Sorting specimen-rich invertebrate samples with cost-effective NGS barcodes: Validating a reverse workflow for specimen processing.

Biologists frequently sort specimen-rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a "reverse workflow" is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next-generation sequencing (NGS) barcoding pipeline that allows for cost-effective high-throughput generation of short specimen-specific barcodes (313 bp of COI; laboratory cost <$0.50 per specimen) through next-generation sequencing of tagged amplicons. We applied our approach to a large sample of tropical ants, obtaining barcodes for 3,290 of 4,032 specimens (82%). NGS barcodes and their corresponding specimens were then sorted into molecular operational taxonomic units (mOTUs) based on objective clustering and Automated Barcode Gap Discovery (ABGD). High diversity of 88-90 mOTUs (4% clustering) was found and morphologically validated based on preserved vouchers. The mOTUs were overwhelmingly in agreement with morphospecies (match ratio 0.95 at 4% clustering). Because of lack of coverage in existing barcode databases, only 18 could be accurately identified to named species, but our study yielded new barcodes for 48 species, including 28 that are potentially new to science. With its low cost and technical simplicity, the NGS barcoding pipeline can be implemented by a large range of laboratories. It accelerates invertebrate species discovery, facilitates downstream taxonomic work, helps with building comprehensive barcode databases and yields precise abundance information.

Synthetic boundary cell in an analytical ultracentrifuge to determine the flotation coefficient of human serum lipoproteins.

Human serum lipoproteins were analyzed by a new modified synthetic boundary cell using an analytical ultracentrifuge. This cell allows the formation of the synthetic boundary at the bottom level of the cell with self-adjusting meniscus and baseline. Thus, the total amount of lipoproteins was seen as a single peak at first. During centrifugation, each component of the lipoproteins was separated according to its flotation characteristics. It was, therefore, possible to determine precisely all lipoprotein components, especially high-density lipoproteins, in the presence of a more rapidly migrating species and to calculate the flotation coefficient of each lipoprotein using the formula for sedimentation coefficient reported by Svedberg (Svedberg, T. (1925) Kolloid-Z. 36, 53-64.

Serum and lymph lipids in rabbits with carbon tetrachloride-induced cirrhosis of the liver.

Lymph flow and the composition of lymph lipids from the hepatic and thoracic ducts of rabbits with cirrhosis of the liver (induced by 46-51 intramuscular injections of a mixture of carbon tetrachloride and olive oil at 4-day intervals) have been compared with those of control animals injected with olive oil only. In cirrhotic animals, the concentration of lymph lipids was not greatly altered, but lymph flow, and consequently the hourly transport of lipids by lymph were greatly increased; the increase in transport of cholesteryl esters, free cholesterol, and phospholipids by way of the thoracic and hepatic duct lymph was particularly striking. The concentration of these lipid fractions in serum from the cirrhotic rabbits was also increased. The differences normally observed between lipid fatty acid compositions of serum and lymph disappeared in cirrhotic animals; this is interpreted as due to increased hepatic permeability to lipoproteins.

Serum prostaglandin D synthase level after coronary angioplasty may predict occurrence of restenosis.

Lipocalin-type prostaglandin D synthase (L-PGDS), which is responsible for the biosynthesis of PGD2, has recently been found to be present in the atherosclerotic plaque of the human coronary artery and also to be secreted in human serum. We measured the serum L-PGDS level and compared it with the expressions of the platelet membrane surface glycoprotein and neutrophil adhesion molecule in patients undergoing PTCA. The L-PGDS level significantly decreased (P < 0.01) and the platelet surface expression of CD62P (P-selectin) significantly increased (P < 0.01) immediately after PTCA in the coronary sinus blood. Both changes were inversely correlated (R = -0.72, P < 0.001). Although the L-PGDS level in the coronary sinus blood remained equivalent to the baseline level in patients who experienced restenosis, the level increased over the baseline level (P < 0.01) at 48 h after PTCA in patients without restenosis. Neutrophil surface expression of CD11b (alpha subunit of Mac-1) significantly increased at 24 h (P < 0.01) to 48 h (P < 0.001) after PTCA in the coronary sinus blood in patients with restenosis but the change showed less significant in patients without restenosis. The changes in the L-PGDS level and the CD11b expression at 48 h after PTCA were inversely correlated (R = -0.55, P < 0.05). An increased serum L-PGDS level at 48 h after PTCA possibly predicts the avoidance of late restenosis. It is suggested that reduction in PGD2 synthesis triggers platelet activation and that a subsequent increase in the PGD2 synthesis suppresses inflammatory reaction at the intervention site indicated by neutrophil activation and inhibits development of restenosis. Pharmacological or biological intervention that increases endogenous PGD2 synthesis should be tested as a new strategy to prevent restenosis.

Progestin binding in vitro by the brain cell nuclei of ovariectomized oestrogen-primed rats.

The uptake and binding of 17 alpha, 21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione, a synthetic progestin, by the hypothalamus and cerebral cortex of ovariectomized oestrogen-primed rats was examined in vitro. Uptake of this steroid by the medial basal hypothalamus was higher than that by the remaining hypothalamus and cerebral cortex. The component in the cytosol from whole hypothalami which bound the radioactive progestin sedimented in the 7S region when centrifuged in a sucrose density gradient. The tritiated progestin was displaced by incubation with non-radioactive progestin or progesterone but not by oestradiol-17 beta, corticosterone or 5 alpha-dihydrotestosterone (1 mumol/l). No 7S binding component was detected in a similar preparation from the cerebral cortex. The nuclear fraction from whole hypothalami extracted by KCl (0.4 mol/l) contained a progestin-binding complex which sedimented at 9S and which was heat-labile and protein in nature. It was concluded that the hypothalamus of ovariectomized oestrogen-primed rats contains progestin-binding material in the cytoplasm and progestin, bound to such material, is transported from the cytoplasm to the nucleus.

Progestin receptor in the thymus of ovariectomized immature rats.

By using the synthetic progestin promegestone (R5020), the location and characteristics of progestin receptors in the thymic cytosols from immature ovariectomized oestrogen-treated rats were determined. Tritiated promegestone bound to the cytosol with high affinity (dissociation constant (Kd) = 2.0 +/- 0.3 nmol/l; promegestone greater than progesterone greater than oestradiol greater than corticosterone testosterone) and low capacity (number of binding sites (Bmax) = 143.0 +/- 13.5 fmol/mg protein). These values were appropriate for progestin receptors. However, an extremely high dose of dexamethasone (10 mumol/l; 1000-fold excess over [3H]promegestone) slightly inhibited the specific binding. Progestin receptors were predominantly located in the reticuloepithelial (RE)-cell fraction, with few in the thymocyte T-cell fraction. The receptor level was raised (24.9 +/- 11.3 (S.E.M.) to 143.0 +/- 13.5 fmol/mg protein) with increased doses of oestrogen (0-30 micrograms) administered in vivo. Using sucrose density gradient ultracentrifugation it was found that the thymic progestin receptor had a sedimentation coefficient of 9S under low-salt conditions. These results clearly suggest that the thymus of the immature female rat contains a specific progestin receptor which is mainly located in the RE cells.

Female sex hormone stimulates cultured human keratinocyte proliferation and its RNA- and protein-synthetic activities.

The present study was carried out to assess the effect of female sex hormones, i.e., estrogen and progesterone, on human keratinocyte proliferation, and its RNA- and protein-synthetic activities in a culture system. The presence of receptors for estrogen and progesterone and their messenger ribonucleic acids (mRNAs) in the cultured cells was also investigated. Human keratinocytes were cultured in the experimental DMEM-Ham's F12 medium containing various concentrations of estrogen or progesterone, which was followed by determining cell yields and [3H]thymidine incorporation. The keratinocytes were also tested for RNA- and protein-synthetic activities by measuring [3H]uridine and [3H]leucine incorporation. Both estrogen and progesterone receptors were determined by the enzyme immunoassay method using monoclonal antibodies, and mRNA expression for these hormone receptors was detected by in situ hybridization. Cell yields and [3H]thymidine incorporation increased gradually until 3 x 10(-10) M of both estrogen and progesterone, decreased thereafter until 3 x 10(-7) M, and peaked at 3 x 10(-10) M. [3H]Uridine and [3H]leucine uptake followed almost the same pattern as the cell proliferation, peaking at 3 x 10(-10) M of both hormones. Small amounts of estrogen and progesterone receptors were present in the cultured cells, and their mRNAs were found to be present in the cell cytoplasm. These results clearly suggest that sex hormones play an important role in human keratinocyte proliferation, and its RNA- and protein-synthetic activities, at least in part, via their hormone receptors.

Comparative study of the asparagine-linked sugar chains of human lipocalin-type prostaglandin D synthase purified from urine and amniotic fluid, and recombinantly expressed in Chinese hamster ovary cells.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaalpha2-->3Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaalpha2-->3Gal and Siaalpha2-->6Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS glycoforms in different tissues.

Autoradiographic and cytochemical localization of androgen in human prostatic cancer cell lines.

For basic studies of receptor dynamics in androgen-responsive tissues and cells, the autoradiographic and cytochemical procedures were applied to cultured tumor cells (DU-145 and PC-3). Uptake and retention of 3H-R1881, a potent synthetic androgen, were observed in DU-145 cells. The radioactive labelling was intense, and solely confined to the nuclei of DU-145 cells. Radioactivity over PC-3 cells was minimal. For assessing binding specificity, DU-145 cells were incubated with 3H-R1881 in the presence or absence of either unlabelled R1881, testosterone, progesterone, estradiol-17 beta, or corticosterone. The displacement of 3H-R1881 with R1881 and testosterone was significant, while no displacement was observed with other steroids. Nuclear localization of cytochemical staining of the dihydrotestosterone-peroxidase conjugate was evident in DU-145 cells. Our results indicate that androgen receptor may reside primarily in target cell nuclei of androgen-responsive tissues and tumors.

Acute and transient increase of lipocalin-type prostaglandin D synthase (beta-trace) level in cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

We measured the concentration of lipocalin-type prostaglandin D synthase (PGDS) in cerebrospinal fluid (CSF) and serum in patients 1, 3, 5, 7, 9, 11, 14 and 17 days after subarachnoid hemorrhage (SAH) due to ruptured cerebral aneurysms. The PGDS level in lumbar CSF increased about two-fold at day 3 (20.85 +/- 2.71 microg/ml, mean +/- SE) and at day 5 (25.24 +/- 3.76), as compared with the level at day 1 (11.25 +/- 1.07). The CSF level gradually decreased and returned to the day 1 level at day 17. The serum PGDS level was much lower than the CSF level (0.39 +/- 0.06 at day 1) and almost unchanged until day 17. The neuron-specific enolase level in CSF, as an index of brain damage, was maximum at day 1 (29.83 +/- 7.32 ng/ml) and decreased at day 3 and at day 5 (18.28 +/- 2.65 and 11.95 +/- 1.82, respectively). These results suggest that the transient and delayed increase in the PGDS level in CSF is due to its induction of PGDS in the arachnoid membrane after SAH.

Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study.

Reinsertion of autogenous nucleus pulposus, an innovative method to delay further disc degeneration, has been proved with an experimental animal model. This study examined whether coculture of nucleus pulposus cells with annulus fibrosus cells (a) activates annulus fibrosus cells and (b) retards disc degeneration when reinserted into the disc in a rabbit model of disc degeneration. Coculture of the two cell types stimulated proliferation of each, as indicated by increased DNA synthesis measured by increases in DNA polymerase alpha expression and uptake of 5-bromo-2'deoxy-uridine assessed by an enzyme-linked immunosorbent assay. In a model of disc degeneration in rabbits, reinsertion of activated nucleus pulposus cells delayed the formation of clusters of chondrocyte-like cells, the destruction of disc architecture, and the elaboration of type-II collagen as measured immunohistochemically compared with no treatment. The direct reinsertion of activated nucleus pulposus cells into the disc offers a promising line of investigation for delaying intervertebral disc degeneration, although these results obtained with notochordal cells may not necessarily apply when mature central nucleus pulposus cells are used.

Autonomous control of expression of genes for insulin-like growth factors during the proliferation and differentiation of C2C12 mouse myoblasts in serum-free culture.

The proliferation and differentiation of skeletal muscle cells in culture are usually controlled by serum components, and the differentiation can be induced by a reduction in the serum concentration. Insulin-like growth factors (IGFs) play a critical role in stimulating myoblast differentiation, and the expression of their genes is controlled by serum factors. We have found that C2C12 myoblasts are capable of proliferation and differentiation even in serum-free medium that does not contain peptide mitogens. During these processes in serum-free medium, the accumulation of mRNAs for IGFs in the cells was observed; and their levels increased with concomitant increases in creatine kinase activity and myotube formation and a decrease in DNA synthesis. Thus, the present results suggest that proliferation and differentiation of C2C12 cells are autonomously controlled and that the increase in the expression of the IGFs may be independent of exogenous components.

Lymph nodal vital staining with newer carbon particle suspensions compared with India ink: experimental and clinical observations.

CH40 and CH1500AA are newly prepared carbon suspensions which were examined as vital staining dyes for their usefulness in visualizing lymphatics at operation and to blacken lymph nodes. In mice, these carbon suspensions at 0.001 ml/g of body weight and India ink were injected subcutaneously into the footpad of the right hindpaw. Regional lymph nodes were visualized and were examined stereomicroscopically to determine how intensely these nodes blackened with carbon suspensions. Compared with India ink, CH40 and CH1500AA blackened the regional lymph nodes much faster and more vividly (1-8 min. after subcutaneous injection). As analyzed by centrifugal particle size distribution, CH40 and CH1500AA are narrowly distributed with a small particle size (150 and 167 nm, respectively, in mean diameter). By contrast, India ink is comprised of widely distributed and relatively large particles in suspension (mean diameter--254 nm). In 10 patients undergoing radical gastrectomy for treatment of stomach cancer, CH40 blackened 69% of regional lymph nodes with metastases (38 of 55) and 76% of those nodes without metastases (387 of 512).

Autoradiographic study on localization of progestin target cells in the chicken testes.

Using an autoradiographic technique, the localization and concentration of progestin (P) in cell constituents of testes from the immature 25-to-70-day-old and mature 120-day-old chickens were investigated. After incubation of the frozen tissue sections with 3H-Promegestone (R5020), a synthetic P, specific radioactivity, was found in the seminiferous epithelium and interstitium in all the animals in the presence or in the absence of various non-radioactive steroids, and the intensity of radioactivity differed with age: high in 25-, 70-and 120-day-old chickens, and low in 50-day-old chickens. Estrogen treatment enhanced the intensity of radioactivity in the cells at all ages. The concentration of radioactivity was higher in the seminiferous epithelium than in the interstitial cells. In the frozen tissue sections, it was not possible to identify the kind of cell taking up the high amount of radioactivity. These results indicate that the testes from chicken during their growing stages contain cell constituents that specifically bind P.

Estrogen receptor in rat thymus cytosol.

Estrogen binding components were characterized in cytosol fractions from the lymphatic tissues of castrated rats. Sucrose gradient analysis revealed specific binding of tritiated estrogen in the thymus. The binding was highly specific since it was easily displaced by unlabeled estrogen, but not by the non-estrogenic steroids used. In thymus cytosols from both males and females, the equilibrium dissociation constant of estrogen binding was--0.3 nM and the saturation binding was 6 fmols/mg of protein. Enzyme- and heat-experiments demonstrated a specific estrogen binder in thymic cytosol which was heat-labile and protein in nature. It was concluded that the rat thymus contains on estrogen receptor which is in part protein and heat-labile.

Effect of sex steroids on the uptake of 3H-leucine by brain tissues of ovariectomized mice.

Effect of the sex steroids, progesterone (P) and testosterone (T), on 3H-leucine uptake by the brain cells of ovariectomized mice were examined. Animals were divided into four groups, i.e. group 1: control animals treated with sesame oil; group 2: animals treated with P; group 3: animals treated with T, and group 4: animals first treated with T and then with P. Animals in each group were given a single i.p. injection of 3H-leucine 24 hr after the last hormonal treatment, and sacrificed 2 hr later. Intensity of the uptake of the radiochemical was measured by counting the number of reduced silver grains over cell bodies in various brain regions using an autoradiographic technique. Group 1 showed a relatively high uptake in the SO, PV and SPh when compared with that in the remaining nuclei examined. Groups 2 and 3 both showed a significant enhancement of the uptake in SCH, ARC and PM when compared with that in group 1. Group 4 showed enhancement of the uptake in most of the nuclei except POL, DM and SPH when compared with that in group 1. However, only the POM, PV, SO and VM revealed a significantly higher uptake than the respective nuclei in groups 2 and 3. The uptake by cells in the EC and CC remained unchanged after the hormonal treatment. The present results suggest that in female mice P or T stimulates protein synthesis in the hypothalamic nuclei and that the effect of P on protein synthesis is greatly influenced by T-priming.

Progestin receptors in ovaries of 4-day cycling rats. An autoradiographic study.

Using an autoradiographic technique we investigated in vitro the localization of cell constituents with concentrations of a radioactive synthetic progestin (P), 3H-R5020 (17 alpha, 21-dimethyl-19-nor-pregna-4, 9-diene-3, 20-dione) and its metabolites in the ovaries of 4-day cycling adult rats. After incubation of the ovarian tissue sections with radioactive R5020 in the presence or absence of various non-radioactive steroids, specific labeling was found in the tissue sections in each phase of the estrous cycle. The intensity of labeling in the ovary during the estrous cycle was highest in proestrus, followed by that in diestrus, metestrus and estrus. The intensity of labeling in cell constituents was as follows: granulosa cells greater than luteal cells greater than thecal cells greater than stromal cells. These results indicate that the rat ovaries contain cell constituents that specifically bind P and that the intensity of binding fluctuates during the estrous cycle.

Biochemical characterization and immunohistochemical localization of progestin and estrogen receptors in castrate rat submandibular gland.

The physicochemical property and immunohistochemical localization of progestin (P) and estrogen (E) receptors (PR and ER) were examined in the submandibular gland (SMG) of 5-8-week-old castrated rats. The localization of epidermal growth factor (EGF) was simultaneously examined in the same tissue. The tissue cytosols from male and female rats specifically bound 3H-promegestone (3H-R5020) and 3H-estradiol-17 beta with high affinity and low capacity; the values were within the range of those reported for other tissues. However, E-treatment suppressed the specific P-binding in the female, whereas it did not in the male. On the contrary, E-treatment did not at all suppress specific E-binding in both sexes. Monoclonal antibodies against PR and ER were mainly located in the epithelium of the excretory duct and granular convoluted tubule, but not in the acinus. The monoclonal antibodies were also located in the large polygonal cell with irregular cell border, probably macrophage in the tissue. The EGF-immunoreactivity was observed in the epithelium of the same tissue region as that in which the monoclonal antibodies were located. The present results clearly suggest that the rat SMG tissue contains specific PR and ER that are mainly located in the epithelium of the duct system where EGF-producing cells are also located. The possibility that P and E may influence EGF-production through their receptors in this tissue was discussed.

Hormone and immune response, with special reference to steroid hormone. 2. Sex steroid receptors in rat thymus.

In this study, we characterized the sex hormone receptors in normal and abnormal rat thymus tissues using biochemical and immunohistochemical techniques. Judging from the experimental results, the progestin and the estrogen receptor-containing cells and the thymulin-producing cells are the same reticuloepithelial (RE) cells. This suggests that the sex steroids mediate immune functions of the thymus through receptors within the RE cells to produce thymulin which induces the thymocyte to differentiate and mature. Whether the sex hormones have any direct effects on either T or B cells is not known at this time. So, further studies are needed to clarify this point. Secondly, using the spontaneously developed thymoma tissues from BUF/Mna rat the present authors have just started to biochemically analyse the existence of sex hormone receptors and to immuno-histochemically identify sex hormone receptor-containing cells and thymulin-producing cells. As a contemporary result, progesterone and estrogen receptors were mainly located in the intact RE cells but not in the neoplastic cells, whereas thymulin-producing cells were in both intact RE cells and neoplastic cells. It seems by now that there is no correlation between steroid hormone receptors and thymulin production in the neoplastic cells, as would be in the intact RE cells. Further study is required to answer this question.

Progestin receptors in adult rat ovary during estrous cycle.

Using a synthetic progestin (P)(i.e. R5020), characteristics of P receptors were determined in the ovarian tissue cytosols from adult estradiol benzoate-treated or 4-day cycling rats. In the estradiol benzoate-treated rats a specific 3H-R5020 binding in the cytosol was found with a number of binding sites, V max = 110 fmol/mg protein and the equilibrium dissociation constant, Kd = 14 nM. In the 4-day cycling rats, specific binding was found in the 6-7 S region with the Vmax which fluctuated during the estrous cycle: the sequence of Vmax (fmol/mg protein) was 395 (proestrus) greater than 122 (diestrus) greater than 96 (late estrus) greater than 62 (metestrus) greater than 40 (early estrus). The Kd value varied during the cycle, the highest (22 nM) in proestrus and the lowest (5 nM) in early estrus. In addition, 3H-R5020 binders were thermolabile and of protein in nature. These results suggest that the rat ovary contains P receptors in its cytoplasm with high affinity and low capacity of P binding, the level of which fluctuates during the estrous cycle.