Machine-guided design of synthetic cell type-specific cis-regulatory elements.

Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing, and stimulus responses, which collectively define the thousands of unique cell types in the body. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for an intended purpose has arisen naturally through evolution. Here, we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell type specificity. We leverage innovations in deep neural network modeling of CRE activity across three cell types, efficient in silico optimization, and massively parallel reporter assays (MPRAs) to design and empirically test thousands of CREs. Through in vitro and in vivo validation, we show that synthetic sequences outperform natural sequences from the human genome in driving cell type-specific expression. Synthetic sequences leverage unique sequence syntax to promote activity in the on-target cell type and simultaneously reduce activity in off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs and demonstrate the required literacy to write regulatory code that is fit-for-purpose in vivo across vertebrates.

Geometrical constraint on stacking of polycyclic aromatic hydrocarbon molecules encapsulated in a single-walled carbon nanotube.

The stacking morphologies of polycyclic aromatic hydrocarbon (PAH) molecules encapsulated in a single-walled carbon nanotube (SWCNT) are investigated by using a molecular-dynamics (MD) method. The encapsulating SWCNTs are of twenty different diameters. For coronene molecules, both conjugate-gradient (CG) energy minimization of the stacked molecules in a SWCNT and dynamics simulation (DS) of encapsulation of the molecules in a SWCNT are performed; while for sumanene molecules, only DS of encapsulation of the molecules in a SWCNT is performed. The tilt angles and intermolecular distances are calculated from the final configurations via CG and DS. On the assumption that the morphologies of the molecules in a SWCNT are determined by the geometrical constraint condition, semi-analytical formulas for the dependence of the tilt angles of the molecules on the SWCNT diameter are derived. These formulas are expressed in terms of the inverse functions of cosine the arguments of which are linear functions of the SWCNT diameter, and successfully agree with the simulation data. Accordingly, they are useful for controlling the tilt angles of the PAH molecules encapsulated in a SWCNT by adjusting the SWCNT diameter. It is also revealed that the stacking geometry of sumanene molecules with small tilt angles in a SWCNT is consistent with that of a sumanene dimer in a free space which Karunarathna and Saebo (Struct. Chem., 2014, 25, 1831) obtained using ab-initio calculations.

Main metabolic pathways of TAK-802, a novel drug candidate for voiding dysfunction, in humans: the involvement of carbonyl reduction by 11ß-hydroxysteroid dehydrogenase 1.

To investigate species differences in the metabolism of TAK-802, in vitro and in vivo metabolic profiles were compared between humans and animals. TAK-802 was mainly metabolized to M-I, M-II, M-III and M-IV in human and animal liver microsomes. Especially the M-IV formation in humans was greater than that in animals. Likewise, M-IV was detected to a lower extent in the plasma and excreta of animals administered with TAK-802, whereas the AUC0-48 h of M-IV was approximately five-fold higher than that of TAK-802 in human plasma. These results indicate that the in vitro metabolic profile reflects the in vivo condition. Thus, to identify the metabolic pathway of TAK-802 in humans, the responsible enzyme to form M-IV was elucidated in vitro. Since M-IV is a reductive metabolite formed in microsomes, the possibility of involvement of 11ß-hydroxysteroid dehydrogenase (11ß-HSD), a carbonyl reductase located in microsomes, was first investigated. Consequently, M-IV formation was confirmed by incubation with human 11ß-HSD1-expressing microsomes and was concentration-dependently inhibited by glycyrrhetinic acid, an inhibitor for 11ß-HSD enzymes, indicating the involvement of 11ß-HSD1 in the M-IV formation. In contrast, little M-IV formation was observed using rat 11ß-HSD1, suggesting species differences between humans and rats. In addition, M-II was formed via M-IV, not via M-I and the CYP identification studies revealed that both M-I formation from TAK-802 and M-II formation from M-IV were mainly catalyzed by CYP3A4. In conclusion, 11ß-HSD1 and CYP3A4 are principally responsible for the metabolism of TAK-802 in humans and 11ß-HSD1 may be responsible for the observed species difference.

Viscosity and drag force involved in organelle transport: investigation of the fluctuation dissipation theorem.

We observed the motion of an organelle transported by motor proteins in cells using fluorescence microscopy. Particularly, among organelles, the mitochondria in PC12 cells were studied. A mitochondrion was dragged at a constant speed for several seconds without pausing. We investigated the fluctuation dissipation theorem for this constant drag motion by comparing it with the motion of Brownian beads that were incorporated into the cells by an electroporation method. We estimated the viscosity value inside cells from the diffusion coefficients of the beads. Then the viscosity value obtained by using the beads was found to be slightly lower than that obtained from the diffusion coefficient for the organelle motion via the Einstein relation. This discrepancy indicates the violation of the Einstein relation for the organelle motion.

Alcohol and aldehyde dehydrogenase polymorphisms and risk for suicide: a preliminary observation in the Japanese male population.

Epidemiological studies have shown that excessive alcohol consumption is a potent risk factor to develop suicidal behavior. Genetic factors for suicidal behavior have been observed in family, twin, and adoption studies. Because alcohol dehydrogenase (ADH1B) His47Arg and mitochondrial aldehyde dehydrogenase (ALDH2) Glu487Lys single nucleotide polymorphisms (SNPs), which affect alcohol metabolism, have been reported to exert significant impacts on alcohol consumption and on the risk for alcoholism in East Asia populations, we explored associations of the two functional SNPs with suicide using a case-control study of 283 completed suicides and 319 control subjects in the Japanese population. We found that the inactive ALDH2 allele (487Lys) was significantly less frequent in the completed suicides (19.3%) than in the controls (29.3%), especially in males, whereas this was not the case in females. The males bearing alcoholism-susceptible homozygotes at both loci (inactive ADH1B Arg/Arg and active ALDH2 Glu/Glu genotypes) have a 10 times greater risk for suicide compared with the males bearing alcoholism-protective homozygotes at both loci. Our data show the genetic impact of the two polymorphisms on suicidal behavior in the Japanese population, especially in males. Because we did not verify the daily alcohol consumption, the association of these SNPs with suicide might be due to alcoholism itself. Further studies using case-control subjects, which verifies the details of current and past alcohol consumption and diagnosis for alcoholism, are required to confirm these findings.

Clinical evaluation of the QuantiFERON-TB Gold test in patients with non-tuberculous mycobacterial disease.

OBJECTIVE: To evaluate the clinical usefulness of the QuantiFERON TB-2G (QFT-2G) test in patients with non-tuberculous mycobacterial (NTM) disease without a previous history of tuberculosis (TB). METHODS: The study consisted of 214 patients with NTM disease who satisfied the diagnostic guidelines of the American Thoracic Society. RESULTS: The causative microorganism was Mycobacterium avium in 83 patients, M. intracellulare in 80, M. kansasii in 33, M. marinum in 12, M. szulgai in 3, M. abscessus in 2 and M. chelonei in 1. The positive response rate of QFT-2G test result was 2% in 163 patients with M. avium-intracellulare complex (MAIC) disease, 52% in 33 with M. kansasii disease, 58% in 12 with M. marinum disease, 33% in 3 with M. szulgai disease, 0% in two with M. abscessus disease and 0% in one with M. chelonei disease. The positivity of the QFT-2G test was 52% in patients with NTM disease, thought to be because NTM possesses common M. tuberculosis-specific antigens. CONCLUSIONS: Although QFT-2G may be a useful diagnostic method to differentiate TB from MAIC disease, there are several problems to be resolved before it can be used as a diagnostic method for NTM disease (M. kansasii disease), including the determination of the positive cut-off level for QFT-2G test.

Indeterminate results of QuantiFERON TB-2G test performed in routine clinical practice.

The present authors assessed risk factors that can promote indeterminate results of QuantiFERON TB-2G (QFT-2G; Cellestis Ltd, Carnegie, Australia) tests. The subjects were 704 patients with suspected tuberculosis (TB) and latent TB infection between January 2005 and December 2007. The QFT-2G test and the tuberculin skin test (TST) were performed for all subjects. If the results of the QFT-2G test were indeterminate, the test was repeated within 1 month. In total, 72 (10.2%) patients showed indeterminate results on the QFT-2G test. Indeterminate results were due to positive control failure in 68 (88.9%) patients and negative control failure in four patients. The results of the TST were negative for 64 patients showing indeterminate results, the remaining eight patients showed a positive response to the TST. Indeterminate results were significantly associated with elderly and immunocompromised patients. Lymphocytopaenia and hypoalbuminaemia were significantly associated with indeterminate laboratory findings. When the QFT-2G test was repeated for all patients showing indeterminate results, 12 (16.7%) patients demonstrated determinate results on the subsequent test. Indeterminate results of the QuantiFERON TB-2G test under routine clinical practice are not infrequent. When scoring QuantiFERON TB-2G test results for elderly and immunocompromised patients, one must be careful because the possibility of obtaining determinate results may be low even if the test is repeated.

A functional polymorphism of the micro-opioid receptor gene is associated with completed suicides.

A recent linkage study suggested that a putative locus for suicidal behavior independent of psychiatric disease phenotypes lies at 5' upstream of the micro-opioid receptor (OPRM1) gene. We explored an association between suicide and genetic variations of the OPRM1 using a case-control study of 183 completed suicides and 374 control subjects. We genotyped four single nucleotide polymorphisms (SNPs) including a common A118G SNP. The genotypic and allelic distributions of the A118G SNP were significantly different between the completed suicide and control groups (P = 0.014 and 0.039, respectively). A dominant model analysis of the A118G SNP showed an enhanced association with suicide (P = 0.0041, Odds ratio 0.575) and this significant association was observed with a logistic regression analysis that takes sex and age factors into account (P = 0.021). Our results raise the possibility that the A118G SNP of the OPRM1 gene is associated with suicide.

Clinical evaluation of QuantiFERON TB-2G test for immunocompromised patients.

The usefulness of the tuberculin skin test (TST) and the QuantiFERON TB-2G (QFT-TB) test were compared in immunocompromised patients. The subjects consisted of 252 immunocompromised patients who were clinically suspected of tuberculosis (TB) infection between April 2005 and December 2006. Regarding the underlying diseases, 74 subjects had malignant diseases, 72 were undergoing immunosuppressive treatment, 52 had diabetes mellitus, 50 had chronic renal failure and four had HIV infection. While the positive rate of the QFT-TB test for the diagnosis of TB infection (TB disease or latent TB infection) was 78.1%, that of TST for TB infection was 50.0%. The QFT-TB test was significantly better than TST. However, 32 (13%) patients had an indeterminate QFT-TB result. Indeterminate findings were significantly more frequent in patients receiving immunosuppressive treatment (28%), especially with lymphocytopaenia in the peripheral blood, than in those who had other underlying diseases. While TST-positive and QFT-TB test-negative results were recognised in immunocompromised patients with bacille Calmette-Guérin vaccination or nontuberculous mycobacterial disease, TST-negative and QFT-TB test-positive results were recognised in immunocompromised patients with a past history of TB infection. It was concluded that the QuantiFERON TB-2G test is a more useful diagnostic method for tuberculosis infection than tuberculin skin test for immunocompromised patients suspected of tuberculosis disease. However, because the results of the QuantiFERON TB-2G test show an indeterminate response for patients receiving immunosuppressive treatment, especially for those with lymphocytopaenia due to severe underlying diseases, care must be taken in the interpretation of the QuantiFERON TB-2G test for these patients.

[Examination of the power of families taking care of patients at home at peace].

OBJECTIVE: We examined the power of families who take care of a patient at home. We wanted to know how much caring power patients needed to stay at home at peace. PATIENTS AND METHODS: The subjects were 150 patients who visit the hospital for day care or are taken care of at home under our management. We examined their age, sex, main disease, the points of their families power for their care, the assessment of how peaceful at home, use of care services, and special things. RESULTS: Most of the patients were in their 80's or 70's. Stroke was the main disease. The most care grade are second grade. Higher care grade are smaller number. About 50% of the families paid for care services. The assessment of low care grade patients did not depend on the power of the families. But the assessment of high care grade patients did depend on that. We concluded that it is difficult to take care at home of high care grade patients who does not have some powers on families for his care. If the patient and his/her family want to use care services, they have to pay 10% charge. It is difficult to alleviate the burden they pose on their family.

Metallothionein immunoreactivity in head and neck carcinomas; special reference to clinical behaviors and chemotherapy responses.

Metallothionein (MT), has selectively binding affinity for heavy metal ions and over expression of MT has a potential against resistance for CDDP anticancer agents and radiation treatment. The role of MT immunoreactivity of squamous cell carcinoma in oral and pharyngeal regions (n = 28) and in the maxillary sinus region (n = 3) was evaluated for distribution patterns of MT and clinicopathologic behaviors. All the sections were examined in 400x and counted for MT positive cells over 5 fields of tumor growing foci. MT immunoreactivity was expressed in both tumor cell cytoplasm and nuclei, and showed heterogeneous localization in tumor epithelial cells and in the stroma. Immunohistochemical localizations showed mosaic patterns as the highest MT staining tumor cells intermingled with negative or low staining cells in neoplastic foci, and in stromal cells. Histiocytic and fibrocytic cells in both peripheral and interstitial stromas were also not stained homogeneously. In oral and pharyngeal carcinomas (n = 28), MT positive cell index in treated cases (n = 11) was 17.85% and that in non treated tumors (n = 17) was 25.19%. In maxillary sinus carcinomas (n = 3), MT positive index was 4.56% and showed lowers levels as compacted to other SCC sites. Among histological grading in oral and pharyngeal SCCs, MT index of well differentiated SCC (n = 9) was 17.04%, of moderately differentiated SCC (n = 13) 21.92% and poorly differentiated SCC (n = 6) was 31.06%. There is no significant correlation of positive index of metallothionein between treated and untreated samples taken in oral and pharyngeal SCCs.

Stimulation of in vivo calcification using collagen membranes cultured with osteoblastic cells in vitro: a preliminary report.

Guided tissue regeneration (GTR) is a useful modality in the management of periodontal disease and for bone augmentation around osseointegrated implants. This study evaluated the in vivo use of atelocollagen membrane (AC) on which osteoblastic cells (OBCs) were cultured in vitro, for application as a GTR membranous material. Osteoblastic cells isolated in our laboratory from mouse calvaria formed a thin film on the AC in vitro which was easily manipulated after 21 days in culture. The AC and OBCs complex material (ACOB) was subjected to freezing and thawing and implanted in mouse subcutaneous tissue for the study of histologic events surrounding the implanted ACOB. Histologic findings in the subcutaneous tissue showed calcification on the ACOB at 28 days postimplantation, while no such finding was evident at the control site, where only AC without OBCs were grafted. The present study suggests the possibility of membrane calcification for GTR through ACOB produced by OBCs on an AC in vitro.

An in vivo evaluation of an osteoinductive implantable material produced by osteoblastic cells in vitro.

Osteoinductive implantable materials have been a subject of basic science research in clinical implantology. This study examined the osteoinductive effect of an implantable material produced by osteoblastic cells that were isolated in the laboratory from mouse calvaria. After 21 days in culture, osteoblastic cells formed a thin film that could be easily manipulated. This thin film was subjected to freezing and thawing and was implanted in mouse muscle tissue. Osteoblastic cells were strongly positive for alkaline phosphatase reactivity and Von Kossa stain in vitro. Collagen type I, osteocalcin (BGP), and alkaline phosphatase were identified at the immunohistochemical electron microscopic level. Histologic findings showed an osteoinductive effect of the implanted material. The results strongly suggest the possibility of producing an osteoinductive implantable material by culturing osteoblastic cells in vitro.

Monkey liver indanol dehydrogenase. Purification, properties, and kinetic mechanism.

Indanol dehydrogenase was purified to apparent homogeneity from monkey liver cytosol. The enzyme was a monomer with a molecular weight of 36,000 and pI of 8.7. The amino acid composition was determined. The enzyme oxidized alicyclic alcohols including transdihydrodiols of benzene and naphthalene in the presence of both NADP+ and NAD+, and reduced several xenobiotic carbonyl compounds in the presence of NADPH, the 4-pro-R hydrogen atom of which was transferred to the substrate. The results of fluorometric binding and kinetic studies are consistent with an ordered sequential mechanism with NADP+ binding first. The enzyme was inhibited competitively versus NADP+ and uncompetitively versus 1-indanol not only by chelating agents such as 1,10-phenanthroline and 2,2'-bipyridine but also by a nonchelating isomer, 4,4'-bipyridine, which suggests hydrophobic interaction of the aromatic compounds with the enzyme, which did not contain zinc. The enzyme was also inhibited by Cibacron blue dye, synthetic estrogens, and delta 4-3-ketosteroids. The inhibition by Cibacron blue was competitive versus NADP+ and noncompetitive versus 1-indanol, whereas those by hexestrol, medroxyprogesterone acetate, and progesterone were uncompetitive versus NADP+ and competitive versus 1-indanol, corraborating the ordered addition of the coenzyme prior to 1-indanol.

Purification and partial characterization of dimeric dihydrodiol dehydrogenase from monkey kidney.

Dihydrodiol dehydrogenase activity was detected in the cytosol of several monkey tissues, among which kidney exhibited the highest activity and contained a high-molecular weight (Mr approximately 65,000) enzyme species. The enzyme species was purified to apparent homogeneity and showed a subunit molecular weight of 39,000. The enzyme oxidized benzene dihydrodiol (Km = 0.9 mM) at a pH optimum of 9.8, and highly reduced vicinal diketones such as camphorquinone (Km = 0.1 mM) and diacetyl (Km = 0.8 mM) around pH 7.5, but alicyclic alcohols, hydroxysteroids and ketosteroids were inactive substrates for this enzyme. Quercitrin, SH-reagents, stilbestrol were inhibitory to the enzyme activity, but other synthetic estrogens, anti-inflammatory agents and 3-ketosteroids were not.

Effect of dietary protein on vitamin E levels in erythrocytes and tissues of rats.

The effect of dietary protein level on the transfer of alpha-tocopherol in blood to tissues including RBC was studied using rats. The first experiment comprised a 10% casein (low protein), 20% casein (normal) and 20% SPI (normal soybean protein) diet groups supplemented with 71.5 mg of alpha-Toc/kg diet. In Exp. 2 the relationship of the tissue alpha-Toc level and protein level in diets, as shown by recovery from vitamin E-deficient status after the administration of alpha-Toc for 3 days, was checked by adjusting the protein level in diets to 10%, 20% and 40% casein. In Exp. 1 alpha-Toc in RBC decreased significantly in the 10% casein and 20% SPI groups compared to the 20% casein group. Moreover, alpha-Toc in kidney, lung and muscle decreased significantly in the 10% casein and 20% SPI groups. alpha-Toc in liver in the 20% SPI group decreased significantly compared to the 20% casein group. In Exp. 2 similar results were observed (Table 4), but alpha-Toc in RBC showed only a tendency to decrease with the low protein diet. In Exp. 1 free cholesterol in RBC increased significantly in the 10% casein group compared with the other two groups.

The influence of marine oil intake upon levels of lipids, alpha-tocopherol and lipid peroxidation in serum and liver of rats.

The effect of dietary intake of marine oils (CLO and SBO) on the lipids, alpha-tocopherol and TBARS in serum and liver was investigated in rats. In the dietary fats (8% in diet), the proportion of marine oils and corn oil was changed at five steps from 100:0 to 0:100. In the groups fed more than 50% CLO, serum cholesterol levels decreased while liver cholesterol increased as compared with the rats fed less than 50% CLO. On the other hand, SBO intake lowered serum cholesterol, TGs and PLs in the group on more than 25% of total fats, while it also induced an elevation of liver cholesterol and total lipids, in slightly higher degree as compared with the case for CLO intake. TBARS levels increased in liver with increasing intake of both marine oils, whereas alpha-tocopherol levels contrarily decreased in serum and liver. Only 0.6 and 0.3% of omega-3 type unsaturated fatty acids (EPA and DHA) were involved in the experimental diets of 50% and 25% marine oil groups respectively, since both marine oils contained about 15% of omega-3 type polyunsaturated fatty acids. Our results show that such a low content of omega-3 type fatty acids affects body lipid metabolism with respect to change in cholesterol, TGs, PLs, alpha-tocopherol and lipid peroxidation.