Kaempferol Inhibits Cervical Cancer Cells by Inducing Apoptosis and Autophagy via Inactivation of the PI3K/AKT/mTOR Signaling Pathway.

BACKGROUND/AIM: Kaempferol, a natural flavonoid, occurs abundantly in fruits and vegetables. It has various bioactivities, with antioxidant, anti-inflammatory, and other beneficial properties. The aim of this study was to investigate the in vitro effects of kaempferol on the proliferation, apoptosis, and autophagy of KB cells, a human cervical cancer cell line, and the corresponding action mechanisms. MATERIALS AND METHODS: The inhibitory efficacy of kaempferol on KB cervical cancer cells was investigated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, migration assay, 4',6-diamidino-2-phenylindole staining, flow cytometry, acridine orange staining and western blotting. RESULTS: Kaempferol reduced KB cell viability and migration in a dose-dependent manner. Additionally, kaempferol-induced apoptosis was confirmed, and kaempferol treatment influenced levels of apoptotic proteins. Autophagy was detected upon visualization of characteristic autophagic vacuoles and acidic vesicular organelles, and verified using western blotting, which revealed elevated levels of autophagy-related proteins. Kaempferol-mediated apoptosis and autophagy were evidently attributable to reduced phosphorylation in the phosphoinositide 3-kinase (PI3K)/serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) pathway. This finding was validated using a pharmacological inhibition assay with the PI3K pathway inhibitor LY294002, which promoted KB cell apoptosis and autophagy. CONCLUSION: Our results suggest that kaempferol induces apoptosis and autophagy by inhibiting the PI3K/AKT/mTOR pathway in human cervical cancer cells, empirically showing the anticancer effects of kaempferol, and thereby presenting it as a potential anticancer therapeutic agent.

Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy.

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

Exploring cellular immunotherapy platforms in multiple myeloma.

Despite major advances in therapeutic platforms, most patients with multiple myeloma (MM) eventually relapse and succumb to the disease. Among the novel therapeutic options developed over the past decade, genetically engineered T cells have a great deal of potential. Cellular immunotherapies, including chimeric antigen receptor (CAR) T cells, are rapidly becoming an effective therapeutic modality for MM. Marrow-infiltrating lymphocytes (MILs) derived from the bone marrow of patients with MM are a novel source of T cells for adoptive T-cell therapy, which robustly and specifically target myeloma cells. In this review, we examine the recent innovations in cellular immunotherapies, including the use of dendritic cells, and cellular tools based on MILs, natural killer (NK) cells, and CAR T cells, which hold promise for improving the efficacy and/or reducing the toxicity of treatment in patients with MM.

Platycodin D induces apoptosis via regulating MAPK pathway and promotes autophagy in colon cancer cell.

Platycodin D (PD) is the main component of triterpene saponins found in Platycodi radix. In this study, we observed a decrease in cell viability, an increase in apoptotic bodies, and an increase in the rate of apoptosis. Also, we observed an increase in cleaved PARP and Bax, a decrease in Bcl-2, and p-ERK, and an increase in p-p38 and p-JNK. Furthermore, a change in cell viability and the expression of p-p38, Bax, and Bcl-2 using the p38 inhibitor revealed a decrease in p-p38 and Bax and an increase in Bcl-2 in the inhibitor treatment group. In addition, we observed an increase in vacuole formation through morphological changes and an increase in acidic vesicular organelles (AVOs). We also observed an increase in the expression of beclin 1, LC 3-I, and -II. There was no significant decrease in cell viability in the group treated with 3-MA, but a decrease in cell viability was noted in the group treated with HCQ. HCQ treatment resulted in an increase in Bax and a decrease in Bcl-2. These findings reveal that in HT-29 colon cancer cells, PD induces apoptosis through the MAPK pathway, thereby exerting anticancer effects. Moreover, autophagy caused by PD inhibits apoptosis by protecting the cells.

Comparative analysis of the aberrant immunophenotype and clinical characteristics in dogs with lymphoma: a study of 27 cases.

Introduction: Aberrant lymphoma phenotypes are frequently found in dogs, but the clinical implications are sparse. Methods: Twenty-seven dogs with aberrant lymphoma diagnosed using flow cytometry between 2017 and 2023 were analyzed. Major paraneoplastic syndromes, prognostic factors, and clinical features of lymphoma were compared to their immunophenotypes. Results: Twenty-seven dogs had aberrant immunophenotypes, with MHCII- (48%) and CD3+/CD21+ (44%) being the most commonly identified aberrancies. In B-cell lymphoma, the most frequent aberrancies were MHC II- (53%), CD3+/CD21+ (41%), CD34+ (24%), and CD79a- (24%). Meanwhile, in T-cell lymphoma, CD3+/CD21+ (63%), CD4-/CD8-(50%), CD5- (50%), and CD45- (50%) were the most common. The platelet-neutrophil ratio was significantly higher in the CD3+/CD21+ group than in the other groups, where either one or both markers were not expressed (55.23 ± 39.64; 18.72 ± 14.95, respectively; p = 0.001). Serum albumin concentration was significantly lower in the MHCII-group (2.59 g/dL, 95% CI 2.31-2.87) than in the MHCII+ group (3.06 g/dL, 95% CI 2.88-3.23; p = 0.009). CD34 expression showed significant correlations with cranial mediastinal mass, WHO clinical substage, and fever (p = 0.028, p = 0.041, and p = 0.047, respectively). MHCII expression was correlated with adverse reactions to chemotherapy, cranial mediastinal masses, and fever (p = 0.009, p = 0.023, and p < 0.001, respectively). No statistically significant differences in the survival period were observed for any of the phenotypic aberrancies. Conclusion: Aberrant lymphomas are common in dogs. Some clinical prognostic factors that significantly correlate with aberrant immunophenotypes have been identified and can be applied clinically.

Intensified NK cell therapy in combination with low-dose chemoradiotherapy against human colorectal cancer.

The therapeutic potential of adoptive natural Killer (NK) cells immunotherapy in combination with chemoradiotherapy, the main treatment modality for colorectal cancer (CRC), has not yet been explored. Here, we aimed to investigate the efficacy of NK cells to potentiate primary tumor control and improve survival outcomes, especially in combination with low-dose chemoradiotherapy. Ex vivo activated NK cells (> 90% purity) from healthy donors were obtained. NK cells were administered intravenously to the CRC-bearing mice and intensified in vivo in combination with low-dose 5-fluorouracil (0.5 mg/kg or 1 mg/Kg) and irradiated tumors with low doses (2 Gy or 4 Gy). Real-time NK cell cytotoxicity demonstrated a synergistic killing effect of a combination of low-dose chemoradiotherapy, mainly through NKp30 and NKG2D, showing a decrease in NK cell degranulation after blocking NKG2D and NKp30. In vivo tumor characteristics after combination treatment showed decreased CD112, CD155, MICA, and MICB expression. Under the combination strategy, 70% of the mice had free lung metastasis and 90% without secondary gross tumors, indicating suppressed distant metastasis to lung and axillary regions. This combination therapy resulted in significantly synergistic antitumor activity against primary solid tumors compared to chemoradiotherapy only. Furthermore, the intensified NK cell administration showed significantly better primary tumor control and survival outcomes than the non-intensified NK cell administration in a human colorectal HT-29 model treated with low-dose chemoradiotherapy. Optimized NK cell therapy combined with low-dose chemoradiotherapy can provide effective therapeutic potential for intractable cold human colorectal cancer.

Bio-conjugated nanoarchitectonics with dual-labeled nanoparticles for a colorimetric and fluorescent dual-mode serological lateral flow immunoassay sensor in detection of SARS-CoV-2 in clinical samples.

Serological detection of antibodies for diagnosing infectious diseases has advantages in facile diagnostic procedures, thereby contributing to controlling the spread of the pathogen, such as in the recent SARS-CoV-2 pandemic. Lateral flow immunoassay (LFIA) is a representative serological antibody detection method suitable for on-site applications but suffers from low clinical accuracy. To achieve a simple and rapid serological screening as well as the sensitive quantification of antibodies against SARS-CoV-2, a colorimetric and fluorescent dual-mode serological LFIA sensor incorporating metal-enhanced fluorescence (MEF) was developed. For the strong fluorescence signal amplification, fluorophore Cy3 was immobilized onto gold nanoparticles (AuNPs) with size-controllable spacer polyethyleneglycol (PEG) to maintain an optimal distance to induce MEF. The sensor detects the target IgG with a concentration as low as 1 ng mL-1 within 8 minutes. The employment of the MEF into the dual-mode serological LFIA sensor shows a 1000-fold sensitivity improvement compared with that of colorimetric LFIAs. The proposed serological LFIA sensor was tested with 73 clinical samples, showing sensitivity, specificity, and accuracy of 95%, 100%, and 97%, respectively. In conclusion, the dual-mode serological LFIA has great potential for application in diagnosis and an epidemiological survey of vaccine efficacy and immunity status of individuals.

Genetic Structure and Genetic Diversity of the Endemic Korean Aucha Perch, Coreoperca herzi (Centropomidae), in Korea.

The Korean endemic aucha perch, Coreoperca herzi, belongs to the family Centropomidae. Thus far, studies on C. herzi have focused on mitochondrial genomes, egg development, and early life history, while studies on their genetic diversity or genetic structure are lacking. We investigated these aspects in this study using mitochondrial DNA data. Haplotypes were divided into the Hangang River, Nakdonggang River, Geumgang River, and southwest region water system populations. A translocated population, the Yangyang Namdaechun Stream, was confirmed to have originated from the Hangang River water system population based on haplotype distribution and genetic structure results. The FST of the mitochondrial DNA indicated distinct genetic differentiation in the Hangang, Nakdonggang, Geumgang, and southwest regions. According to COI and analyses, the analysis of molecular variance revealed a higher variance in the four water system groups (98.41%) than in the southwest region water system versus the Hangang River water system (80.27%) groups. This study presents basic data for conservation by providing extensive information on the genetic diversity, genetic structure, and translocation population of C. herzi.

Helixor-M Suppresses Immunostimulatory Activity through TLR4-Dependent NF-κB Pathway in RAW 264.7 Cells.

Inflammation causes a protective immune response, which can be observed by examining the inflammatory responses of macrophages. Macrophages release various immunostimulatory factors when destroying external pathogens. We induced lipopolysaccharides (LPS) in RAW 264.7 cells, a macrophage cell line, to determine whether Helixor-M can cause immuno-suppression. Helixor-M is known to have anticancer and immune effects. However, an indicator that regulates immunity has not been clearly confirmed. To this end, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to confirm Helixor-M was not cytotoxic. Western blotting and real-time polymerase chain reaction (RT-PCR) confirmed the anti-inflammatory effects. Additionally, immunofluorescence assay confirmed the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, a representative inflammatory pathway. Helixor-M was found to be non-cytotoxic, induce the NF-κB pathway, and reduce the levels of pro-inflammatory cytokine and mitogen-activated protein kinase (MAPK). We found Helixor-M affected the PI3K/AKT/JNK pathway. Therefore, we confirmed Helixor-M acts as an anti-inflammatory agent through NF-κB, TLR4 and PI3K inhibition and that it could be an effective immunosuppressive drug.

Chrysin Induces Apoptosis via the MAPK Pathway and Regulates ERK/mTOR-Mediated Autophagy in MC-3 Cells.

Chrysin is a flavonoid found abundantly in substances, such as honey and phytochemicals, and is known to exhibit anticancer effects against various cancer cells. Nevertheless, the anticancer effect of chrysin against oral cancer has not yet been verified. Furthermore, the mechanism underlying autophagy is yet to be clearly elucidated. Thus, this study investigated chrysin-mediated apoptosis and autophagy in human mucoepidermoid carcinoma (MC-3) cells. The change in MC-3 cell viability was examined using a 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide cell viability assay, as well as 40,6-diamidino-2-phenylindole, annexin V, and propidium iodide staining. Western blotting was used to analyze the proteins related to apoptosis and the mitogen-activated protein kinase (MAPK) pathway. In addition, the presence or absence of autophagy and changes in the expression of related proteins were investigated using acridine orange staining and Western blot. The results suggested that chrysin induced apoptosis and autophagy in MC-3 oral cancer cells via the MAPK/extracellular signal-regulated kinase pathway. Moreover, the induced autophagy exerted a cytoprotective effect against apoptosis. Thus, the further reduced cell viability due to autophagy as well as apoptosis induction highlight therapeutic potential of chrysin for oral cancer.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Myricetin induces apoptosis through the MAPK pathway and regulates JNK­mediated autophagy in SK­BR­3 cells.

Myricetin, a flavonoid found in fruits and vegetables, is known to have antioxidant and anticancer effects. However, the anticancer effects of myricetin on SK­BR­3 human breast cancer cells have not been elucidated. In the present study, the anticancer effects of myricetin were confirmed in human breast cancer SK­BR­3 cells. As the concentration of myricetin increased, the cell viability decreased. DAPI (4',6­diamidino­2­phenylindole) and Annexin V/PI staining also revealed a significant increase in apoptotic bodies and apoptosis. Western blot analysis was performed to confirm the myricetin­induced expression of apoptosis­related proteins. The levels of cleaved PARP and Bax proteins were increased, and that of Bcl­2 was decreased. The levels of proteins in the mitogen­activated protein kinase (MAPK) pathway were examined to confirm the mechanism of myricetin­induced apoptosis, and it was found that the expression levels of phosphorylated c­Jun N­terminal kinase (p­JNK) and phosphorylated mitogen­activated protein kinases (p­p38) were increased, whereas that of phosphorylated extracellular­regulated kinase (p­ERK) was decreased. It was also demonstrated that myricetin induced autophagy by promoting autophagy­related proteins such as microtubule­associated protein 1A/1B­light chain 3 (LC 3) and beclin 1. In addition, 3­methyladenine (3­MA) was used to evaluate the association between cell viability and autophagy in cells treated with myricetin. The results showed that simultaneous treatment with 3­MA and myricetin promoted the apoptosis of breast cancer cells. Furthermore, treatment with a JNK inhibitor reduced cell viability, promoted Bax expression, and reduced the expression of p­JNK, Bcl­2, and LC 3­II/I. These results suggest that myricetin induces apoptosis via the MAPK pathway and regulates JNK­mediated autophagy in SK­BR­3 cells. In conclusion, myricetin shows potential as a natural anticancer agent in SK­BR­3 cells.

A case of leukaemia cutis in a dog with T-cell chronic lymphocytic leukaemia.

Leukaemia cutis (LC) is the infiltration of neoplastic leukocytes into the skin, characterised by haemorrhagic papules, nodules, and plaques. LC has been reported in human leukaemia patients, but it is extremely rare in dogs. A 13-year-old spayed female Golden Retriever that was previously diagnosed with chronic lymphocytic leukaemia was managed with chlorambucil (20 mg/m2 orally, every 2 weeks) and prednisolone (2 mg/kg orally, every other day) for 8 months; however, immunosuppression was temporarily discontinued because of a bacterial urinary tract infection. Cutaneous signs, including multifocal ecchymosis and white plaques, appeared 1 month after cessation of chemotherapy. Histopathological examination revealed small- to intermediate-sized lymphocytes with mild atypia in a perivascular to interstitial pattern within the superficial dermis. The bands of atypical cells within the superficial dermis were strongly and extensively positive for CD3 on immunohistochemistry. Polymerase chain reaction analysis of the biopsied skin revealed clonal rearrangement of the T-cell receptor gamma locus gene. Given the evidence of clinical signs, peripheral immunophenotyping, histopathology, immunohistochemistry, and clonal gene arrangement, LC was diagnosed. The lesions disappeared when chemotherapy was restarted but were occasionally observed when chemotherapy was stopped. To the authors' best knowledge, this is the first case report of LC in a dog.

Safety analysis of ex vivo-expanded canine natural killer cells in a xenogeneic mouse model of graft-versus-host disease.

Canine natural killer (NK) cells are large, granular lymphocytes that are neither B lymphocytes nor T lymphocytes. However, it has been reported that canine NK cells share some of the phenotypic characteristics of T lymphocytes, such as CD3 and CD5. Studies are needed to assess the safety of canine NK cells for immunotherapy, especially because the safety of using allogeneic NK cells as an immunotherapy for dogs has yet to be shown. In this study, the safety of cultured canine NK cells was assessed using a xenogeneic mouse model of graft-versus-host disease (GVHD). Mice were injected with either canine peripheral blood mononuclear cells (PBMCs) or cultured NK cells for 2 or 3 weeks. Data were then collected on changes in mice body weights, disease severity scores, and survival rates. Histopathological and immunohistochemical evaluations were also performed. All mice injected with canine PBMCs died within 45 days after injection. Severe clinical signs were caused by GVHD. The histopathological and immunohistochemical evaluations showed that mice injected with canine PBMCs had multiple lesions, including necrosis in their lungs, livers, kidneys, and stomachs, and the injected cells were present around the lesions. By contrast, no mice injected with cultured NK cells without removing the CD3+ TCR- cells exhibited any clinical abnormalities. Moreover, they all survived the 90-day experimental period without exhibiting any histopathological changes. Accordingly, the results of this study suggest that canine NK cells do not cause significant side effects such as GVHD and allogeneic NK cells can safely be used for cancer immunotherapy in dogs.

Comparison of clinical and inflammatory parameters in dogs with pyometra before and after ovariohysterectomy.

This study aimed to identify potential biomarkers of canine pyometra and their correlations with clinical parameters. First, 90 dogs with pyometra and 26 healthy female dogs were compared. Then, paired samples (before and after ovariohysterectomy) from 22 dogs with pyometra and 9 healthy controls from the initial cohort were compared. Concentrations of acute inflammatory proteins, C-reactive protein (CRP) and serum amyloid A (SAA), and cell-free DNA (cfDNA), were significantly higher in dogs with pyometra than in clinically healthy dogs. Cell-free DNA was the most sensitive biomarker for systemic inflammation, based on the receiver operating characteristic curve analysis (area under the curve = 0.959). In addition, cfDNA and CRP were significantly associated with inflammation and organ injury-related clinical parameters. Following the surgical removal of the inflamed uterus, interleukin-6 (IL-6), high-mobility group box 1 (HMGB1), and procalcitonin (PCT) significantly decreased, whereas changes in CRP, SAA, and cfDNA were not significant. These findings indicate that cfDNA, CRP, and SAA are potential clinical biomarkers of systemic inflammation in dogs with pyometra and PCT, IL-6, and HMGB1 are potential biomarkers of clinical recovery.

Cette étude visait à identifier les biomarqueurs potentiels du pyomètre canin et leurs corrélations avec les paramètres cliniques. Tout d'abord, 90 chiens avec pyomètre et 26 chiennes en bonne santé ont été comparés. Ensuite, des échantillons appariés (avant et après ovariohystérectomie) de 22 chiens avec pyomètre et neuf témoins sains de la cohorte initiale, ont été comparés.Les concentrations des protéines inflammatoires aiguës, protéine C réactive (CRP) et amyloïde sérique A (SAA), et d'ADN acellulaire (cfDNA), étaient significativement plus élevées chez les chiens atteints de pyomètre que chez les chiens cliniquement sains. L'ADN acellulaire était le biomarqueur le plus sensible pour l'inflammation systémique, sur la base de l'analyse de la courbe caractéristique de fonctionnement du récepteur (aire sous la courbe = 0,959). De plus, le cfDNA et la CRP étaient significativement associés à l'inflammation et aux paramètres cliniques liés aux lésions aux organes.Après l'ablation chirurgicale de l'utérus enflammé, l'interleukine-6 (IL-6), la protéine HMGB1 (« high-mobility groupe box 1 ¼) et la procalcitonine (PCT) ont significativement diminué, alors que les changements de CRP, SAA et cfDNA n'étaient pas significatifs. Ces résultats indiquent que cfDNA, CRP et SAA sont des biomarqueurs cliniques potentiels de l'inflammation systémique chez les chiens avec pyomètre et PCT, IL-6 et HMGB1 sont des biomarqueurs potentiels de récupération clinique.(Traduit par Docteur Serge Messier).

Characterization of the complete mitochondrial genome of Aphyocypris chinensis (Cypriniformes, Xenocyprididae), and its phylogenetic position in Cypriniformes.

Aphyocypris chinensis Günther, 1868 is a small freshwater fish of the family Xenocyprididae (Cypriniformes). In this study, we determined its complete mitochondrial genome and phylogenetic position in Cypriniformes. The complete mitochondrial genome is 16,608 bp in size, containing 13 protein-coding genes, two RNAs, 22 tRNAs, and a control region. It has the typical vertebrate mitochondrial gene arrangement. Our mitogenomic phylogeny revealed that A. chinensis belongs to Xenocyprididae, rather than Danionidae. This mitogenome information could play an essential role in resolving the conflict over its current taxonomic status in Cypriniformes.

Safety and immunological effects of recombinant canine IL-15 in dogs.

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays pivotal roles in innate and adaptive immunity. It is also a promising cytokine for treating cancer. Despite growing interest in its use as an immunotherapeutic, its safety and immunological effects in dogs have not been reported. In this study, healthy dogs were given recombinant canine IL-15 (rcIL-15) intravenously at a daily dose of 20 µg/kg for 8 days and monitored for 32 days to determine the safety and immunological effects of rcIL-15. The repeated administration of rcIL-15 was well tolerated, did not cause any serious side effects, and promoted the selective proliferation and activation of canine anti-cancer effector cells, including CD3+CD8+ cytotoxic T lymphocytes, CD3+CD5dimCD21-, and non-B/non-T NK cell populations, without stimulating Treg lymphocytes. The rcIL-15 injections also stimulated the expression of molecules and transcription factors associated with the activation and effector functions of NK cells, including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, granzyme B, Ly49, T-bet, and Eomes. These results suggest that rcIL-15 might be a valuable therapeutic adjuvant to improve immunity against cancer in dogs.

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo.

BACKGROUND AIMS: Tracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression. METHODS: Ex vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The authors administered 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry. RESULTS: The fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45+CD56+CD3- NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D. CONCLUSIONS: Administered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45+CD56+CD3- NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.

An integrative approach reveals a new species of flightless leaf beetle (Chrysomelidae: Suinzona) from South Korea.

The leaf beetle genus Suinzona, consisting of over 20 species, is mainly known for species from Southwest China, and its closely related genus Potaninia, with only two species, is widely distributed in South China and upper Southeast Asia. Despite recent morphological studies, the status of these taxa has long been controversial. Here, we investigated the taxonomic status and phylogenetic relationships of the genera Suinzona and Potaninia within Chrysomelinae using molecular, morphological and ecological data. Molecular phylogenetic analysis supported that they should be regarded as distinct genera, which is consistent with morphological evidence, e.g., well-developed/reduced hind wings. Based on combined evidence from examination of larval and adult morphology, host plants and mitochondrial genomes, we demonstrate that P. cyrtonoides should be placed in the genus Suinzona and that specimens from South Korea represent a new species. Suinzona borowieci sp. nov., occurring in narrow strips of habitat, shows high levels of genetic divergence and distantly related host plants between populations. The population differentiation seems to be correlated with its non-functional wings causing reduced dispersal ability and genetic isolation. Several populations have declined dramatically over the last few decades due to loss of habitat and thus are in need of protection as conservation units.

Complete mitochondrial genome sequence of Squalidus chankanensis tsuchigae (Cypriniformes: Cyprinidae).

We determined the complete mitochondrial genome sequence of Squalidus chankanensis tsuchigae (Cypriniformes: Cyprinidae). It is 16,603 bp long, with 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNA genes, and 1 control region, which is 927 bp long, located between tRNApro and tRNAphe. The overall base composition is as follows: 29.98% A, 16.86% G, 25.44% T, and 27.72% C, with a slight AT bias. These results provide necessary data for phylogenetic studies on Squalidus species.