Single-cell transcriptome analysis of endometrial tissue.

STUDY QUESTION: How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER: By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY: Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION: The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells, respectively. In addition, we performed a gene ontology analysis of the differentially expressed genes and found that these genes are mainly related to cell cycle, translational processes and metabolism. LIMITATIONS, REASONS FOR CAUTION: Although CD9-positive single epithelial cells sorting was successfully established in our laboratory, the amount of transcriptome data per individual epithelial cell was low, complicating further analysis. This step most likely failed due to the high dose of RNases that are released by the cells' natural processes, or due to rapid turnaround time or the apoptotic conditions in freezing- or single-cell solutions. Since only the cells from the late-secretory phase were subject to more focused analysis, further studies including larger sample size from the different time-points of the natural menstrual cycle are needed. The methodology also needs further optimization to examine different cell types at high quality. WIDER IMPLICATIONS OF THE FINDINGS: The symbiosis between clinical biopsy and the sophisticated laboratory and bioinformatic protocols described here brings together clinical diagnostic needs and modern laboratory and bioinformatic solutions, enabling us to implement a precise analytical toolbox for studying the endometrial tissue even at the single-cell level.

Suitcase lab measurement of digital cellphone interference levels on hearing aids.

A low-cost, "real-life" method for measuring the interference caused by digital wireless (cell-phones) telephones in hearing aids is proposed. Data would be valid for specific telephone and hearing aid models. The estimated equipment cost is $500.

The effect of different dose levels of degradable starch microspheres (Spherex) on the distribution of a cytotoxic drug after regional administration to tumour-bearing rats.

Tumour uptake of a radiolabelled anticancer drug, tauromustine ([14C]TCNU), was investigated, using two different routes of administration in rats with colon adenocarcinomas implanted in the liver. Intra-arterial administration produced higher concentrations of the drug and/or its metabolites in tumour tissue and lower concentrations in normal tissue compared to intravenous administration. The investigation of co-injection of [14C]TCNU intra-arterially with degradable starch microspheres (DSM) indicated that a high dose of DSM (30 mg/kg) resulted in a high concentration of radioactivity in normal liver tissue, and in adjacent organs. This unfavourable pattern was not observed with the low dose of DSM (12.5 mg/kg), which produced a significant increase in tumour uptake. The results demonstrate the efficacy of partial vascular blockade, as elicited by the low dose of DSM. This regime caused [14C]TCNU to be preferentially retained in the active peripheral regions of the tumour.

Hepatic intra-arterial administration of doxorubicin and degradable starch microspheres. A pharmacokinetic study in the rat.

Urinary concentration of doxorubicin and its active metabolite doxorubicinol was followed in two groups of rats after hepatic intra-arterial injection of a single dose of 6 mg/kg doxorubicin or the same dose combined with 30 mg/kg Spherex (DSM). Urinary samples were collected during five days following administration. The concomitant DSM administration caused a significant decrease of the cumulative urinary excretion of intact doxorubicin; average five days of cumulative values were 73 and 103 micrograms respectively in the group with and without DSM. This indicates a DSM-induced reduction in systemic drug exposure. The elimination rates of doxorubicin or its active metabolite were not influenced by DSM. The decrease in excretion of the parent drug was not due to an interaction between the drug and DSM as evident by examining the binding of doxorubicin to DSM in vitro. Instead regionally altered drug distribution seems a plausible explanation to the reduction.

The effect of different dosages of degradable starch microspheres (Spherex) on the distribution of doxorubicin regionally administered to the rat.

To increase the effectiveness of intra-arterial treatment, embolizing agents are used. DSM (Spherex) is a starch microsphere suspension--mean diameter 45 mu--which can be used repeatedly to produce a transient vascular occlusion for about half an hour. It has been used experimentally and clinically in the treatment of liver cancer to increase the efficacy of drugs such as doxorubicin. In the present study the effect of DSM on the distribution of 14C-labelled doxorubicin was investigated in rats with liver tumours. A previously used pharmacological dose of 30 mg/kg DSM was compared to a lower dose of 12.5 mg/kg, especially regarding the intrahepatic distribution of co-injected drug at two and twelve hours after injection. The lower dose was to found to give a more effective tumour targeting as well as less regional toxicity. The results of the present study show that in experimental models lower doses of DSM should be beneficial.

Degradable starch microspheres in cytostatic treatment of a liver carcinoma; experimental studies in rats with 5-fluorouracil, tauromustine, carmustine, doxorubicin and RSU-1069.

The degradable starch microspheres (DSM) used have a size of 45 microns and are dissolved by amylase in blood. After intraarterial administration of a mixture of DSM and cytostatic drugs the coinjected drugs remain for a longer time in the target tissue/tumor. A transient hypoxia occurs. Systemic exposure of drugs is decreased. Rats with a carcinoma implanted into the liver were given DSM and drugs via the hepatic artery. DSM did not significantly increase the incorporation of 5-fluorouracil (5-FU) into liver tumor RNA. The incorporation of 5-FU into intestinal and bone marrow RNA increased. DSM increased the antitumor effect of doxorubicin, tauromustine, carmustine and RSU-1069 (aziridine 2-nitroimidazole). Side effects, such as liver and gastric necroses and body weight loss, appeared in some rats. The toxic overspill to the stomach seemed to be reduced by giving the DSM in two parts, with all the cytotoxic drug in the first part. The effect on liver and tumor was not decreased by this procedure. DSM alone had no anti-tumor effect. DSM alone decreased liver UDP-glucuronic acid in tumor-free rats, given either by the hepatic artery or, in the double dose, by the portal vein. DSM alone did not increase liver NADPH-cytochrome c reductase activity or serum ASAT (aspartate-aminotransferase) or ALAT (alanine-aminotransferase), indicating that the DSM are inert to the liver, when infused into the tributary vessels.

Compression in the Time Domain.

High input levels can cause saturation and "fast pumping" effects in hearing aids. The resulting distortion products and changes in speech amplitude envelope are examined in the time domain. Spectrograms reveal changes in speech elements not apparent with time domain methods.

Reduction by norepinephrine of the side effects induced by combined hepatic arterial administration of degradable strach microspheres and adriamycin in rats with a liver adenocarcinoma.

In previous studies we found degradable starch microspheres (DSM) to increase the antitumor effect of adriamycin injected by the hepatic artery in rats with a liver adenocarcinoma. Increased side effects also appeared, namely body weight loss and liver necroses. In the present paper, norepinephrine in four different protocols was added to the injection of adriamycin + DSM to decrease the drug flow to normal tissues. In two protocols norepinephrine decreased the body weight loss. There was also a non-significant decrease in liver necroses but also in antitumor effect. In these experiments we also observed that some rats given adriamycin + DSM got gastric necroses. This was not found when norepinephrine was added. Addition of propranolol to norepinephrine did not decrease side effects. Vasoactive drugs may therefore be of value to diminish adverse side effects of the combination cytostatic agent + DSM, probably decreasing overspill into normal tissues.

Improved antitumor effect of the nitrosourea drugs tauromustine (TCNU) and carmustine (BCNU) on a rat liver adenocarcinoma after hepatic arterial administration with degradable starch microspheres.

The effect of the cytostatic nitrosourea drugs TCNU and BCNU on tumor growth was studied in rats with a transplantable colon adenocarcinoma implanted into the liver. The drugs were given as a single dose into the hepatic artery alone or together with degradable starch microspheres (DSM). The effect of the treatment on tumor growth was measured 7 days later. Compared to control animals, TCNU decreased tumor growth. BCNU alone did not significantly decrease tumor growth. The antitumor effect of both drugs was significantly improved by DSM. The addition of DSM to the drugs caused liver damage in a few rats. There were no differences between different treatment groups regarding body weight changes. DSM may protect bone marrow against BCNU toxicity. Injection of DSM alone had no effect either on tumor growth or on survival.

Indomethacin and pancreatic blood flow. An experimental study in pigs.

Indomethacin has been reported to decrease pancreatic blood flow. The drug has been used as an analgesic in acute pancreatitis. As decreased blood flow to the pancreas may detrimentally affect the outcome of acute pancreatitis, we investigated the effects of indomethacin on blood flow in the normal porcine pancreas. Regional blood flows, with special reference to the pancreatic flow, were studied with radioactively labelled microspheres in ketamine-anesthetized pigs before and after intravenous administration of indomethacin 2 mg/kg during 10 min. A transient decrease of cardiac output was seen during the infusion. Basal pancreatic blood flow was significantly increased 10 and 30 min after administration of indomethacin. No significant changes were found in small-intestinal or renal blood flow. We conclude that indomethacin does not reduce blood flow in normal porcine pancreas.

Enhanced effect of adriamycin on a rat liver adenocarcinoma after hepatic artery injection with degradable starch microspheres.

Rats with solitary liver tumors were treated with adriamycin administered via the hepatic artery with and without degradable starch microspheres. Tumor growth inhibition was significantly greater, and tumors were decreased in size 7 days after, following treatment with adriamycin + microspheres. The bone marrow seemed to be protected. However, the addition of microspheres to adriamycin gave a body weight loss and evidence of increased liver damage. Possible interrelations between liver damage, antitumor effect and body weight loss are indicated.

Continuous wound irrigation in the pig.

It was demonstrated in the pig that full-thickness wounds in the skin can be continuously treated by irrigation for several days. Irrigation was accomplished through a porous, occlusively applied dressing having two ports, one for supply and one for drainage. The fluid, delivered by means of an iv set, was sucked through the inert dressing, leaving it partially saturated, and with fluid spread evenly through its pores. Particulate matter was removed along narrow paths converging on the outlet, not showing any significant diversion. The apparatus for fluid supply and drainage was arranged to move with the animal, thus eliminating the risk of obstruction of the tubes as a result of twisting. The fluid was eliminated by one suction line, and the vacuum in the dressing maintained by another. This allowed the suction to be adjusted to a level near the atmospheric pressure.

Input stimuli for obtaining frequency responses of automatic gain control hearing aids.

Developing a family of frequency response curves for AGC types of hearing instruments using swept pure tones at varying input levels often produces erroneous results. This problem is caused by exceeding the threshold for activating the AGC circuit at some frequencies but not at other frequencies during the pure-tone sweep, thereby producing a different frequency response from that which would be obtained with a complex input signal such as speech-shaped noise. This measurement artifact may be minimized by ensuring that the threshold for activating the AGC circuit is either always exceeded or never exceeded during the development of a frequency response curve. Three input signals are compared for developing a family of frequency responses for an AGC hearing aid: (1) swept pure tone, (2) swept pure tone with bias tone added, and (3) shaped broad-band noise. The shaped broad-band noise appears to be the input signal of choice.

Pharmacokinetics of intra-arterial mitomycin C with or without degradable starch microspheres (DSM) in the treatment of non-resectable liver cancer.

The effects of degradable starch microspheres (DSM) on mitomycin C pharmacokinetics and bone marrow toxicity were studied in a phase II multicenter study. Sixty-three patients with non-resectable primary or secondary liver cancer were randomized to receive either i.a. mitomycin C 15 mg/m2 first, followed 5 weeks later by mitomycin C 15 mg/m2 plus DSM 360 mg administered into the hepatic artery (group I) or the same treatments in the opposite sequence (group II). In 36 out of 47 patients who received at least 2 treatments, peripheral venous blood samples were analyzed for mitomycin C pharmacokinetics on a minimum of 2 paired courses. In all patients, the area under the concentration time curve (AUC) was significantly lower when the drug was co-administrated with DSM, but the terminal half-life (t1/2) of mitomycin C was unchanged. In group I the addition of DSM resulted in a significantly lowered AUC, but not in group II. The discrepancy between the 2 groups is probably due to differences in DSM-induced intra-hepatic shunting. The addition of DSM resulted in significantly higher platelet nadir values, but unchanged white blood cell count nadir value. In conclusion, DSM reduce the systemic exposure of mitomycin C and seem to lessen the haematologic toxicity judged from a less pronounced decrease in platelets.

Influence of degradable starch microspheres (Spherex) on the retention of pertechnetate in a solitary rat liver tumor.

In a model of secondary liver cancer in the rat an evaluation was made of the influence of degradable starch microspheres (Spherex) on the drug retention in tumor and liver tissue. Sodium pertechnetate was used as a drug model substance and was injected into the hepatic artery alone or with degradable starch microspheres (DSM) in a dose of 6 or 12 mg. The distribution of pertechnetate was measured by a gamma-camera equipped with a high resolution collimator. In rats with liver tumor the total elimination of pertechnetate from the liver was delayed when compared to rats without tumor. The tumor concentration of pertechnetate was higher than that of the surrounding liver tissue, irrespective of the presence of DSM. With a DSM dose of 12 mg there was a significantly higher retention of pertechnetate in the tumor during the whole observation period compared to pertechnetate only. The results of this study indicates that DSM can be of value in regional liver chemotherapy to increase liver tumor drug exposure and to reduce systemic toxicity.

Hepatic artery administration of degradable starch microspheres: II. Effects on incorporation of uridine and uracil into RNA of an adenocarcinoma transplanted to rat liver.

Effects of degradable starch microspheres administered via the hepatic artery were examined in rats in which an adenocarcinoma was transplanted into the liver. 3H-uridine or 3H-uracil with cold uridine and uracil, respectively, in amounts corresponding to therapeutic doses of these two pyrimidines as fluoro compounds, were administered with or without microspheres. Labeling of the acid-soluble fraction and RNA of tumor, liver, small intestine, spleen, kidney, and bone marrow was examined after 3 and 60 min after injection. When microspheres were added, the specific radioactivity of tumor RNA was significantly higher at both 3 min (P less than 0.05) and 60 min (P less than 0.01) in the rats given uridine, and in rats given uracil it was higher at 60 min after injection (P less than 0.05). There were no such differences in the labeling of the normal tissues. The results indicate that arterial administration of cytostatic drugs, such as 5-fluoropyrimidines, together with degradable starch microspheres might increase the cytotoxic effect on tumors nourished by the artery.

5-Fluorouracil incorporation into RNA of a rat liver adenocarcinoma after hepatic artery injection together with degradable starch microspheres.

The effect of degradable starch microspheres (DSM) on the cellular incorporation of 5-fluorouracil (FUra) was studied in rats with solitary liver tumours. 3H-labelled FUra [0.78 mg (6000 nmol)/kg b.wt.] was injected with saline or mixed with DSM, into the hepatic artery. Labelling of the acid soluble fraction (ASF), RNA and DNA of tumour, liver, bone marrow and small intestine was measured 60 minutes after injection. The DSM had no significant effect on the incorporation of FUra into the ASF or RNA, neither in tumour nor liver tissue. Regarding the tumour/normal tissue ratios of specific radioactivities, there was with DSM a higher tumour/liver and a higher tumour/bone marrow ratio in the ASF, indicating an increased tumour drug exposure with DSM. However, this was not accompanied by any significant increase in drug anabolism.

The influence of degradable starch microspheres on liver uptake of 5-fluorouracil after hepatic artery injection in the rat.

The effect of degradable starch microspheres (DSM) on the distribution of 5-fluorouracil (5-FU) after hepatic artery injection was studied in normal rats. Carbon-14-labelled 5-FU was injected separately or together with DSM into the hepatic artery. Radioactivity was measured in liver tissue, bile, peripheral blood, and urine. When microspheres were added, the liver uptake of 5-FU was increased; its peak concentration in peripheral blood decreased, as did the early urinary excretion of radioactivity. The addition of degradable starch microspheres to hepatic artery injections of cytostatic drugs might be of value in increasing the drug concentration in tumour tissue and reducing systemic toxicity.