HwMR is a novel magnesium-associated protein.

Microbial rhodopsins (MRho) are vital proteins in Haloarchaea for solar light sensing in extreme living environments. Among them, Haloquadratum walsbyi (Hw) is a species known to survive high MgCl2 concentrations, with a total of three MRhos identified, including a high-acid-tolerance light-driven proton outward pump, HwBR, a chloride-insensitive chloride pump, HwHR, and a functionally unknown HwMR. Here, we showed that HwMR is the sole magnesium-sensitive MRho among all tested MRho proteins from Haloarchaea. We identified at least D84 as one of the key residues mediating such magnesium ion association in HwMR. Sequence analysis and molecular modeling suggested HwMR to have an extra H8 helix in the cytosolic region like those in signal-transduction-type MRho of deltarhodopsin-3 (dR-3) and Anabaena sensory rhodopsin (ASR). Further, HwMR showed a distinctly prolonged M-state formation under a high concentration of Mg2+. On the other hand, an H8 helix truncated mutant preserved photocycle kinetics like the wild type, but it led to missing M-state structure. Our findings clearly suggested not only that HwMR is a novel Mg2+-associated protein but that the association with both Mg2+ and the H8 domain stabilizes M-state formation in HwMR. We conclude that Mg2+ association and H8 are crucial in stabilizing HwMR M state, which is a well-known photoreceptor signaling state.

Hydroxygenkwanin Increases the Sensitivity of Liver Cancer Cells to Chemotherapy by Inhibiting DNA Damage Response in Mouse Xenograft Models.

Molecules involved in DNA damage response (DDR) are often overexpressed in cancer cells, resulting in poor responses to chemotherapy and radiotherapy. Although treatment efficacy can be improved with the concomitant use of DNA repair inhibitors, the accompanying side effects can compromise the quality of life of patients. Therefore, in this study, we identified a natural compound that could inhibit DDR, using the single-strand annealing yeast-cell analysis system, and explored its mechanisms of action and potential as a chemotherapy adjuvant in hepatocellular carcinoma (HCC) cell lines using comet assay, flow cytometry, Western blotting, immunofluorescence staining, and functional analyses. We developed a mouse model to verify the in vitro findings. We found that hydroxygenkwanin (HGK) inhibited the expression of RAD51 and progression of homologous recombination, thereby suppressing the ability of the HCC cell lines to repair DNA damage and enhancing their sensitivity to doxorubicin. HGK inhibited the phosphorylation of DNA damage checkpoint proteins, leading to apoptosis in the HCC cell lines. In the mouse xenograft model, HGK enhanced the sensitivity of liver cancer cells to doxorubicin without any physiological toxicity. Thus, HGK can inhibit DDR in liver cancer cells and mouse models, making it suitable for use as a chemotherapy adjuvant.

An unusual uterine papillary serous carcinoma with post therapy disseminating metastasis presenting as primary renal malignancy: a case report.

Introduction: Uterine papillary serous carcinoma (UPSC) is a highly aggressive endometrial carcinoma that often presents as a high-stage disease. UPSC has a high propensity for metastasis and recurrence, even with little or no myometrial invasion. It usually metastasizes to the pelvis, retroperitoneal lymph nodes, upper abdomen, or peritoneum. However, renal metastasis of UPSC is extremely rare. Case presentation: The authors reported a unique UPSC case in a 75-year-old unmarried woman. Twenty years ago, she had a history of right breast cancer and underwent a modified radical mastectomy. Three years ago, she was diagnosed with endometrial carcinoma, and six courses of chemotherapy and radiotherapy were administered. Computed tomography and retrograde pyelography revealed a right renal pelvic tumor, and a right nephroureterectomy was performed. Renal metastatic UPSC was diagnosed. The patient was administered adjuvant chemotherapy. Clinical discussion: Metastatic UPSCs initially presenting at distant sites are uncommon manifestations. This tumor should be differentially diagnosed in patients presenting with metastatic high-grade serous papillary carcinoma of unknown primary origin. Conclusion: Diagnosing metastatic renal UPSC, based on preoperative and imaging examinations, is often challenging. Thus, a review of the past history, histopathology, and immunohistochemical evaluation plays a crucial and valuable role in the definite and differential diagnosis of this tumor type.

Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway.

BACKGROUND: Melanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. OBJECTIVE: To explore whether IMQ could induce melanogenesis in melanoma cells. METHODS: The mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. RESULTS: We demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. CONCLUSIONS: Low dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

Incidental finding of multiple diffuse large B-cell lymphomas masquerading as jejunojejunal intussusception with unexplained pleural effusion: a case report.

Primary non-Hodgkin's lymphoma of the gastrointestinal (GI) tract is rare. It is aggressive and necessitates early diagnosis and management. Simultaneous primary GI lymphomas are unusual with rarely reported cases. Case presentation: This novel case report describes an 84-year-old man with multiple primary diffuse large B-cell lymphomas (DLBCLs) of the jejunum with disseminating pleural and multiple regional lymph nodes involvement presenting as intestinal obstruction and segments of jejunojejunal intussusception. The patient underwent surgical intervention and adjuvant chemotherapy. Unfortunately, the patient suffered from multiple organ failure and died 4 months after surgery. Clinical discussion: Obstruction and perforation are rare and life-threatening complications of GI lymphoma. Multiple DLBCLs of the jejunum are rare. Moreover, primary GI-DLBCL that initially presents with pleural effusion or with intestinal perforation is uncommon. This report aims to remind clinicians that lymphoma should be considered when assessing the cause of unexplained pleural effusion, especially when the available examination data cannot be confirmed by clinical manifestations. Conclusion: Through this case report, the authors learn that clinical manifestations, morphological characteristics, immunophenotypes, and molecular biological characteristics are vastly different and important. This poses the biggest challenge before surgery and should not be ignored.

Unusually aggressive primary testicular diffuse large B-cell lymphoma initially presenting as systemic disseminating metastases in older adult men: a case report.

Primary testicular lymphoma (PTL) accounts for 1-2% of all nonHodgkin lymphomas (NHL), 4% of extranodal nonHodgkin lymphomas, and ~9% of testicular malignancies. A rare subtype of PTL is primary testicular diffuse large B-cell lymphoma (PT-DLBCL), which may initially present as disseminating metastasis in older adult males and has a poor prognosis. Case presentation: Herein, the authors describe the case of a 64-year-old man with the chief complaint of a painless unilateral scrotal mass. Computed tomography scans of the abdomen and a pelvic examination demonstrated a left testicular tumor with multiple lymphadenopathies partially aggregated in the para-aortic area and disseminated to multiple soft tissues and organs. Subsequently, the patient underwent a left radical orchiectomy. Pathological and immunohistochemical examinations confirmed the diagnosis of left PT-DLBCL with systemic disseminating metastases. Clinical discussion: PTL often aggressively spreads to other extranodal organs, such as the contralateral testis, central nervous system, lung, pleura, Waldeyer's ring, and soft tissues. In men over 60 years of age, PT-DLBCL is the most common testicular malignancy. However, extensive systemic metastasis as the initial presentation is extremely rare. PT-DLBCL has a dismal prognosis and requires radical orchiectomy followed by multimodal therapy and central nervous system prophylaxis or systemic intervention to improve survival. Conclusion: The diagnosis of PT-DLBCL through preoperative and imaging examinations is often challenging. Thus, histopathology and immunohistochemical markers play a crucial and valuable role in the definite diagnosis and differential diagnosis of PTLs.

Incidental finding of metastatic prostatic adenocarcinoma of frontotemporal bone presenting as subdual hematoma: A case report and review of literature.

INTRODUCTION: and important: Prostatic cancer is often prone to metastasis and bone invasion. Skull metastasis in prostate cancer is uncommon, accounting for less than 2% of all metastases. However, frontotemporal bone metastasis without dural or brain metastasis is rare. CASE PRESENTATION: Herein, we report the case of a 91-year-old male patient who presented with a sudden-onset dizziness, a fall to the ground, and gradual loss of consciousness. Computed tomography (CT) of the brain revealed an aggressive bony lesion secondary to locally advanced metastatic malignancy and subdural hematoma. Subsequently, he underwent decompressive craniectomy. Histopathological and immunohistochemical (IHC) examinations demonstrated metastatic prostatic adenocarcinoma (PCa). Although after treatment by a multidisciplinary team, unfortunately, the patient expired two months after the surgery and could no longer be traced. CLINICAL DISCUSSION: In the majority of reported cases, CT scans of the brain are often mistaken for subdural hematoma or meningioma. The present case suggests is a preliminary incidental case of a single frontotemporal bony lesion. This is the first case described in the literature of incidental finding of metastatic PCa presenting with asymptomatic characteristics. CONCLUSION: Awareness of the possibility of metastatic PCa involving the skull bones, as well as histopathological and IHC examinations, are important to arrive at a correct initial diagnosis.

A rare primary pericardial DLBCL masquerading as an unexplained malignant pleural effusion in an elderly woman: A case report.

Introduction: Primary cardiac lymphoma (PCL) is an extremely rare and fatal heart neoplasm. Primary cardiac non-Hodgkin lymphoma (NHL) is uncommon, considering the rarity of pericardial diffuse large B-cell lymphoma (DLBCL) with advanced pleural metastasis. Case presentation: We reported an 86-year-old female with primary pericardial DLBCL diagnosed initially by pleural effusion cytology. The chest imaging study revealed multiple pericardial lobulated infiltrative masses and epicardial invasion. Subsequently, she underwent an emergent pericardial window with a pericardial mass biopsy. The final histopathological and immunohistochemical (IHC) stain confirmed primary pericardial DLBCL, initially showing unexplained malignant pleural effusion. Clinical discussion: The presence and extent of tumour invasion in the heart can be confirmed by echocardiography, computerised tomography (CT), or magnetic resonance imaging (MRI). However, the final histopathological diagnosis requires an examination of the endocardial, myocardial, pericardial window and biopsy or pericardial and pleural effusion cytology. This is the first case report of primary pericardial DLBCL diagnosed by metastatic malignant pleural effusion cytology per the literature review. Conclusion: The definitive diagnosis for primary pericardial DLBCL is based on effusion cytology, histopathological and IHC evaluation, and clinical characteristics and image feature correlation.

Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells.

BACKGROUND: Lysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused lysosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells. OBJECTIVE: To determine whether lysosomes are involved in IMQ-induced apoptosis. METHODS: The human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluorescence caspase-8 kit and determined by flow cytometry and confocal microscopy. RESULTS: IMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo. CONCLUSIONS: IMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.

Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway.

Hepatocellular carcinoma (HCC), a hepatic malignancy, has a poor prognosis and contributes to cancer-related death worldwide. Cellular senescence is an anticancer therapeutic strategy that causes irreversible cell cycle arrest and enables immune-mediated clearance of cancer cells. Atorvastatin, an HMG-CoA reductase inhibitor, has been shown to inhibit tumor growth and induce apoptosis or autophagy in malignant tumors. However, whether atorvastatin can induce HCC cell senescence and the mechanisms involved are poorly understood. The effects of atorvastatin-induced senescence were examined in both HCC cells and mouse xenograft models. The phenomenon and mechanism of senescence were examined by cell cycle analysis, senescence-associated ß-galactosidase (SA-ß-gal) staining and western blotting in HCC cells, and HCC tissues from mice were analyzed by immunohistochemical (IHC) staining. We demonstrated that atorvastatin induced cell growth inhibition and G0/G1 phase cell cycle arrest, leading to senescence in HCC cells. Atorvastatin-induced senescence was independent of p53, p14, and p16, and atorvastatin not only decreased the secretion of IL-6, a major senescence-associated secretory phenotype (SASP) factor, and the phosphorylation of STAT3 but also inhibited the expression of hTERT, a catalytic subunit of telomerase. Supplementation with exogenous IL-6 reversed both atorvastatin-induced suppression of STAT3 phosphorylation and hTERT expression and atorvastatin-induced senescence. Overexpression of constitutively activated STAT3 rescued HCC cells from atorvastatin-induced hTERT suppression and senescence. Moreover, atorvastatin decreased tumor growth in mouse xenograft models. Consistent with these results, atorvastatin decreased the IL-6, p-STAT3, and hTERT levels and increased ß-gal expression in tumor sections. Taken together, these data indicate that atorvastatin can induce atypical cellular senescence in HCC cells to inhibit tumor growth, an effect mediated by downregulation of hTERT through suppression of the IL-6/STAT3 pathway.

Antibody formation in pregnant women with maternal-neonatal human platelet antigen mismatch from a hospital in northern Taiwan.

Neonatal alloimmune thrombocytopenia (NAIT) is a clinical syndrome that resembles hemolytic disease of the newborn, affecting the platelets only. The thrombocytopenia results from the maternal alloantibodies reacting with specific human platelet antigens (HPAs) on the fetal platelets. Forty-four maternal plasma samples were screened for platelet alloantibodies using qualitative solid phase enzyme-linked immunosorbent assay (ELISA) commercial kit (LIFECODES Pakplus, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA), and both the maternal and the corresponding cord blood samples were genotyped (LIFECODES ThromboType, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA). HPA genotyping results correlated with the genetic frequencies in the Taiwan population. A total of 34 newborns (77.3%) had partial HPA genotyping mismatches with the corresponding mothers. The most common partial mismatches between mothers and neonates in HPA genotypes were 13 (29.5%) in both HPA-3b and HPA-15a, followed by 12 (27.3%) in HPA-15b, and 8 (18.2%) in HPA-3a. The frequencies of homozygotic mother with heterozygotic neonate were 15.9% in both HPA-3a and HPA-15b, 9.1% in HPA-15a, 6.8% in HPA-3b, and 2.3% in both HPA-2a and HPA-6a. In this study, maternal HPA antibodies were found in five samples, whereas HLA class I antibodies were found in seven maternal plasma samples from the antibody screen. The results from this study have demonstrated that HPA mismatch is not the main cause for the production of HPA alloantibodies.

Mcl-1 determines the imiquimod-induced apoptosis but not imiquimod-induced autophagy in skin cancer cells.

BACKGROUND: Imiquimod had been shown to induce apoptosis and autophagy in several skin cancer cells, especially basal cell carcinoma (BCC) cells. OBJECTIVE: We evaluate the molecular mechanisms of imiquimod-induced apoptosis and autophagy in skin cancer cell lines. METHODS: The Mcl-1, Bcl-2 and Bcl-xL proteins were determined by immunoblotting. The Mcl-1 mRNA level was examined by RT-PCR and real-time PCR. The mechanisms of imiquimod-induced decrease in Mcl-1 protein were evaluated by addition of cycloheximide, MG132 proteasome inhibitor or pan-caspase inhibitor. The phosphorylation of eIF4E, 4E-BP1 and eEF2 in imiquimod treated cells were examined by immunoblotting. The imiquimod-induced apoptosis and autophagy were evaluated in Mcl-1-overexpressing cells by XTT test, mitochondrial membrane potential measurement, DNA content assay, LC3 immunoblotting, EGFP-LC3 puncta formation and quantification of acidic vesicular organelle with acridine orange staining. RESULTS: The decrease in the Mcl-1 protein level was faster and stronger than the decrease in Bcl-2 and Bcl-xL in imiquimod-treated skin cancer cells. The imiquimod-induced decrease in Mcl-1 protein was not caused by blocked transcription or the promotion of degradation but was associated with inactivation of translation factors in BCC cells. The Mcl-1-overexpressing BCC cells were more resistant to intrinsic cellular apoptosis than control BCC cells during imiquimod treatment. Mcl-1 overexpression in BCC cells resulted in the basal activation of autophagy but did not modulate imiquimod-induced autophagy or rescue imiquimod-induced autophagic cell death in BCC cells. CONCLUSIONS: Imiquimod may rapidly downregulate Mcl-1 protein levels by inhibiting translation in skin cancer cells. Mcl-1 may act to protect against apoptosis but not autophagy and autophagic cell death during imiquimod treatment in skin cancer cells.

Modification of alternative splicing of Mcl-1 pre-mRNA using antisense morpholino oligonucleotides induces apoptosis in basal cell carcinoma cells.

Myeloid cell leukemia-1 (Mcl-1, Mcl-1L) is an anti-apoptotic protein of the Bcl-2 family that acts as a critical molecule in apoptosis control. Mcl-1 pre-mRNA can undergo alternative splicing to yield the short isoform, Mcl-1S, which resembles BH3-only pro-apoptotic proteins and induces apoptosis. Overexpression of Mcl-1 may play a role in various human tumors, and Mcl-1 may serve as a target in cancer therapy. In this study, we found an imbalance between the expression levels of Mcl-1L and Mcl-1S in the skin basal cell carcinoma (BCC) cell line when compared with primary keratinocytes. We showed that overexpression of Mcl-1S induces apoptosis in BCC cells. Finally, we showed that Mcl-1 antisense morpholino oligonucleotides (AMOs) can specifically target Mcl-1 pre-mRNA and shift the splicing pattern from Mcl-1L to Mcl-1S mRNA and protein. This shift increases the level of pro-apoptotic Mcl-1S and reduces the level of anti-apoptotic Mcl-1L, which induces apoptosis in BCC cells and AGS cells, a human gastric adenocarcinoma epithelial cell line. Thus, this report provides a strategy for cancer therapy in which AMOs change the alternative splicing pattern of Mcl-1 pre-mRNA and thereby induce apoptosis.

Protective immunity of E. coli-synthesized NS1 protein of Japanese encephalitis virus.

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.