD8 T cell trafficking to the small intestine and generated tissue resident memory T (Trm) cells. To overcome the need for multiple doses of Am80, which can cause tissue toxicity at the injection site and hinder clinical translation, we developed a polymeric nanocarrier that showed equivalent imprinting efficacy upon a single injection, lowering the cumulative dose to one-third. Furthermore, we also developed a modified vaccination strategy that could augment effector and memory T cell responses not only in the small intestine, but also in the cecum, colon and rectum, conferring protection to the entire intestinal tract. We conclude that supplementation with mucosal imprinting signals is a promising approach to enhance parenteral vaccines by generating mucosal protection against GI infections. Supported by the Bill & Melinda Gates Foundation

A common framework of monocyte-derived macrophage activation.

Macrophages populate every organ during homeostasis and disease, displaying features of tissue imprinting and heterogeneous activation. The disconnected picture of macrophage biology that has emerged from these observations is a barrier for integration across models or with in vitro macrophage activation paradigms. We set out to contextualize macrophage heterogeneity across mouse tissues and inflammatory conditions, specifically aiming to define a common framework of macrophage activation. We built a predictive model with which we mapped the activation of macrophages across 12 tissues and 25 biological conditions, finding a notable commonality and finite number of transcriptional profiles, in particular among infiltrating macrophages, which we modeled as defined stages along four conserved activation paths. These activation paths include a "phagocytic" regulatory path, an "inflammatory" cytokine-producing path, an "oxidative stress" antimicrobial path, or a "remodeling" extracellular matrix deposition path. We verified this model with adoptive cell transfer experiments and identified transient RELMɑ expression as a feature of monocyte-derived macrophage tissue engraftment. We propose that this integrative approach of macrophage classification allows the establishment of a common predictive framework of monocyte-derived macrophage activation in inflammation and homeostasis.

Tissue Imprinting on 2D Nanoflakes-Capped Silicon Nanowires for Lipidomic Mass Spectrometry Imaging and Cancer Diagnosis.

Spatially resolved tissue lipidomics is essential for accurate intraoperative and postoperative cancer diagnosis by revealing molecular information in the tumor microenvironment. Matrix-free laser desorption ionization mass spectrometry imaging (LDI-MSI) is an emerging attractive technology for label-free visualization of metabolites distributions in biological specimens. However, the development of LDI-MSI technology that could conveniently and authentically reveal molecular distribution on tissue samples is still a challenge. Herein, we present a tissue imprinting technology by retaining tissue lipids on 2D nanoflakes-capped silicon nanowires (SiNWs) for further mass spectrometry imaging and cancer diagnosis. The 2D nanoflakes were prepared by liquid exfoliation of molybdenum disulfide (MoS2) with nitrogen-doped graphene quantum dots (NGQDs), which serve as both intercalation agent and dispersant. The obtained NGQD@MoS2 nanoflakes were then decorated on the tip of vertical SiNWs, forming a hybrid NGQD@MoS2/SiNWs nanostructure, which display excellent lipid extraction ability, enhanced LDI efficiency and molecule imaging capability. The peak number and total ion intensity of different lipids species on animal lung tissues obtained by tissue imprinting LDI-MSI on NGQD@MoS2/SiNWs were ∼4-5 times greater than those on SiNWs substrate. As a proof-of-concept demonstration, the NGQD@MoS2/SiNWs nanostructure was further applied to visualize phospholipids on sliced non small cell lung cancer (NSCLC) tissue along with the adjacent normal tissue. On the basis of selected feature lipids and machine learning algorithm, a prediction model was constructed to discriminate NSCLC tissues from the adjacent normal tissues with an accuracy of 100% for the discovery cohort and 91.7% for the independent validation cohort.

Molecular and microscopic identification of Eomarteilia granula infection in Manila clam Ruditapes philippinarum off the south coast of Korea.

A report on the new species Eomarteilia (=Marteilia) granula infecting Manila clam Ruditapes philippinarum from Japan in 2014 suggests the possibility of E. granula infecting other Manila clam populations in the Northwest Pacific region, including Korea. In this study, we report the first infections by E. granula in Manila clams off the south coast of Korea. Histology revealed Marteilia-like plasmodia in the digestive tubule epithelia. Tissue imprints demonstrated that each parasite sporangium enclosed 4 spores and transmission electron microscopy (TEM) revealed ultrastructure of primary cells enclosing secondary cells, which contained spores. Mature spores consisted of 3 sporoplasms: outermost, intermediate, and innermost. The outermost sporoplasm showed a peripheral electron-dense monolayer characteristic of E. granula. The 18S rDNA amplified from the Marteilia-like parasite yielded 1784-bp PCR amplicon sequences which were 99.8% similar to that of E. granula previously reported (as M. granula) from Japan. In the molecular phylogenetic analysis, the novel Marteilia-like organism formed a well-supported clade with E. granula. Accordingly, we concluded that the novel Marteilia-like parasite that we found infecting some Korean Manila clams is Eomarteilia granula. Field surveys revealed that the infection was limited to clams of the south coast of Korea, with the prevalence ranging from 3.3 to 5.0%.

Monolithic Gold Nanoparticles/Thiol-β-cyclodextrin-Functionalized TiO2 Nanowires for Enhanced SALDI MS Detection and Imaging of Natural Products.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has been successfully applied in the analysis of various small molecules. In this work, gold nanoparticles/thiol-β-cyclodextrin-functionalized TiO2 nanowires (AuNPs/SH-β-CD-TiO2 NWs) were prepared to enhance the performance of SALDI MS and mass spectrometry imaging (MSI). A monolithic TiO2 film was first grown on an indium tin oxide (ITO) glass slide via a modified sol-gel method and treated in an alkaline environment to form nanowires. TiO2 NWs were chemically modified by SH-β-CD for immobilizing AuNPs densely and strongly. Compared with the conventional organic matrix 2,5-dihydroxybenzoic acid (DHB), the prepared AuNPs/SH-β-CD-TiO2 NWs showed superior performances on detection sensitivity, repeatability, and analyte coverage. Analytes typically detectable with negative-ion matrix-assisted laser desorption/ionization (MALDI) MS could also be observed using AuNPs/SH-β-CD-TiO2 NWs in the positive ion mode. Its successful usage efficiently enhanced the SALDI MS detection of various small molecules such as carbohydrates, fatty acids, and bile acids in the positive ion mode. The developed SALDI substrate was further used to characterize and discriminate the natural and in vitro cultured Calculus Bovis, as well as natural and artificial Moschus. Furthermore, the spatial distribution of several natural products in spearmint leaves and potato tubers was explored by tissue imprinting and deposition on the AuNPs/SH-β-CD-TiO2 NW surface for SALDI MSI in dual-polarity mode, respectively. The wide application and satisfied detection sensitivity make AuNPs/SH-β-CD-TiO2 NWs ideal for SALDI MS and MSI of various natural products.

Impact of molecular surgical margin analysis on the prediction of pancreatic cancer recurrences after pancreaticoduodenectomy

BackgroundPancreatic cancer is one of the lethal cancers among solid malignancies. Pathological diagnosis of surgical margins is sometimes unreliable due to tissue shrinkage, invisible field cancerization and skipped lesions like tumor budding. As a result, tumor recurrences sometimes occur even from the pathologically negative surgical margins.MethodsWe applied molecular surgical margin (MSM) analysis by tissue imprinting procedure to improve the detection sensitivity of tiny cancerous cells on the surgical specimen surface after pancreatoduodenectomy. Surgical specimens were collected from 45 pancreatic cancer cases who received subtotal stomach preserving pancreatoduodenectomy at Nagoya University Hospital during 2017–2019. Quantitative methylation-specific PCR (QMSP) of the original methylation marker panel (CD1D, KCNK12, PAX5) were performed and analyzed with postoperative survival outcomes.ResultsAmong 45 tumors, 26 cases (58%) were QMSP-positive for CD1D, 25 (56%) for KCNK12 and 27 (60%) for PAX5. Among the 38 tumors in which at least one of the three markers was positive, CD1D-positive cancer cells, KCNK12-positive cancer cells, and PAX5-positive cancer cells were detected at the surgical margin in 8 cases, 7 cases and 10 cases, respectively. Consequently, a total of 17 patients had at least one marker detected at the surgical margin by QMSP, and these patients were defined as MSM-positive. They were associated with significantly poor recurrence-free survival (p = 0.002) and overall survival (p = 0.005) than MSM-negative patients. Multivariable analysis showed that MSM-positive was the only significant independent factor for worse recurrence-free survival (hazard ratio: 3.522, 95% confidence interval: 1.352–9.179, p = 0.010). On the other hand, a significant proportion of MSM-negative cases were found to have received neoadjuvant chemotherapy (p = 0.019).ConclusionPancreatic cancer-specific methylation marker panel was established to perform MSM analysis. MSM-positive status might represent microscopically undetectable cancer cells on the surgical margin and might influence the postoperative long-term outcomes.

Memory CD8+ T cells exhibit tissue imprinting and non-stable exposure-dependent reactivation characteristics following blood-stage Plasmodium berghei ANKA infections.

Experimental cerebral malaria (ECM) is a severe complication of Plasmodium berghei ANKA (PbA) infection in mice, characterized by CD8+ T‐cell accumulation within the brain. Whilst the dynamics of CD8+ T‐cell activation and migration during extant primary PbA infection have been extensively researched, the fate of the parasite‐specific CD8+ T cells upon resolution of ECM is not understood. In this study, we show that memory OT‐I cells persist systemically within the spleen, lung and brain following recovery from ECM after primary PbA‐OVA infection. Whereas memory OT‐I cells within the spleen and lung exhibited canonical central memory (Tcm) and effector memory (Tem) phenotypes, respectively, memory OT‐I cells within the brain post‐PbA‐OVA infection displayed an enriched CD69+CD103− profile and expressed low levels of T‐bet. OT‐I cells within the brain were excluded from short‐term intravascular antibody labelling but were targeted effectively by longer‐term systemically administered antibodies. Thus, the memory OT‐I cells were extravascular within the brain post‐ECM but were potentially not resident memory cells. Importantly, whilst memory OT‐I cells exhibited strong reactivation during secondary PbA‐OVA infection, preventing activation of new primary effector T cells, they had dampened reactivation during a fourth PbA‐OVA infection. Overall, our results demonstrate that memory CD8+ T cells are systemically distributed but exhibit a unique phenotype within the brain post‐ECM, and that their reactivation characteristics are shaped by infection history. Our results raise important questions regarding the role of distinct memory CD8+ T‐cell populations within the brain and other tissues during repeat Plasmodium infections.

Commission test of the efficacy of the supramolecular complex of ivermectin against gastrointestinal strongylatoses of horses

The purpose of the researchis a commission test of the efficacy of the supramolecular complex of ivermectin at gastrointestinal strongylatoses of horses.Materials and methods. The commission experiment was performed in June 2019 in the North Caucasian Federal District of the Chechen Republic. The efficacy of the supramolecular complex of ivermectin was evaluated on 20 horses of different breeds in the Seradin Equestrian Center. To determine the rate of gastrointestinal nematode infection in horses, we collected fresh feces in the morning, made tissue imprints from perianal folds and examined them by the Fülleborn's method using a saturated solution of sodium chloride.Results and discussion. The coproovoscopic examination determined a 100% infection of horses with Strongylates and 40.0% infection with Oxyuris sp., with 488.7±24.4 strongylates eggs and 16.9±0.84 Oxyuris sp. eggs found in 1 g of feces, respectively. Seven and 15 days after the supramolecular complex of ivermectin administered to the horses from the first test group at a dose of 0.15 mg/kg for the active substance (7.5 mg/kg for the drug) in a mixture with mixed feed, we did not find any Strongylata eggs or Oxyuris sp. eggs in feces or on tissue imprints. Insufficient efficacy was obtained in the horses from the second test group after being treated with the active substance of ivermectin at a dose of 0.15 mg/kg. The strongylate egg number decreased on average by 244.3±12.21, namely, by 50.1%, and no Oxyuris sp. eggs were found. The efficacy against Oxyuris sp. was 100%. The horses consumed the mixture of drugs with feed readily. There were no side effects after the drugs applied. Thus, we established the high efficacy of the supramolecular complex of ivermectin against gastrointestinal strongylatoses, and the convenience of its use in a mixture with feed to untamed herd horses individually and by group feeding.

First evidence of fish nocardiosis in Mexico caused by Nocardia seriolae in farmed red drum (Sciaenops ocellatus, Linnaeus).

Between August and December 2013, the offshore cages of a commercial marine farm culturing red drum Sciaenops ocellatus in Campeche Bay Mexico were affected by an outbreak of an ulcerative granulomatous disease with up to 70% cumulative mortality. Thirty-one adults displaying open ulcers on the skin were submitted for diagnosis. At necropsy, multiple white-yellowish nodules (0.1-0.5cm in diameter) were present in all internal organs, where the kidney and the spleen were the most severely affected. Histopathology evinced typical systemic granulomatous formations. Gram and Ziehl-Neelsen stains on tissue imprints, bacterial swabs and tissue sections revealed Gram-positive, acid-fast, branching beaded long rod filamentous bacteria. Tissue samples resulted positive for nocardiosis with a Nocardia genus-specific nested PCR. Definite identification at the species level and taxonomic positioning of the fastidious pathogen were achieved through a specific Nocardia seriolae PCR and by sequencing the gyrB gene of pure isolates. After administration of antibiotics during fry production, a posterior follow-up monitoring (from 2014 to 2017) detected mild but recurrent outbreaks of the bacteria with no seasonality pattern. To the extent of our knowledge, this is the first report of piscine nocardiosis in Mexico and the first time this disease is detected in red drum.