Flavonol dioxygenase chemistry mediated by a synthetic nickel superoxide.

Nature exploits transition metal centers to enhance and tune the oxidizing power of natural oxidants such as O2 and H2O2. The design and interrogation of synthetic metallocomplexes with similar reactivity to metalloproteins provides one strategy for gaining insight into the mechanistic underpinnings of oxygen-activating enzymes such as oxidases, oxygenases, and dioxygenases like Ni-quercetinase (Ni-QueD). Ni-QueD catalyzes the oxidative ring opening of the polyphenol quercetin, a natural product with antioxidant properties. Herein, we report the synthesis and characterization of Ni(13-DOB), a Ni(II) species complexed by an N4-macrocycle that has been characterized by single crystal X-ray crystallography. Ni(13-DOB) forms a Ni-superoxide intermediate (Ni(13-DOB)O2•-) upon treatment with H2O2 and Et3N, as verified by resonance Raman spectroscopy. We demonstrate through UV/vis and LCMS that Ni(13-DOB)O2•- is capable of the 1-electron oxidation of flavonols, including both 3-hydroxyflavone (3-HF, the simplest flavonol) and quercetin itself. Incorporation of two O-atoms into the flavonol radical via superoxide from Ni(13-DOB)O2•- precedes oxidative cleavage of the flavonol scaffold in each case, consistent with quercetinase ring cleavage by Ni-QueD in Streptomyces sp. FLA. Conversion of 3-HF into 2-hydroxybenzoylbenzoic acid was accomplished with catalytic turnover of Ni(13-DOB) at ambient temperature, as confirmed by HPLC timecourses and GCMS analysis of isotopic labeling studies. The Ni(13-DOB)-mediated oxidative cleavage of quercetin to the corresponding biomimetic phenolic ester was also verified through 18O-isotopic labeling studies. Through the HPLC characterization of both on- and off-pathway products of flavonol dioxygenation by Ni(13-DOB)O2•-, the stringent reaction pathway control provided by enzyme active sites is highlighted.

Tactile stimulation in the delivery room: past, present, future. A systematic review.

In current resuscitation guidelines, tactile stimulation is recommended for infants with insufficient respiratory efforts after birth. No recommendations are made regarding duration, onset, and method of stimulation. Neither is mentioned how tactile stimulation should be applied in relation to the gestational age. The aim was to review the physiological mechanisms of respiratory drive after birth and to identify and structure the current evidence on tactile stimulation during neonatal resuscitation. A systematic review of available data was performed using PubMed, covering the literature up to April 2021. Two independent investigators screened the extracted references and assessed their methodological quality. Six studies were included. Tactile stimulation management, including the onset of stimulation, overall duration, and methods as well as the effect on vital parameters was analyzed and systematically presented. Tactile stimulation varies widely between, as well as within different centers and no consensus exists which stimulation method is most effective. Some evidence shows that repetitive stimulation within the first minutes of resuscitation improves oxygenation. Further studies are warranted to optimize strategies to support spontaneous breathing after birth, assessing the effect of stimulating various body parts respectively within different gestational age groups.

The FIREBIRD-II CubeSat mission: Focused investigations of relativistic electron burst intensity, range, and dynamics.

FIREBIRD-II is a National Science Foundation funded CubeSat mission designed to study the scale size and energy spectrum of relativistic electron microbursts. The mission consists of two identical 1.5 U CubeSats in a low earth polar orbit, each with two solid state detectors that differ only in the size of their geometric factors and fields of view. Having two spacecraft in close orbit allows the scale size of microbursts to be investigated through the intra-spacecraft separation when microbursts are observed simultaneously on each unit. Each detector returns high cadence (10 s of ms) measurements of the electron population from 200 keV to >1 MeV across six energy channels. The energy channels were selected to fill a gap in the observations of the Heavy Ion Large Telescope instrument on the Solar, Anomalous, and Magnetospheric Particle Explorer. FIREBIRD-II has been in orbit for 5 years and continues to return high quality data. After the first month in orbit, the spacecraft had separated beyond the expected scale size of microbursts, so the focus has shifted toward conjunctions with other magnetospheric missions. FIREBIRD-II has addressed all of its primary science objectives, and its long lifetime and focus on conjunctions has enabled additional science beyond the scope of the original mission. This paper presents a brief history of the FIREBIRD mission's science goals, followed by a description of the instrument and spacecraft. The data products are then discussed along with some caveats necessary for proper use of the data.

Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells.

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.

Images of God used by self-injurious burn patients.

Suicide by burning and other forms of self-injurious behaviors which involve burning are sometimes considered to have religious overtones. The ritual death of widows upon their husband's funeral pyre is closely associated with Hindu beliefs. Buddhists have used self-immolation as a form of protest. The Judaeo-Christian traditions have imagery of fire as cleansing and purifying; there is also secular imagery associating fire with images of condemnation and evil. Previous studies have described religiosity as a common theme among survivors. The present study describes the ways in which persons who inflicted self-injurious behaviors through burning, including attempted suicide, imagine the Divinity and use religious language to give meaning to their experience.

The complex realities of primary prevention for HIV infection in a "just do it" world.

Primary prevention of HIV requires behavior changes to decrease the risk of sexual, drug using, and occupational exposure to infection. The difficulties associated with behavior change are related to social, cultural, and political constraints that reinforce heterosexism, sexism, and racism. These problems are compounded by concurrent epidemics of violence, poverty, and drug use, and are made more difficult by the absolutist mentality. Use of theory-based interventions, awareness of community attributes, and a change to pragmatic political agendas can help in the development of effective prevention programs.

Interleukin-1beta-induced cyclooxygenase-2 expression requires activation of both c-Jun NH2-terminal kinase and p38 MAPK signal pathways in rat renal mesangial cells.

The inflammatory cytokine interleukin-1beta (IL-1beta) induces cyclooxygenase-2 (Cox-2) expression with a concomitant release of prostaglandins from glomerular mesangial cells. We reported previously that IL-1beta rapidly activates the c-Jun NH2-terminal/stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein kinase (MAPK) and also induces Cox-2 expression and prostaglandin E2 (PGE2) production. The current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 JNK2/SAPKbeta reduces Cox-2 expression and PGE2 production stimulated by IL-1beta. Similarly, overexpression of the kinase-dead form of p38 MAPK also inhibits IL-1beta-induced Cox-2 expression and PGE2 production. These results suggest that activation of both JNK/SAPK and p38 MAPK is required for Cox-2 expression after IL-1beta activation. Furthermore, our experiments confirm that IL-1beta activates MAP kinase kinase-4 (MKK4)/SEK1, MKK3, and MKK6 in renal mesangial cells. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta- induced Cox-2 expression with inhibition of both JNK/SAPK and p38 MAPK phosphorylation. Overexpression of the kinase-dead form of MKK3 or MKK6 demonstrated that either of these two mutant kinases inhibited IL-1beta-induced p38 MAPK phosphorylation and Cox-2 expression but not JNK/SAPK phosphorylation and activation. This study suggests that the activation of both JNK/SAPK and p38 MAPK signaling cascades is required for IL-1beta-induced Cox-2 expression and PGE2 synthesis.

N-(4-hydroxyphenyl)retinamide-induced death in human lymphoblastoid cells: 50 kb DNA breakage as a means of distinguishing apoptosis from necrosis.

Experimental studies of N-(4-hydroxyphenyl)retinamide, a potential cancer chemopreventive agent, have primarily involved breast cancer and neuroblastoma cell populations together with an investigation of myeloid leukemia cells and have principally been concerned with the induction of apoptosis. This investigation of N-(4-hydroxyphenyl)retinamide-induced apoptosis using T-cell-derived human lymphoblastoid lines extends these studies by indicating distinctive features associated with this drug. The induction of apoptosis is restricted to a limited concentration range, which, if exceeded, results in cell death by necrosis. While morphological changes typical of apoptosis induced by many agents are readily demonstrable after treatment of lymphoblastoid cells with 3 microM N-(4-hydroxyphenyl)retinamide, distinctive features evident using the retinoid include the absence of cell cycle arrest along with the mode and pattern of DNA breakage. Analysis by conventional gel electrophoresis indicated that internucleosomal fragmentation of DNA was an unreliable indicator of apoptosis. On the other hand, higher order DNA breakage was consistently detected during drug-induced apoptosis, but not as a result of treatment causing necrosis.

Methodological aspects of the interval trapping method with comments on nocturnal activity patterns in house mice living in outdoor enclosures.

Activity periods in small rodents are influenced by a variety of factors including climatic conditions, photoperiod, resource availability, and competitors. The timing of nocturnal activity was examined in free-living house mice inhabiting large outdoor enclosures. Live-traps were checked at 2-h intervals on specified nights. Mice were more active just after dusk and less active just before dawn. There was no significant differential timing of activity by age or sex. Dominant males were more active in the latter half of the night and subordinate males were more active in the first half of the night. Questions pertaining to the availability of traps, the proportion of mice still not captured, density effects on capture rates, and human activity influencing mouse activity were also tested. On average, more than 86% of the traps remained open even for the 2-h interval nearest dawn. A minimum of 70% of the trappable mice were not captured on a given trap night. The rate of mouse captures did not increase with increasing density. There was no difference in the overall mean number of mice caught on nights with and without interval trapping. However, capture rates were significantly lower in the period after a 2-h trapping interval than in the periods before or during that interval. Adult males and adult non-reproductive females were caught again sooner if they spent more time in the trap. There was no effect of the amount of time spent in the trap on the time to recapture for adult reproductive females or male or female juveniles.

HIV infection: challenges for dialysis personnel.

Patients with human immunodeficiency viral (HIV) infection who also require dialysis present a complex constellation of challenges for dialysis personnel. The major challenges that health care providers (HCPs) face when working with HIV-infected patients are related to overcoming personal fears and to adapting to HIV-specific patient care needs. Dialysis personnel who encounter these concerns do so with a background of experience that has prepared them to cope effectively with HIV disease in the dialysis setting. This article explores the problems of occupational exposure to HIV and chronic health care issues common to patients with HIV infection and renal disease.

Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats.

Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, i.p.) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 microns) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments ( < 4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.

Enhanced expression of bax in small preantral follicles during 4-vinylcyclohexene diepoxide-induced ovotoxicity in the rat.

4-Vinylcyclohexene diepoxide (VCD) destroys small preantral (25-100 microns) ovarian follicles after repeated dosing in mice and rats. A previous study determined this follicular destruction is via apoptosis (physiological cell death). The purposes of this study were to examine the effects of VCD on amounts of mRNA for several genes that might be involved in this ovotoxic response and to determine the specificity of this response for small preantral follicles. The genes of interest were bax, a cell death gene; three forms of the antioxidant enzyme, superoxide dismutase (mitochondrial manganese-containing or MnSOD, cytosolic copper/zinc-containing or Cu/ZnSOD, and secreted or secSOD); and microsomal epoxide hydrolase (mEH), involved in detoxification of VCD. Female Fischer 344 rats were administered daily doses (10 days) of vehicle control (sesame oil) or VCD (80 mg/kg, ip). Four hours after the last injection, livers and ovaries were removed. Small (25-100 microns) and large (100-250 microns) preantral follicles were separated from the ovaries by gentle dissociation and collected by mouth pipeting. Total RNA was extracted from all tissues, reverse transcribed into first-strand cDNA, and amplified by polymerase chain reaction using oligonucleotide primers specific for each gene. Relative levels of mRNA were visualized by agarose gel electrophoresis and autoradiography and quantified by densitometric analysis. Coamplification of ribosomal protein L19 (constitutively expressed in ovarian tissue) was used for normalization in each sample. Increased levels of mRNA for bax (172 +/- 20% of control, p < 0.05), MnSOD (248 +/- 70% of control, p < 0.05), and mEH (352 +/- 120% of control, p < 0.05) were measured in 25- to 100-microns follicles collected from VCD-treated compared with control rats. Unlike 25- to 100-microns follicles (the targets of ovotoxicity), in 100- to 250-microns follicles (nontargets) there were no changes (p > 0.05) in mRNA levels for bax or MnSOD in VCD-treated rats; however, mRNA levels for mEH were significantly decreased (79 +/- 4% of control, p < 0.05), compared with control. No changes in levels of mRNA for mEH were observed in liver from VCD-treated rats relative to control. Additionally, in liver VCD caused a significant decrease in mRNA levels for bax (31 +/- 5% of control, p < 0.05) and Cu-ZnSOD (56 +/- 17% of control, p < 0.05). In summary, dosing of rats with VCD enhanced expression of mRNA encoding several genes that might respond during the induction of ovotoxicity. The selective increase in bax in the population of follicles destroyed by repeated dosing with VCD may reflect their susceptibility to apoptosis.

Follicular mechanisms associated with 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats.

The mechanism of 4-vinylcyclohexene diepoxide (VCD)-induced oocyte destruction in small preantral follicles of rats and mice has not been elucidated. This study examined the effects of daily dosing of female rats with VCD on protein synthesis and follicle viability. An investigation of granulosa cells as a target for VCD was also made. Small preantral follicles (25 to 100 microns) isolated from untreated immature rats (day 28) as well as from rats injected daily for 10 d with VCD (0.57 mmol/kg, IP) or vehicle control (sesame oil) were incubated for 3, 6, or 10 h in vitro with or without VCD. Viability (trypan blue dye exclusion) or protein synthesis (3H-leucine incorporation) in follicles was measured. Large preantral follicles (100 to 250 microns), isolated oocytes or granulosa cells from small preantral follicles, hepatocytes, and adrenal cells served as controls. Viability was not compromised in small follicles isolated from untreated or VCD-injected rats. However, following in vitro incubation of small preantral follicles with VCD, there was a significant decrease in viability by 6 h. This loss in viability was observed in granulosa cells and was even greater in follicles from dosed as compared with undosed animals. The various cell types were incubated in vitro with or without VCD for 3 h and the rate of protein synthesis was measured by 3H-leucine incorporation during the last hour of incubation. Incubation of small preantral follicles from untreated animals with VCD for 3 h produced significant inhibition in the rate of protein synthesis. This effect was reversed and significantly stimulated after 6 and 10 h of incubation with VCD. Follicles from animals that had been dosed daily with VCD for 10 d demonstrated similar inhibition of protein synthesis following 3 h in vitro incubation with VCD; however, unlike those from undosed rats, follicles from dosed rats did not recover from this inhibition after 6 or 10 h of in vitro incubation with VCD. In vitro incubation with VCD stimulated the rate of protein synthesis in large preantral follicles; however, no effect on the rate of protein synthesis was observed in isolated oocytes and granulosa cells, hepatocytes, or adrenal cells. These observations suggest that VCD affects follicular viability via an effect on granulosa cells and that daily dosing of rats with VCD makes small preantral follicles more susceptible to ovotoxicity by VCD.

Patient education for behavior change: help from the Transtheoretical and Harm Reduction models.

Promoting healthy behaviors at all levels of prevention is an important component of nursing care in the HIV epidemic. This paper explores two models that can be used to support behavior change efforts: the Transtheoretical Model, which describes stages in the change process, and the Harm Reduction Model, which offers an incremental approach to helping clients move toward safer and healthier habits. A review of the literature shows that these holistic and caring models can be combined effectively to assist clients who are dealing with HIV-related issues. The resulting theory-based approach can help nurses maintain consistency and direction in educational interventions for behavior change.