All-polarization-maintaining Er-doped dual comb fiber laser using single-wall carbon nanotubes.

We demonstrated a bi-directional, Er-doped dual comb fiber laser consisting of all-polarization-maintaining fiber devices. Polyimide films in which single-wall carbon nanotubes (SWNTs) were dispersed were used as the in-line saturable absorber. In order to avoid synchronization of the two combs and associated damage to the SWNT film, a two-branch configuration with two SWNT films was employed. Soliton pulses with almost the same optical spectra were generated stably in each direction, and dual comb beats were observed simply by overlapping the two outputs. The repetition frequency was 28 MHz, and the frequency difference was 105-140 Hz. Thanks to the small frequency difference, dual comb beats corresponding to the whole optical spectrum were observed without any overlapping. Fourier transform spectroscopy using the developed dual comb source was examined, and the characteristics of an optical filter were successfully obtained.

Mitochondrial dynamics and their intracellular traffic in porcine oocytes.

Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.

Possible selection of viable human blastocysts after vitrification by monitoring morphological changes.

PURPOSE: Morphological assessment of human blastocysts has been effective for selecting embryos with high potential. However, they often show repeated shrinkage and expansion toward their hatching. Here we assessed whether capturing morphological changes over time of vitrified-warmed blastocysts could lead to a better selection of viable embryos from shrunken blastocysts. METHODS: The implantation rates of vitrified-warmed blastocysts that were shrunken or expanded (developing) at the time of loading for transfer were compared among 2,729 cycles that were subjected to single blastocyst transfer. Vitrified (107) and fresh blastocysts (17) were donated for the experimental study. To assess the relationship between morphology (expanded vs. shrunken) and the mitochondrial respiration of blastocysts, the oxygen consumption rate (OCR) was analyzed for 55 specimens using an uncoupler of oxidative phosphorylation. The remaining 69 blastocysts were used for recording morphological changes every 15 min for 48 h after warming. RESULTS: Because there were no surplus embryos, 7 % of the vitrified-warmed blastocysts were shrunken and transferred. The shrunken embryos had sufficient implantation ability (40 %). The OCR of the shrunken embryos was significantly lower than that of their expanded counterparts. Upon exposure to the uncoupler, the OCR of some shrunken embryos increased to levels similar to the expanded specimens. Time-lapse images revealed some shrunken embryos which formed blastocoel by 5 h following warming exhibited developmental competence to the hatched stage. CONCLUSIONS: Data of the present study suggest a group of shrunken blastocysts contains many viable and clinically available embryos and time-lapse observation of vitrified-warmed blastocysts is a potential method to distinguish viable embryos from shrunken blastocysts.

Evaluation of Delivered Doses in Proton Beam Therapy for Prostate Cancer Using Positron Emission Tomography/Computed Tomography Imaging.

AIMS: Proton beams deposit energy along their paths and stop abruptly without penetrating the opposite side, making it difficult to detect their actual paths. However, confirming the path may lead to evaluating the actual doses to organs at risk in proton therapy for prostate cancer. As proton beams produce positron emitters through nuclear fragmentation reactions, theoretically, proton beam paths can be measured by positron emission tomography/computed tomography (PET/CT). Therefore, this study investigated whether conducting PET/CT examinations immediately after proton beam therapy helps to assess the doses delivered to the rectal and urinary bladder walls, which are the major sites of radiation-related toxicity. MATERIALS AND METHODS: Between June 2022 and June 2023, 51 consecutive patients with prostate cancer who underwent proton beam therapy were enrolled and imaged with PET/CT to measure these radioactive particles and validate the actual dose delivered to the rectal and urinary bladder walls. RESULTS: The delivered doses assessed using PET/CT after proton beam therapy strongly correlated with the planned volume for proton beam treatment. CONCLUSIONS: PET/CT exhibited potential as a valuable tool for validating the irradiated dose to organs at risk.

Glomerular hyperfiltration with hyperglycemia in the spontaneously diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model.

Glomerular hyperfiltration is observed in an early stage of kidney diseases including diabetic nephropathy. A better understanding of pathophysiological changes in glomerular hyperfiltration is essential for development of new therapies to prevent kidney disease progression. In this study, we investigated glomerular changes including glomerular filtration rate (GFR) and glomerular size in the Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model, and we also evaluated pharmacological effects of the sodium glucose cotransporter 2 inhibitor dapagliflozin on the renal lesions. Dapagliflozin was administered to SDT fatty rats from 5 to 17 weeks of age. Blood and urinary biochemical parameters were periodically measured. GFR was determined by transdermal GFR monitor at 16 weeks of age and histopathological analysis was performed at 17 weeks of age. SDT fatty rat developed severe hyperglycemia and exhibited pathophysiological abnormalities in the kidney, such as an increased GFR, glomerular hypertrophy and tissue lesions. Dapagliflozin achieved good glycemic control during the experimental period, inhibited the increase in GFR, and improved histopathological abnormalities in tubules. These results suggest that the SDT fatty rat is a useful model for analyzing the pathogenesis of diabetic nephropathy during its early stage and dapagliflozin improves not only hyperglycemia but also glomerular hyperfiltration and tubule lesions in SDT fatty rat.

Impaired immune response to voluntary arm-crank ergometer exercise in patients with cervical spinal cord injury.

STUDY DESIGN: Non-randomized study. OBJECTIVE: The mechanism underlying exercise-induced argumentation of natural killer cell cytotoxic activity (NKCA) in humans remains unclear. To address this, NKCA responses were studied during and after exercise in persons with cervical spinal cord injury (CSCI) and dysfunctional sympathetic nervous system. SETTING: Kibikogen Rehabilitation Center for Employment Injuries. METHODS: We examined the NKCA responses to 20-min arm-crank ergometer exercise at 60% of maximum oxygen consumption in eight persons with CSCI (between C6 and C7) and six able-bodied subjects. NKCA, adrenaline, and cortisol were measured before, immediately after exercise, 1 h after exercise, and 2 h after exercise. RESULTS: In able-bodied subjects, NKCA increased immediately after exercise (P<0.01) and then decreased to below the pre-exercise level 1 h after exercise, before recovering to the baseline level at 2 h after exercise. Plasma adrenaline concentrations increased significantly immediately after exercise (P<0.01) and returned to the baseline level 1 h after exercise. The plasma cortisol level did not change throughout the study. In contrast, NKCA, plasma concentrations of adrenaline, and cortisol did not change throughout the study in subjects with CSCI. CONCLUSION: In subjects with CSCI, the lack of response in NKCA throughout the experiment is probably mainly due to a dysfunctional sympathetic nervous system.

A novel orexin antagonist from a natural plant was discovered using zebrafish behavioural analysis.

OBJECTIVE: Phenotypic screening is one of the most practical approaches to the identification of mediators of behaviour, since it is difficult to model brain function in vitro, at a cellular level. We used a zebrafish (Danio rerio) behavioural assay to discover novel, natural, neuroactive compounds. MATERIALS AND METHODS: A zebrafish behavioural assay was performed for seven natural compounds, obtained from plants. The behavioural profiles were compared to those of known psychoactive drugs. We characterised a natural compound exhibiting a behaviour profile similar to that of suvorexant, using in silico, in vitro and microarray expression analysis. RESULTS: The behavioural analysis performed in this study classified central nervous system drugs according to their mechanism. Zebrafish treated with a natural compound, 8b-(4'-Hydroxytigloyloxy) costunolide (8b), showed behaviour profiles similar to those of zebrafish treated with suvorexant, a known orexin antagonist. This behavioural assay was validated using in silico and in vitro assays, which revealed that the new compound was a dual orexin receptor antagonist. In addition, transcriptome analysis suggested that 8b might regulate the nuclear factor-κB (NF-κB) related pathway. CONCLUSIONS: We conclude that zebrafish phenotypic screening, combined with in silico assays and gene expression profiling, is a useful strategy to discover and characterize novel therapeutic compounds, including natural products.

Protection of cattle against rinderpest with vaccinia virus recombinants expressing the HA or F gene.

Rinderpest is a highly contagious ruminant viral disease manifested by a rapid course and greater than 90% mortality. Infectious vaccinia virus recombinants were constructed that express either the hemagglutinin or the fusion gene of rinderpest virus. All cattle vaccinated with either recombinant or with the combined recombinants produced neutralizing antibodies against rinderpest virus and were protected against the disease when challenged with more than 1000 times the lethal dose of the virus.

RNA-seq analysis of diet-driven obesity and anti-obesity effects of quercetin glucoside or epigallocatechin gallate in Drosophila adults.

OBJECTIVE: High-fat diet (HFD) feeding stimulates fat accumulation in mammals and Drosophila. In the present study, we examined whether simultaneous feeding of familiar anti-obesity drugs, quercetin glycosides (QG) and epigallocatechin gallate (EGCG), to Drosophila has the same suppressive effect on fat accumulation as previously reported in rats and mice. To understand the underlying molecular mechanisms of HFD diet-induced obesity and the suppression effect of the drugs, we performed transcriptome analyses. MATERIALS AND METHODS: We induced extra fat accumulation by feeding Drosophila fly food containing 20% coconut oil and quantified the triglyceride accumulated in flies. The effects of anti-obesity drugs were also evaluated. We isolated total RNA from each sample and performed RNA-seq analyses and quantitive Real Time-Polymerase Chain Reaction (qRT-PCR) to investigate altered gene expression. RESULTS: The mRNA levels of several genes involved in lipid metabolism, glycolysis/gluconeogenesis, and anti-oxidative stress changed in HFD-fed adults. Moreover, the levels altered in those fed an HFD with QG or EGCG. The qRT-PCR further confirmed the RNA-seq data, suggesting that the expression of five essential genes for lipid metabolism changed in HFD-fed flies and altered in the flies treated with anti-obesity drugs. The most remarkable alteration was observed in the dHSL gene encoding a lipase involved in lipid-storage after HFD feeding and HFD with QG or EGCG. These alterations are consistent with HFD-induced fat accumulation as well as the anti-obesity effects of the drugs in mammals, suggesting that the genes play an important role in anti-obesity effects. CONCLUSIONS: These are the first reports to date of entire profiles of altered gene expression under the conditions of diet-induced obesity and its suppression by anti-obesity drugs in Drosophila.

POU3F2 participates in cognitive function and adult hippocampal neurogenesis via mammalian-characteristic amino acid repeats.

POU3F2/BRN-2 is a transcription factor that is mainly expressed in the central nervous system and plays an important role in brain development. The transactivation domain of POU3F2 includes multiple mammalian-characteristic tandem amino acid repeats (homopolymeric amino acid repeats). We previously generated knock-in mice (Pou3f2Δ/Δ mice) in which all three homopolymeric amino acid repeats were deleted from the Pou3f2 transactivation domain and identified phenotypic impairments in maternal behavior and pup recognition. Yet, the exact biological implications of homopolymeric repeats are not completely understood. In this study, we investigated cognitive function and hippocampal neurogenesis in Pou3f2Δ/Δ mice. Pou3f2Δ/Δ mice exhibited cognitive impairment in object recognition and object location tests. Immunohistochemistry for doublecortin, a marker of immature neurons, showed a lower number of newborn neurons in the dentate gyrus of adult Pou3f2Δ/Δ mice compared with wild-type mice. Consistent with this observation, adult Pou3f2Δ/Δ mice had lower numbers of 5-bromo-2'-deoxyuridine (BrdU) and NeuN double-positive cells at 4 weeks after BrdU injection compared with control mice, indicating the decreased generation of mature granule cells in Pou3f2Δ/Δ mice. Taken together, these results suggest that POU3F2 is involved in cognitive function as well as adult hippocampal neurogenesis, and that homopolymeric amino acid repeats in this gene play a functional role.

Contaminated oral intubation equipment associated with an outbreak of carbapenem-resistant Pseudomonas in an intensive care unit.

Twenty intensive care patients were diagnosed as infected or colonized by Pseudomonas aeruginosa within a one-month period; a rate three to four times higher than the typical background frequency of this infection in the intensive care unit (ICU). Patients with positive respiratory specimens were mechanically ventilated, which included re-used disinfected bite blocks during intubation. Fourteen specimens from 20 positive patients originated in the respiratory tract. Seven clonal variants were isolated and identified as originating from the same strain by pulsed-field analysis. These isolates were also matched to the strain detected on the re-used bite blocks, which had been disinfected with 140ppm sodium hydrochloride. Notably, Staphylococcus aureus was also detected on bite blocks sterilized with ethylene dioxide, indicating incomplete disinfection. In immunocompromised patients, re-use of bite blocks during intubation must be prohibited. Single-use kits or intubation without the use of bite blocks is recommended.

Purification and properties of acid ribonucleases in human serum and leukocytes.

Acid RNase was purified from normal human serum about 2400-fold by chromatography on phosphocellulose and Sephadex G-75 and rechromatography on Sephadex G-75. Assayed with yeast RNA as substrate, the enzyme showed the maximal activity at about pH 6.5 with sodium phosphate buffer. The reaction was activated by Na+, K+, and spermine, but it was not affected greatly by Mg2+, Co2+, and EDTA. Ca2+, Fe2+, Zn2+, and Cu2+ inhibited the reaction. Among the synthetic substrates examined, the enzyme preferentially hydrolyzed pyrimidine nucleotides, with a higher affinity for polycytidylate than for polyuridylate. The enzyme was thermolabile, but it stabilized with bovine plasma albumin. The molecular weight was approximately 15,000, estimated gel filtration on Sephadex G-75, and its isoelectric pH was above 11.0. From normal human leukocytes, acid RNase was purified about 400-fold by the same procedure described previously except that rechromatography on Sephadex G-75 was omitted. The properties of leukocytic RNase were found to be similar to those of serum acid RNase, but the latter enzyme differed in substrate specificity substantially from leukocytic RNase, preferring polyuridylate to polycytidylate. This evidence shows that serum RNase is not of leukocytic origin under normal physiological conditions.

Serum acid ribonuclease in myelogenous leukemia.

Acid and alkaline RNase activities in serum were measured with yeast RNA as the substrate in normal subjects and in leukemic patients pretreatment and posttreatment, and the acid/alkaline ratios of activities were 0.63 +/- 0.08 (S.D.) (N, 12), 2.28 +/- 0.82 (N, 8), and 0.60 +/- 0.13 (N, 9), respectively. The mean value for the ratio in the pretreated leukemia was significantly higher than that in the other 2 groups (p less than 0.01). By separating these acid and alkaline RNases from normal and leukemic sera by phosphocellulose chromatography, it was further confirmed that acid RNase alone increased markedly in leukemic serum. From serum and leukocytes of leukemic patients, acid RNases were purified about 2000-fold and 300-fold, respectively, by phosphocellulose and Sephadex G-75 chromatography. Both enzymes displayed properties nearly identical with those of normal serum and leukocytes, except that leukemic serum acid RNase had about a 2.4-fold greater affinity for polyuridylate than for polycytidylate as substrate, in contrast to normal serum acid RNase that degraded polycytidylate exclusively. On the other hand acid RNases from serum leukocytes of leukemia showed a similar substrate preference. These results suggest that the high RNase levels of leukemic sera are due to an excessive leakage of acid RNase into the blood stream from abnormal leukocytes.

A calcium/calmodulin-dependent cyclic adenosine monophosphate phosphodiesterase from Drosophila heads.

A Ca2+-activated cycl AMP phosphodiesterase from Drosophila melanogaster heads was studied. The enzyme accounted for approx. 40% of the total, soluble cyclic AMP phosphodiesterase activity in heads. After gel filtration, Ca2+ stimulation of the enzyme was no longer apparent, but Ca2+ activation could be restored by the addition of boiled Drosophila extract to the column-fractionated phosphodiesterase. The protein responsible for restoring Ca2+ activation was purified and shown to have some characteristics of calmodulin. In addition, porcine calmodulin was able to activate the Drosophila phosphodiesterase. Thus, the phosphodiesterase-calmodulin system in Drosophila appears analogous to similar systems in mammals.

Comparison of biliary excretion and metabolism of lithocholic acid and its sulfate and glucuronide conjugates in rats.

Biliary excretion and biotransformation of tracer doses of [14C]lithocholic acid and its sulfate and glucuronide intravenously injected into bile-drainaged rats were compared. Biliary excretion efficiency was in the order of unconjugate sulfate glucuronide and all conjugates were completely excreted into bile within 60 min after injection. Only tracer doses of radioactivity were found in the liver and urine. About 90% of radiolabeled bile acids in bile were conjugated with taurine immediately after injection of lithocholic acid, whereas lithocholic acid-glucuronide was only partly conjugated with taurine all the time (less than 6%) and excreted into bile mainly as native compound. In the first 10 min, 66% of lithocholic acid-sulfate was conjugated with taurine and it gradually proceeded up to 87%. Hydroxylation at C-6 and C-7 positions of lithocholic acid proceeded time-dependently up to 45%. No hydroxylation was observed with lithocholic acid-sulfate or glucuronide. Differences of biliary excretion rate of these conjugates may be one of the reasons for the delayed decrease of sulfated and glucuronidated bile acids in serum after bile drainage to patients with obstructive jaundice of during the recovery of acute hepatitis than non-esterified bile acids.

Cytotoxic effect and uptake mechanism by isolated rat hepatocytes of lithocholate and its glucuronide and sulfate.

The hepatotoxicity and uptake mechanism of lithocholate and its glucuronide and sulfate were studied using isolated rat hepatocytes. Cytotoxicity was in the order of lithocholate greater than lithocholate-glucuronide greater than lithocholate-sulfate; their 50% cytotoxic concentrations on hepatocytes were 50, 150 and 700 microM, respectively. Thus, glucuronidation as well as sulfation acted to detoxify lithocholate, not relating to the previously reported higher cholestatic effect of lithocholate-glucuronide than lithocholate. Lithocholate uptake was linear up to 50 microM, whereas the uptakes of lithocholate-glucuronide and sulfate were saturable with an apparent Km and Vmax of 32 microM and 6.4 nmol/min per 10(6) cells for lithocholate-glucuronide and 26 microM and 11.8 nmol/min per 10(6) cells for lithocholate-sulfate. Na+ replacement by choline+ had no effect on the uptake of lithocholate and lithocholate-glucuronide, whereas it slightly inhibited lithocholate-sulfate uptake. Lithocholate-glucuronide uptake was inhibited by lithocholate-sulfate and sulfobromophthalein, whereas lithocholate-glucuronide and sulfobromophthalein had no effect on lithocholate-sulfate uptake. These data indicate that hepatic lithocholate uptake is mediated by simple diffusion, and that hepatic uptake of lithocholate-glucuronide and sulfate is mainly mediated by a Na(+)-independent carrier.

Binding of lithocholate and its glucuronide and sulfate by human serum albumin.

In the present study, the binding affinities of lithocholate sulfate and glucuronide by human serum albumin were compared to that of lithocholate by equilibrium dialysis, and the binding sites of these bile acids on those of various fluorescent probes and bilirubin were also studied. The dissociation constants for the primary binding sites for lithocholate sulfate and glucuronide on human serum albumin were 0.057 and 0.24 microM, respectively, which were lower than that for lithocholate (0.82 microM). Lithocholate sulfate and glucuronide, as well as lithocholate, did not simply inhibit the binding of the site II and III fluorescent probes or bilirubin to albumin. Inhibition by these bile acids of the site I fluorescent probe binding to albumin suggested that the secondary binding sites of these bile acids are equal to site I. The results of simultaneous equilibrium dialysis using [3H]lithocholate and [14C]lithocholate sulfate indicated that these compounds have the same primary binding site. In conclusion, sulfation and glucuronidation may increase the binding affinities of bile acids to human serum albumin without changing their binding site.

Purification and properties of alkaline ribonuclease from human serum.

1. Five alkaline ribonucleases (EC 3.1.4.22) were purified about 140- to 1900-fold from human serum by phosphocellulose and DEAE-cellulose chromatographies and Sephadex G-75 filtration, with a total recovery of 22%. These were designated as RNAases 1-5. 2. Optimum activities were observed at pH 8.5-8.7 for RNAases 1-4, and at pH 7.5 for RNAase 5. The molecular weights of these enzymes were estimated by gel filtration as 45 000, 32 000, 20 000, 13 000 and 8500, respectively. 3. These RNAases were found to be heat-labile proteins but are markedly stabilized with bovine plasma albumin. The reaction was activated by Na+, K+, Mg2+ and Ca2+, and inhibited by Co2+, Fe2+, Cu2+ and Zn2+. EDTA had little effect on the velocity of the reaction. Spermine caused 2- to 7-fold activation. 4. Among the substrates examined, these RNAases preferentially hydrolyzed pyrimidine bodies and except for RNAase 5 had a higher affinity for poly(C) than poly(U) as substrate. Each enzyme was free from other nucleolytic enzymes and hydrolyzed only RNA.

Purification and properties of deoxyribonuclease from human urine.

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.

Cholestasis induced by lithocholate and its glucuronide: their biliary excretion and metabolism.

We studied the effects of the infusion of lithocholate and lithocholate-3-sulfate and 3-glucuronide in rats (0.29 mumol/min per 100 g body weight for 40 min) on bile flow, together with their biliary excretion and metabolism. Lithocholate-glucuronide had a higher cholestatic effect than lithocholate, whereas lithocholate-sulfate had almost no effect on bile flow. Lithocholate was mainly converted to taurine or glucuronide conjugates in the bile, serum and liver and hydroxylation of the tauro-conjugate proceeded. Lithocholate-sulfate was almost completely excreted in the bile, mainly as tauro-conjugate. Lithocholate-glucuronide was excreted in bile almost without conjugation, while some taurine conjugation occurred in the serum and liver. These results suggest that the poor biotransformation of lithocholate-glucuronide is related to its higher cholestatic potency than lithocholate.