Potentiated antitumor effects of APS001F/5-FC combined with anti-PD-1 antibody in a CT26 syngeneic mouse model.

APS001F is a strain of Bifidobacterium longum genetically engineered to express cytosine deaminase that converts 5-fluorocytosine (5-FC) to 5-fluorouracil. In the present study, antitumor effects of APS001F plus 5-FC (APS001F/5-FC) in combination with anti-PD-1 monoclonal antibody were investigated using a CT26 syngeneic mouse model. Both of dosing of APS001F/5-FC before and after anti-PD-1 mAb in the combination dosing exhibited antitumor effects as well as prolonged survival over the nontreated control. The survival rate in the combination therapy significantly increased over the monotherapy with APS001F/5-FC and that with anti-PD-1 mAb. Regulatory T cells among CD4+ T cells in tumor decreased in the combination therapy, while the ratio of CD8+ T cells was maintained in all groups. Taken these results together, APS001F/5-FC not only demonstrates a direct antitumor activity, but also immunomodulatory effects once localized in the hypoxic region of the tumor, which allows anti-PD-1 mAb to exert potentiated antitumor effects.

A case of ezetimibe-effective hypercholesterolemia with a novel heterozygous variant in ABCG5.

Sitosterolemia is caused by homozygous or compound heterozygous gene mutations in either ATP-binding cassette subfamily G member 5 (ABCG5) or 8 (ABCG8). Since ABCG5 and ABCG8 play pivotal roles in the excretion of neutral sterols into feces and bile, patients with sitosterolemia present elevated levels of serum plant sterols and in some cases also hypercholesterolemia. A 48-year-old woman was referred to our hospital for hypercholesterolemia. She had been misdiagnosed with familial hypercholesterolemia at the age of 20 and her serum low-density lipoprotein cholesterol (LDL-C) levels had remained about 200-300 mg/dL at the former clinic. Although the treatment of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors was ineffective, her serum LDL-C levels were normalized by ezetimibe, a cholesterol transporter inhibitor. We noticed that her serum sitosterol and campesterol levels were relatively high. Targeted analysis sequencing identified a novel heterozygous ABCG5 variant (c.203A>T; p.Ile68Asn) in the patient, whereas no mutations were found in low-density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9), or Niemann-Pick C1-like intracellular cholesterol transporter 1 (NPC1L1). While sitosterolemia is a rare disease, a recent study has reported that the incidence of loss-of-function mutation in the ABCG5 or ABCG8 gene is higher than we thought at 1 in 220 individuals. The present case suggests that serum plant sterol levels should be examined and ezetimibe treatment should be considered in patients with hypercholesterolemia who are resistant to HMG-CoA reductase inhibitors.

Identification of a homozygous frameshift variant in RFLNA in a patient with a typical phenotype of spondylocarpotarsal synostosis syndrome.

Spondylocarpotarsal synostosis syndrome, a rare syndromic skeletal disorder characterized by disrupted vertebral segmentation with vertebral fusion, scoliosis, short stature, and carpal/tarsal synostosis, has been associated with biallelic truncating mutations in the filamin B gene or monoallelic mutations in the myosin heavy chain 3 gene. We herein report the case of a patient with a typical phenotype of spondylocarpotarsal synostosis syndrome who had a homozygous frameshift mutation in the refilin A gene (RFLNA) [c.241delC, p.(Leu81Cysfs*111)], which encodes one of the filamin-binding proteins. Refilins, filamins, and myosins play critical roles in forming perinuclear actin caps, which change the nuclear morphology during cell migration and differentiation. The present study implies that RFLNA is an additional causative gene for spondylocarpotarsal synostosis syndrome in humans and a defect in forming actin bundles and perinuclear actin caps may be a critical mechanism for the development of spondylocarpotarsal synostosis syndrome.

In situ delivery and production system of trastuzumab scFv with Bifidobacterium.

A monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2), trastuzumab has become a standard treatment for HER2-positive breast cancer. Recent advancements in antibody engineering have enabled the efficient generation of the trastuzumab single-chain variable fragment (scFv). In this study, we genetically engineered Bifidobacterium, a bacterial strain shown to accumulate safely and selectively in hypoxic tumor sites by intravenous (iv) injection, to express and secrete the trastuzumab scFv. The recombinant scFv bound to cell surface HER2 and inhibited in vitro growth of HER2-positive human cancer cells. Moreover, iv-injected recombinant bacteria specifically localized and secreted trastuzumab scFv in xenografted human HER2-positive tumors and consequently inhibited tumor growth. The development and results of this novel in situ delivery and production system for trastuzumab scFv with Bifidobacterium represents a promising avenue for future application in cancer treatment.

Identification of a novel heterozygous mutation of the Aggrecan gene in a family with idiopathic short stature and multiple intervertebral disc herniation.

Aggrecan is a critical proteoglycan component of the extracellular matrix of the growth plates and articular cartilage and has a key role in the biophysical and biomechanical properties of cartilage. Recently, heterozygous mutations in the ACAN gene, which encodes aggrecan, have been identified in patients with short stature and accelerated bone age. We herein report another family with a heterozygous ACAN mutation associated with idiopathic short stature along with accelerated bone age and early-onset herniation of the lumbar discs at the levels of L1/2 through L5/S1. Whole-exome sequencing identified a novel heterozygous frameshift mutation in the ACAN gene (c.1744delT; p.Phe582fs*69) in all of the affected family members but not in the unaffected one, providing further evidence that ACAN haploinsufficiency causes short stature with advanced bone maturation. In addition, we advocate early-onset multiple disc herniation as a novel phenotype associated with ACAN haploinsufficiency.

Cross-calibration of pencil-beam (DPX-NT) and fan-beam (QDR-4500C) dual-energy X-ray absorptiometry for sarcopenia.

Sarcopenia, defined as the loss of muscle mass accompanied by weakness, is an important factor leading to frailty and is a growing concern in the aging Japanese society. Muscle mass can be calculated by dual-energy X-ray absorptiometry (DXA), but results differ between devices produced by different manufactures. Thus, cross-calibration is needed to compare body composition results in multicenter trials or when scanners are replaced. The purpose of this study was to perform an in vivo calibration of total body scans between pencil-beam (DPX-NT, GE Healthcare) and fan-beam (QDR-4500C, Hologic Inc.) DXA units. A total 30 subjects (15 women, 15 men, mean age = 35 years, range 22-49 years) were recruited. The lumbar bone mineral density (BMD), femoral neck BMD, appendicular fat and lean body mass, and the appendicular skeletal muscle mass index (ASMI) were highly correlated (r = 0.979-0.993, r(2) = 0.889-0.977). The conversion formulas were as follows: lumbar BMD, Y = -0.08 + 1.16X (X = QDR-4500C, Y = DPX-NT), femoral neck BMD, Y = -0.015 + 1.11X, and ASMI Y = 0.92 + 0.90X. There is excellent comparability between the DPX-NT and the QDR-4500C DXA units. However, cross-calibration equations are required to assess muscle volume, fat, and ASMI in multicenter studies investigating sarcopenia.

Intracranial calcification and psychotic symptoms after irradiation in a patient with Fanconi anemia: A case report.

Background: Patients with Fanconi anemia (FA) are at high risk for the development of malignancies, and are often treated with radiation therapy. Radiation therapy during childhood can cause intracranial calcification after a latent period, which has been associated with psychiatric symptoms. Despite the high sensitivity of patients with FA to radiation, intracranial calcification has rarely been reported in these patients. Case Presentation: A 17-year-old girl presented with psychiatric symptoms and cognitive impairment. She had been diagnosed with FA at 3 years old, and had received a bone marrow transplant at 5 years old with a preparative regimen that included total body irradiation. Results of IQ tests revealed a characteristic pattern of decline between the ages of 15 and 17 years. Computed tomography indicated multiple intracranial calcifications in regions associated with psychotic symptoms, including the frontal lobe and thalamus. The patient's psychiatric symptoms improved with the administration of blonanserin. Limitations: The patient did not have regular intracranial imaging, making it difficult to confirm a direct relationship between intracranial calcification, psychiatric symptoms, and cognitive impairment. It is unclear whether the intracranial calcification in this case can be explained entirely by irradiation. Conclusion: This case suggests a link between FA, intracranial calcification, and psychosis, in which intracranial calcification may have caused psychiatric symptoms. At present, evidence regarding the characteristics of psychiatric symptoms of intracranial calcification and its treatment is lacking. The current case will be helpful for elucidating the pathogenesis of this disorder and developing appropriate treatment protocols.

Rapidly Progressive Kidney Failure Associated with Perirenal Capsular Lesion Due to IgG4-Related Disease.

A 71-year-old Japanese man with progressive kidney failure was referred to our hospital. Laboratory tests showed elevated IgG4 levels. Contrast-enhanced computed tomography (CT) revealed soft tissue surrounding the left kidney and right atrophic kidney. A histopathological examination revealed inflammation and fibrosis with rich IgG4-positive cells in the thickened kidney capsule, but not in the kidney parenchyma. Poor enhancement in the left kidney on contrast-enhanced CT and wrinkling of glomerular capillaries in pathological tissues were also observed. These findings indicated IgG4-related perirenal lesions leading to low renal perfusion and kidney failure. The perirenal lesions and kidney failure were improved by corticosteroid therapy.

Brothers with novel compound heterozygous mutations in COL27A1 causing dental and genital abnormalities.

COL27A1 encodes a collagen type XXVII alpha 1 chain. It is the product of this gene that provides the structural support of connective tissue and is reported to be the causative gene of Steel syndrome (OMIM #615155). The primary symptoms of patients with this defect are consistent with systemic bone disease; however, recent reports note findings of intellectual disability and hearing loss. In this study, we identified novel COL27A1 compound heterozygous variants in two brothers with rhizomelia and congenital hip dislocation as well as dental and genital abnormalities that have not yet been reported in Steel syndrome. This variant, of maternal origin, caused an amino acid substitution of arginine for glycine, c.2026G>C or p.G676R, in the collagen helix domain, which is assumed to damage the structure of the helix. The paternally transmitted variant, c.2367G>A, is located at the 3' end of exon 12, and cDNA analysis revealed a splicing alteration. These novel, compound heterozygous COL27A1 variants might indicate an association of the gene with tooth and genital abnormalities.

Compound heterozygous variants in the ABCG8 gene in a Japanese girl with sitosterolemia.

Sitosterolemia is an autosomal recessive disorder that affects lipid metabolism and is characterized by elevated serum plant sterol levels, xanthomas, and accelerated atherosclerosis. In this study, we report a novel nonsense single-nucleotide variant, c.225G > A (p.Trp75*), and an East Asian population-specific missense multiple-nucleotide variant, c.1256_1257delTCinsAA (p.Ile419Lys), in the ABCG8 gene in a compound heterozygous state observed in a Japanese girl with sitosterolemia.

A case of sitosterolemia due to compound heterozygous mutations in ABCG5: clinical features and treatment outcomes obtained with colestimide and ezetimibe.

Sitosterolemia is a rare, autosomal recessively inherited disorder of lipid metabolism caused by mutations in the "ATP-binding cassette, subfamily G" member 5 and 8 proteins (encoded by the ABCG5 and ABCG8 genes, respectively), which play critical roles in the intestinal and biliary excretion of plant sterols. We report the clinical features and treatment outcomes of an 18-month-old Japanese girl with sitosterolemia, who presented with multiple linear and intertriginous xanthomas around the joint areas. Serum lipid analyses revealed elevated levels of total cholesterol (T-Chol: 866 mg/dL), low density lipoprotein-cholesterol (LDL-C: 679 mg/dL), and plant sterols (sitosterol: 24.6 mg/dL, campesterol: 19.2 mg/dL, stigmasterol: 1.8 mg/dL). Compound heterozygous mutations (p.R419H and p.R389H) were identified in ABCG5. The patient was placed on a low cholesterol/low plant sterol diet and treated with colestimide (a bile acid sequestrant) and ezetimibe (an NPC1L1 inhibitor). Serum T-Chol and LDL-C levels decreased to normal within 2 mo, and plant sterol levels decreased by 30% within 4 mo. The xanthomas regressed gradually, and almost completely disappeared after 1.5 yr of treatment. No further reductions of plant sterol levels were observed. Long-term follow-up is important to verify appropriate therapeutic goals to prevent premature atherosclerosis and coronary artery disease.

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage-specific manner.

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.

Draft Genome Sequence of Streptomyces incarnatus NRRL8089, which Produces the Nucleoside Antibiotic Sinefungin.

A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.

Direct electron transfer type disposable sensor strip for glucose sensing employing an engineered FAD glucose dehydrogenase.

The FAD-dependent glucose dehydrogenase (FADGDH) from Burkholderia cepacia has several attractive features for glucose sensing. However, expanding the application of this enzyme requires improvement of its substrate specificity, especially decreasing its high activity toward maltose. A three-dimensional structural model of the FADGDH catalytic subunit was generated by homology modeling. By comparing the predicted active site with that of glucose oxidase, the two amino acid residues serine 326 and serine 365 were targeted for site-directed mutagenesis. The single mutations that produced the highest glucose specificity were combined, leading to the creation of the S326Q/S365Y double mutant, which was virtually nonreactive to maltose while retaining high glucose dehydrogenase activity. The engineered FADGDH was used to develop a direct electron transfer-type, disposable glucose sensor strip by immobilizing the enzyme complex onto a carbon screen-printed electrode. While the electrode employing wild-type FADGDH provided dangerously flawed results in the presence of maltose, the sensor employing our engineered FADGDH showed a clear glucose concentration-dependent response that was not affected by the presence of maltose.