PCSK9 inhibitor use during pregnancy in a case of familial hypercholesterolemia complicated with coronary artery disease.

Limited data have been reported on the use of proprotein convertase subtilisin/kexin type 9 (PCSK 9) inhibitors during pregnancy in women with familial hypercholesterolemia (FH). Here, we present the first case of initiating evolocumab (PCSK9 inhibitor) in a compound heterozygous FH mother. The patient was a 34-year-old primipara with severe dyslipidemia and a history of coronary artery bypass surgery. An elevated low-density lipoprotein cholesterol (LDL-C) level of 420 mg/dL was detected in the first trimester and persistently increased throughout pregnancy. Evolocumab was administered at 31 and 35 weeks of gestation, showing a positive effect on stabilizing LDL-C levels. Planned delivery with labor analgesia was performed at 38 + 4 weeks. Both the mother and infant were discharged without any notable complications. Hence, evolocumab, an IgG2 monochromatic antibody with little placental permeability, may be an alternative medication with limited influence on infants. Further studies are needed to assess the safety of evolocumab administration during pregnancy.

Reductive Retrocyclization of a Mangana(II)cyclopentasilane to Form Manganese(0) Bis(η2-disilene) Complexes.

Ligand-exchange reactions on a mangana(II)cyclopentasilane complex that contains two THF ligands with aryl isocyanides led to the formation of manganese(0) bis(η2-disilene) complexes via a retrocyclization. In stark contrast, ligand-exchange reactions with CNtBu, an N-heterocyclic carbene, or pyridine-based ligands furnished manganese(II) complexes wherein the manganacyclopentasilane framework remained intact. The thermolysis of the obtained bis(η2-disilene) complex in the presence of mesityl isocyanide led to the formation of a cyclotetrasilane via the formal dimerization of the two η2-disilene moieties. The insertion of a mesityl isocyanide into the Mn-Siß bond results in the formation of a manganese(II) complex supported by a [SiCSi]-type tridentate ligand scaffold.

FOXP4 inhibits squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre-existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4-positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1-derived HPV 16-positive W12 cells via an ELF3-dependent pathway. In organotypic raft cultures, FOXP4-downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte-derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3-dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.

Antibody Screening System Using a Herpes Simplex Virus (HSV)-Based Probe To Identify a Novel Target for Receptor-Retargeted Oncolytic HSVs.

Herpes simplex virus (HSV) is a promising tool for developing oncolytic virotherapy. We recently reported a platform for receptor-retargeted oncolytic HSVs that incorporates single-chain antibodies (scFvs) into envelope glycoprotein D (gD) to mediate virus entry via tumor-associated antigens. Therefore, it would be useful to develop an efficient system that can screen antibodies that might mediate HSV entry when they are incorporated as scFvs into gD. We created an HSV-based screening probe by the genetic fusion of a gD mutant with ablated binding capability to the authentic HSV entry receptors and the antibody-binding C domain of streptococcal protein G. This engineered virus failed to enter cells through authentic receptors. In contrast, when this virus was conjugated with an antibody specific to an antigen on the cell membrane, it specifically entered cells expressing the cognate antigen. This virus was used as a probe to identify antibodies that mediate virus entry via recognition of certain molecules on the cell membrane other than authentic receptors. Using this method, we identified an antibody specific to epiregulin (EREG), which has been investigated mainly as a secreted growth factor and not necessarily for its precursor that is expressed in a transmembrane form. We constructed an scFv from the anti-EREG antibody for insertion into the retargeted HSV platform and found that the recombinant virus entered cells specifically through EREG expressed by the cells. This novel antibody-screening system may contribute to the discovery of unique and unexpected molecules that might be used for the entry of receptor-retargeted oncolytic HSVs.IMPORTANCE The tropism of the cellular entry of HSV is dependent on the binding of the envelope gD to one of its authentic receptors. This can be fully retargeted to other receptors by inserting scFvs into gD with appropriate modifications. In theory, upon binding to the engineered gD, receptors other than authentic receptors should induce a conformational change in the gD, which activates downstream mechanisms required for viral entry. However, prerequisite factors for receptors to be used as targets of a retargeted virus remain poorly understood, and it is difficult to predict which molecules might be suitable for our retargeted HSV construct. Our HSV-based probe will allow unbiased screening of antibody-antigen pairs that mediate virus entry and might be a useful tool to identify suitable pairs for our construct and to enhance our understanding of virus-cell interactions during infection by HSV and possibly other viruses.

Ultra-Sensitive Isopropanol Biochemical Gas Sensor (Bio-Sniffer) for Monitoring of Human Volatiles.

Our groups have previously developed a biochemical gas sensor to measure isopropanol (IPA) in exhaled air and have applied it for breath IPA investigation in healthy subjects and diabetes patients. In this study, the original bio-sniffer was modified with a series of components that improved the limit of detection (LOD). First, the modified IPA bio-sniffer used a C8855-type photomultiplier tube (PMT) that performed well in the photon sensitivity at the peak wavelength of nicotinamide adenine dinucleotide (NADH) fluorescence. Second, the multi-core bifurcated optical fiber, which incorporated 36 fibers to replace the previous dual-core type, enhanced the fluorescence collection. Third, the optical fiber probe was reinforced for greater width, and the flow-cell was redesigned to increase the area of the enzyme-immobilized membrane in contact with the air sample. These modifications lowered the detection limit to 0.5 ppb, a significant increase over the previous 1.0 ppb. Moreover, the modified bio-sniffer successfully analyzed the IPA concentration in exhaled air from a volunteer, which confirmed its capability for real-world sample detection. The modified bio-sniffer is more applicable to breath measurement and the detection of other extremely-low-concentration samples.

Twin anemia-polycythemia sequence with blood chimerism in monochorionic dizygotic opposite-sex twins.

We report a case of twin anemia-polycythemia sequence with blood chimerism in monochorionic dizygotic twins born to a 30-year-old woman who conceived via ovulation induction. The neonates developed twin anemia-polycythemia sequence; the female twin had anemia and the male had polycythemia. We detected blood chimerism using fluorescence in situ hybridization (FISH). Twin anemia-polycythemia sequence carries not only perinatal risks, but also genetic and immunological implications due to blood chimerism. Although previous reports have described twin-to-twin transfusion syndrome in monochorionic dizygotic twins, we report the first case of twin anemia-polycythemia sequence in monochorionic dizygotic twins.

Identification of Lycopodium Alkaloids Produced by an Ultraviolet-Irradiated Strain of Paraboeremia, an Endophytic Fungus from Lycopodium serratum var. longipetiolatum.

12- epi-Lycopodine (1), a Lycopodium alkaloid, along with lycopodine (2) and huperzine A (3), were discovered in the mycelium of Paraboeremia sp. Lsl3KI076, a UV-irradiated strain of Paraboeremia sp. Lsl3, an endophytic fungus from Lycopodium serratum Thunb. var. longipetiolatum Spring. Additionally, a trace of 1 was isolated from Phlegmariurus nummulariifolius (Blume) Ching, and the structure was elucidated on the basis of spectroscopic data. This is the first report proving that a new naturally occurring Lycopodium alkaloid can be obtained from an endophytic fungus.

New Metabolites Produced by Cylindrocarpon sp. SY-39 from a Driftwood.

Four novel compounds, cylindropyrone A (1), 10'-hydroxyilicicolinic acid D (3), cylindrolactones A (4) and B (5), together with known dihydroinfectopyrone (2) were isolated from the culture of Cylindrocarpon sp. SY-39 from a driftwood. Their structures were elucidated using 1D- and 2D-NMR spectroscopy. Compound 3 showed antimicrobial activity against Staphylococcus aureus with MIC value of 5.0 µg/mL.

Biochemical Gas Sensors (Biosniffers) Using Forward and Reverse Reactions of Secondary Alcohol Dehydrogenase for Breath Isopropanol and Acetone as Potential Volatile Biomarkers of Diabetes Mellitus.

This study describes two biosniffers to determine breath acetone and isopropanol (IPA) levels and applies them for breath measurement in healthy subjects and diabetic patients. Secondary alcohol dehydrogenase (S-ADH) can reduce acetone and oxidize nicotinamide adenine dinucleotide (NADH to NAD+) in a weak acid environment. NADH can be excited by 340 nm excitation lights and subsequently emit 490 nm fluorescence. Therefore, acetone can be measured by the decrease in NADH fluorescence intensity. S-ADH can also oxidize IPA and reduce NAD+ to NADH when it is in an alkaline environment. Thus, IPA can be detected by the increase of fluorescence. The developed biosniffers show rapid response, high sensitivity and high selectivity. The breath acetone and IPA analysis in healthy subjects shows that the mean values were 750.0 ± 434.4 ppb and 15.4 ± 11.3 ppb. Both acetone and IPA did not show a statistical difference among different genders and ages. The breath acetone analysis for diabetic patients shows a mean value of 1207.7 ± 689.5 ppb, which was higher than that of healthy subjects (p < 1 × 10-6). In particularly, type-1 diabetic (T1D) patients exhaled a much higher concentration of acetone than type-2 diabetic (T2D) patients (p < 0.01). The breath IPA also had a higher concentration in diabetic patients (23.1 ± 20.1 ppb, p < 0.01), but only T2D patients presented a statistical difference (23.9 ± 21.3 ppb, p < 0.01). These findings are worthwhile in the study of breath biomarkers for diabetes mellitus diagnosis. Additionally, the developed biosniffers provide a new technique for volatolomics research.

Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus.

Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors. IMPORTANCE: Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

Induction of necroptosis in multinucleated giant cells induced by conditionally replicating syncytial oHSV in co-cultures of cancer cells and non-cancerous cells.

Viral modifications enabling syncytium formation in infected cells can augment lysis by oncolytic herpes simplex viruses (oHSVs) which selectively kill cancer cells. In the case of receptor-retargeted oHSVs (RR-oHSVs) that exclusively enter and spread to cancer cells, anti-tumor effects can be enhanced in a magnitude of >100,000-fold by modifying the virus to a syncytial type (RRsyn-oHSV). However, when syncytia containing non-cancerous cells are induced by conditionally replicating syncytial oHSV (CRsyn-oHSV), syncytial death occurs at an early stage. This results in limited anti-tumor effects of the CRsyn-oHSV. Here, we investigated whether necroptosis is involved in death of the syncytia formed by the fusion of cancer cells and non-cancerous cells. Mixed-lineage kinase domain-like (MLKL), a molecule executing necroptosis, was expressed in all murine cancer cell lines examined, while receptor-interacting protein kinase 3 (RIPK3), which phosphorylates MLKL, was absent from most cell lines. In contrast, RIPK3 was expressed in non-cancerous murine fibroblast cell lines. When a CRsyn-oHSV-infected RIPK3-deficient cancer cell line was co-cultured with the fibroblast cell line, but not with the cancer cells themselves, MLKL was phosphorylated and syncytial death was induced. These results indicate that early necroptosis is induced in multinucleated giant cells formed by CRsyn-oHSV when they also contain non-cancerous cells.

Very low-dose lenalidomide therapy for elderly multiple myeloma patients.

Lenalidomide treatment for refractory or relapsed multiple myeloma in elderly patients may be feasible in an outpatient setting. However, difficulties have been associated with the management of adverse effects. Therefore, a dose reduction in lenalidomide has been recommended in some cases. In this report, we encountered the successful treatment of myeloma in 6 elderly patients (aged above 70 years) with very low-dose lenalidomide (5 mg daily). Four patients exhibited more than a partial response with an 8.6 months median follow-up period, which was comparable with previous findings. The major adverse effect observed was infection, which occurred during the first several cycles. Others were less toxic, especially the absence of grade 3/4 toxicities for hematological adverse effects.Although a dose reduction in lenalidomide therapy for elderly patients is controversial, a very low dose could be safe and effective. Our group is currently conducting a multi-center prospective trial to evaluate the efficacy of low-dose lenalidomide therapy.

Cell-Free Production and Regeneration of Cofactors.

Cofactors, such as adenosine triphosphate, nicotinamide adenine dinucleotide, and coenzyme A, are involved in nearly 50% of enzymatic reactions and widely used in biocatalytic production of useful chemicals. Although commercial production of cofactors has been mostly dependent on extraction from microbial cells, this approach has a theoretical limitation to achieve a high-titer, high-yield production of cofactors owing to the tight regulation of cofactor biosynthesis in living cells. Besides the cofactor production, their regeneration is also a key challenge to enable continuous use of costly cofactors and improve the feasibility of enzymatic chemical manufacturing. Construction and implementation of enzyme cascades for cofactor biosynthesis and regeneration in a cell-free environment can be a promising approach to these challenges. In this chapter, we present the available tools for cell-free cofactor production and regeneration, the pros and cons, and how they can contribute to promote the industrial application of enzymes.

Bing-Neel syndrome with a paravertebral mass.

Bing-Neel syndrome, a rare neurological complication of Waldenström macroglobulinemia, is caused by the direct infiltration of malignant lymphoplasmacytic cells into the central nervous system. We report a patient who presented with back pain, weakness, lower extremity numbness, and gait disturbance accompanied by immunoglobulin M paraproteinemia and lymphoplasmacytic lymphoma in the bone marrow. Thoracic and lumbar magnetic resonance imaging revealed a long paravertebral mass around the spinal column, but the direct infiltration could not be proven. The patient was diagnosed with possible Bing-Neel syndrome and managed with bendamustine and rituximab. After chemotherapy, the patient's neurological and radiological findings improved. Magnetic resonance imaging should be considered when the Bing-Neel syndrome diagnosis is unclear.

The 2,3-epoxy naphthoquinol produced by endophyte Arthrinium marii M-211.

A novel 2,3-epoxy naphthoquinol, named (6R,7R,8R)-theissenone A (1), possessing an oxatricyclo[5.4.0.03,5]undeca-trien-2-one skeleton, together with two known compounds, (6S,7R,8R)-theissenone (2) and arthrinone (3), were produced by an endophytic fungus, Arthrinium marii M-211, which was isolated from mangrove plants. The structure of 1, including the absolute stereochemistry, was elucidated by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data and time-dependent density functional theory (TDDFT) calculations of electronic circular dichroism (ECD) spectra. Additionally, the absolute structure of 2 was deduced as a diastereomer of 1 using ECD spectral data analysis. Compounds 1, 2 and 3 exhibited cytotoxic activity against the H4IIE rat hepatoma cells, with IC50 values of 67.5, 46.6 and 13.4 µM, respectively.

Laeverin/aminopeptidase Q induces indoleamine 2,3-dioxygenase-1 in human monocytes.

Human extravillous trophoblast (EVT) invades the maternal endometrium and reconstructs uterine spiral arteries cooperatively with maternal immune cells. Although EVT has allogeneic paternal antigens, the maternal immune system does not reject it. Here, we found that laeverin (LVRN), an EVT-specific cell surface peptidase, interacts with monocytes to produce indoleamine 2,3-dioxygenase-1 (IDO1). LVRN-transfected Swan71 cells, a cytotrophoblast-derived cell line, and increased IDO1 expression in PBMC under cell-to-cell interacting conditions. Soluble recombinant LVRN (r-LVRN) interacted with CD14-positive monocytes and induced their IDO1 expression without the intervention of other immune cell populations. LVRN-induced IDO1 production was promoted in PMA-activated monocyte-like THP-1 cells. Furthermore, r-LVRN decreased the tryptophan level and increased the kynurenine/tryptophan ratio in the culture media of the PMA-treated THP-1 cells. These findings suggest that LVRN is one of the key molecules that mediate the interaction between EVT and monocytes/macrophages and creates an immunosuppressive environment at the maternal-fetal interface in the uterus.

Spheroid formation induces chemokine production in trophoblast-derived Swan71 cells.

PROBLEM: In the cell column of anchoring villi, the cytotrophoblast differentiates into extravillous trophoblast (EVT) and invades the endometrium in contact with maternal immune cells. Recently, chemokines were proposed to regulate the decidual immune response. To investigate the roles of chemokines around the anchoring villi, we examined the expression profiles of chemokines in the first-trimester trophoblast-derived Swan71 cells using a three-dimensional culture model. METHOD OF STUDY: The gene expressions in the spheroid-formed Swan71 cells were examined by microarray and qPCR analyses. The protein expressions were examined by immunochemical staining. The chemoattractant effects of spheroid-formed Swan71 cells were examined by migration assay using monocyte-derived THP-1 cells. RESULTS: The expressions of an EVT marker, laeverin, and matrix metalloproteases, MMP2 and MMP9, were increased in the spheroid-cultured Swan71 cells. Microarray and qPCR analysis revealed that mRNA expressions of various chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CXCL10, in the spheroid-cultured Swan71 cells were up-regulated as compared with those in the monolayer-cultured Swan71 cells. These expressions were significantly suppressed by hypoxia. Migration assay showed that culture media derived from the spheroid-formed Swan71 cells promoted THP-1 cell migration. CONCLUSION: This study indicated that chemokine expressions in Swan71 cells increase under a spheroid-forming culture and the culture media have chemoattractant effects. Since three-dimensional cell assembling in the spheroid resembles the structure of the cell column, this study also suggests that chemokines play important roles in the interaction between EVT and immune cells in their early differentiation stage.

Exocrine Pancreatic Insufficiency Possibly Related to Atypical Chronic Graft-versus-Host Disease.

We report the case of a 66-year-old woman who presented with diarrhea and weight loss approximately 14 months after unrelated allogeneic bone marrow transplantation for acute myeloid leukemia. Her early post-transplant course was notable for mild acute skin graft-versus-host disease (GVHD) and biopsy-proven upper gastrointestinal (GI) acute GVHD, both of which resolved with treatment. She then developed weight loss and diarrhea treated with prednisolone for what was thought to be GI late acute GVHD. However, her diarrhea and weight loss persisted. Colonoscopy showed a grossly intact mucosa, and stool studies only confirmed steatorrhea. However, an atrophic pancreas was found on an abdominal computed tomography (CT) scan. Exocrine pancreatic enzymes, such as lipase and pancreatic amylase, were markedly decreased, yet pancreatic endocrine function remained intact. The patient's diarrhea and weight loss improved upon treatment with pancrelipase. Therefore, we suggest that her exocrine pancreatic insufficiency was likely partly caused by atypical chronic GVHD.

Androgen promotes squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

BACKGROUND: Since the human papillomavirus vaccines do not eliminate preexisting infections, nonsurgical alternative approaches to cervical intraepithelial neoplasia (CIN) have been required. We previously reported that FOXP4 (forkhead box transcription factor P4) promoted proliferation and inhibited squamous differentiation of CIN1-derived W12 cells. Since it was reported that FOXP expressions were regulated by the androgen/androgen receptor (AR) complex and AR was expressed on the CIN lesions, in this study we examined the effects of androgen on CIN progression. METHODS: Since AR expression was negative in W12 cells and HaCaT cells, a human male skin-derived keratinocyte cell line, we transfected AR to these cell lines and investigated the effects of dihydrotestosterone (DHT) on their proliferation and squamous differentiation. We also examined the immunohistochemical expression of AR in CIN lesions. RESULTS: DHT reduced the intranuclear expression of FOXP4, attenuating cell proliferation and promoting squamous differentiation in AR-transfected W12 cells. Si-RNA treatments showed that DHT induced the expression of squamous differentiation-related genes in AR-transfected W12 cells via an ELF3-dependent pathway. DHT also reduced FOXP4 expression in AR-transfected HaCaT cells. An immunohistochemical study showed that AR was expressed in the basal to parabasal layers of the normal cervical epithelium. In CIN1 and 2 lesions, AR was detected in atypical squamous cells, whereas AR expression had almost disappeared in the CIN3 lesion and was not detected in SCC, suggesting that androgens do not act to promote squamous differentiation in the late stages of CIN. CONCLUSION: Androgen is a novel factor that regulates squamous differentiation in the early stage of CIN, providing a new strategy for nonsurgical and hormone-induced differentiation therapy against CIN1 and CIN2.

Characterization of D-xylose isomerase from Shinella zoogloeoides NN6 and its application for producing D-allulose and two D-ketopentoses in a one-pot multi-step transformation.

Researchers continue to search for efficient processes to reduce the production costs of rare sugars. In this paper, we report a novel D-xylose isomerase from Shinella zoogloeoides NN6 (SzXI) and its application for efficient rare sugar production. Purified SzXI did not show remarkable properties when compared with those of a previously reported D-xylose isomerase. However, NN6 was found to express inducible SzXI and constitutive D-allulose 3-epimerase (SzAE) when cultivated with D-xylose as the sole carbon source. These two enzymes were partially purified and immobilized onto HPA25L, an anion exchange resin. The co-immobilized SzXI and SzAE (i-XA) showed optimal activity at 65°C in sodium phosphate buffer (pH 7.5) and 90°C in sodium phosphate buffer (pH 6.5), respectively. i-XA produced D-ribulose via D-xylulose from D-xylose at a conversion ratio of D-xylose:D-xylulose:D-ribulose of 72:18:10. Furthermore, D-allulose was also produced via D-fructose using D-glucose as the substrate, with a D-allulose yield of 11.2%. This is the first report describing a bacterium expressing D-xylose isomerase and D-allulose 3-epimerase that converts readily available sugars such as D-glucose and D-xylose to rare sugars.