Food for all? Wildfire ash fuels growth of diverse eukaryotic plankton.

In December 2017, one of the largest wildfires in California history, the Thomas Fire, created a large smoke and ash plume that extended over the northeastern Pacific Ocean. Here, we explore the impact of Thomas Fire ash deposition on seawater chemistry and the growth and composition of natural microbial communities. Experiments conducted in coastal California waters during the Thomas Fire revealed that leaching of ash in seawater resulted in significant additions of dissolved nutrients including inorganic nitrogen (nitrate, nitrite and ammonium), silicic acid, metals (iron, nickel, cobalt and copper), organic nitrogen and organic carbon. After exposure to ash leachate at high (0.25 g ash l-1) and low (0.08 g ash l-1) concentrations for 4 days, natural microbial communities had 59-154% higher particulate organic carbon concentrations than communities without ash leachate additions. Additionally, a diverse assemblage of eukaryotic microbes (protists) responded to the ash leachate with taxa from 11 different taxonomic divisions increasing in relative abundance compared with control treatments. Our results suggest that large fire events can be important atmospheric sources of nutrients (particularly nitrogen) to coastal marine systems, where, through leaching of various nutrients, ash may act as a 'food for all' in protist communities.

Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector.

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors.

An experimental protocol for fertility preservation in prepubertal boys recently diagnosed with cancer: a report of acceptability and safety.

BACKGROUND: Gonadal damage is a consequence of therapy for pediatric malignancies. Prepubertal males have no semen or mature spermatozoa, posing a challenge for fertility preservation. Testicular tissue cryopreservation is a potential option but is still experimental. We report on a pilot protocol that offered testicular biopsy cryopreservation to families of prepubertal boys with newly diagnosed malignancy. The aims were to determine the acceptability and safety of this procedure. METHODS: Parents of prepubertal boys with diagnoses at highest risk for treatment-related gonadal damage were offered the option of testicular cryopreservation. Half of the biopsy was frozen for the subject's potential future use and the remainder used for research. Data on negative intraoperative and/or 7 day post-operative sequelae of testicular biopsies were assessed. Two to four weeks later, parents were asked to complete a questionnaire on factors influencing their decision to have the biopsy or not. RESULTS: Since January 2008, 24 boys have met the eligibility criteria but three required immediate treatment and were excluded. Sixteen of 21 families (76%) consented to testicular biopsy, indicating the prospective acceptability of this option to parents of boys aged 3 months to 14 years; 14 underwent the procedure without any negative intra- or post-operative sequelae. Although the time at diagnosis is stressful, families can give thoughtful consideration to this option. Factors such as religion, finance, ethics and the experimental nature of cryopreservation did not play a major role in decision-making. CONCLUSIONS: Parents of prepubertal boys with cancer are willing to pursue testicular tissue cryopreservation at diagnosis, and testicular biopsy caused no acute adverse effects.

Changes in Ocean Heat, Carbon Content, and Ventilation: A Review of the First Decade of GO-SHIP Global Repeat Hydrography.

Global ship-based programs, with highly accurate, full water column physical and biogeochemical observations repeated decadally since the 1970s, provide a crucial resource for documenting ocean change. The ocean, a central component of Earth's climate system, is taking up most of Earth's excess anthropogenic heat, with about 19% of this excess in the abyssal ocean beneath 2,000 m, dominated by Southern Ocean warming. The ocean also has taken up about 27% of anthropogenic carbon, resulting in acidification of the upper ocean. Increased stratification has resulted in a decline in oxygen and increase in nutrients in the Northern Hemisphere thermocline and an expansion of tropical oxygen minimum zones. Southern Hemisphere thermocline oxygen increased in the 2000s owing to stronger wind forcing and ventilation. The most recent decade of global hydrography has mapped dissolved organic carbon, a large, bioactive reservoir, for the first time and quantified its contribution to export production (∼20%) and deep-ocean oxygen utilization. Ship-based measurements also show that vertical diffusivity increases from a minimum in the thermocline to a maximum within the bottom 1,500 m, shifting our physical paradigm of the ocean's overturning circulation.

DNA replication of first-generation adenovirus vectors in tumor cells.

A major role of the early gene 1A and 1B products (E1A and E1B) in adenovirus infection is to create a cellular environment appropriate for viral DNA replication. This is, in part, achieved by inactivation of tumor suppressor gene products such as pRb or p53. The functions of these same cellular proteins are also frequently lost in tumor cells. Therefore, we hypothesized that tumor cell lines with deregulated p53 and/or pRb pathways might support replication of E1A/E1B-deleted, first-generation adenovirus vectors (AdE1(-)). Here, we analyzed the impact of virus uptake, cell cycling, and the status of cell cycle regulators on AdE1(-) DNA synthesis. Cellular internalization of AdE1(-) vectors varied significantly among different tumor cell lines, whereas nuclear import of incoming viral DNA appeared to be less variable. Replication assays performed under equalized infection conditions demonstrated that all analyzed tumor cell lines supported AdE1(-) synthesis to varying degrees. There was no obvious correlation between the efficiency of viral DNA replication and the status of p53, pRb, and p16. However, the amount of virus attached and internalized changed with the cell cycle, affecting the intracellular concentration of viral DNA and thereby the replication efficacy. Furthermore, infection with AdE1 - vectors caused a partial G(2)/M arrest or delay in cell cycle progression, which became more pronounced in consecutive cell cycles. Correspondingly, vector DNA replication was found to be enhanced in cells artificially arrested in G(2)/M. Our findings suggest that cell cycling and thus passing through G(2)/M supports AdE1(-) DNA replication in the absence of E1A/E1B. This has potential implications for the use of first-generation adenovirus vectors in tumor gene therapy.

Blood pressure load--a better determinant of hypertension.

Noninvasive ambulatory blood pressure monitoring was used to evaluate the diagnosis of hypertension in 168 untreated patients with essential hypertension. On the basis of overall office blood pressure--the mean of 12 measurements, 2 in each of three positions (supine, sitting, and standing) on 2 consecutive days--133 patients were diagnosed as having hypertension (diastolic blood pressure of 90 mm Hg or higher) and 35 as having borderline hypertension (diastolic blood pressure of less than 90 mm Hg). The mean blood pressures for those with hypertension and borderline hypertension were 149/99 and 135/87 mm Hg, respectively. The mean ambulatory diastolic blood pressure was 90 mm Hg or higher in 123 patients during awake hours and in 91 patients during 24 hours. The diastolic blood pressure loads (percentage of ambulatory diastolic blood pressures more than 90 mm Hg) in patients with hypertension and borderline hypertension, respectively, were 69% and 43% during awake hours and 59% and 35% during 24 hours. The systolic blood pressure loads (percentage of systolic readings more than 140 mm Hg) during awake and 24 hours were 56% and 48%, respectively, in patients with established hypertension and 31% and 26%, respectively, in those with borderline hypertension. Thus, ambulatory blood pressure monitoring and blood pressure load provide useful information for diagnosing hypertension.

Allergic reactions to latex among health-care workers.

With the emergence of the acquired immunodeficiency syndrome (AIDS) epidemic and the practice of protecting health-care workers from all body fluids, the use of rubber gloves has increased, as has occupational allergy to latex among health-care workers. During 1991, 49 Mayo Medical Center employees sought assessment and treatment of rhinitis, conjunctivitis, contact urticaria, contact dermatitis, asthma, or eczema thought to be related to exposure to latex. Most of these persons had a history of atopy and worked in areas where rubber gloves were used and changed frequently. Of the 49 subjects, 34 had positive results of skin tests to latex products, and the sera from 19 of 35 persons tested contained increased latex-specific IgE antibodies. Employees with sensitivity to latex (and co-workers in the immediate areas) should use vinyl gloves and should notify their own health-care providers of their sensitization. Changes in job assignment may be necessary for some persons.

Antihypertensive efficacy of lisinopril. Ambulatory blood pressure monitoring.

Noninvasive automatic ambulatory blood pressure monitoring during 24 hours in eight patients with moderate hypertension was used to determine the blood pressure response to lisinopril, an angiotensin-converting enzyme inhibitor. Office, 24-hour ambulatory, awake ambulatory, and sleep ambulatory diastolic blood pressures were decreased from 108 +/- 3, 98 +/- 8, 101 +/- 7, and 87 +/- 14 mm Hg, respectively, at baseline to 83 +/- 4 (P less than or equal to 0.0001), 82 +/- 7 (P less than 0.0001), 84 +/- 7 (P less than 0.0001), and 73 +/- 9 mm Hg (P less than 0.005), respectively, after 20 weeks of lisinopril treatment (dose range, 40 to 80 mg once daily). The diastolic blood pressure loads (percentages of ambulatory diastolic blood pressures more than 90 mm Hg) during 24 hours and during awake hours were 74% +/- 19% and 83% +/- 15%, respectively, at baseline and 24% +/- 19% (P less than 0.0001) and 29% +/- 21% (P less than 0.0001), respectively, during treatment. Heart rate was not altered by lisinopril. In conclusion, lisinopril is an effective antihypertensive agent for the treatment of moderate hypertension, and ambulatory blood pressures and diastolic blood pressure loads are useful for evaluating therapy for hypertension.

Lack of RB protein correlates with increased sensitivity to UV-radiation-induced apoptosis in human breast cancer cells.

The underlying causes for different apoptotic responses in neoplastic cells are still not fully understood. We demonstrate here that a human breast cancer cell line, MDA-MB-468, which lacks the retinoblastoma protein (RB), is particularly sensitive to low doses of ultraviolet (UV) radiation. These cells are 15-20-fold more sensitive to UV radiation than RB-positive cell lines, as measured by both apoptosis and clonogenic assays. In addition, a prostate cancer cell line that lacks functional RB, DU-145, was found to have a similar apoptotic response to low doses of UV radiation. Based on these data, we hypothesized that the lack of RB is responsible for the extreme sensitivity of these cells to UV-radiation-induced apoptosis. To further examine the role of RB in apoptosis, cells of RB-positive human breast cancer and normal cell lines were infected with the human papilloma virus type 16 (HPV-16) E7 and assessed for UV-radiation sensitivity. The HPV-16 E7 protein is known to decrease levels of free RB in cells. Infection of RB-positive human breast cancer or normal cells with E7 resulted in a 4-5-fold increase in sensitivity to UV radiation compared to controls. The above data suggest a role for the RB protein in protecting cells from undergoing apoptosis in response to UV radiation.

Correlation between verapamil plasma concentration and P-R prolongation in essential hypertension.

Plasma verapamil concentration was correlated with serial electrocardiographic P-R intervals in patients with essential hypertension receiving immediate-release (80 to 120 mg three times a day) or sustained-release (240 mg daily) verapamil. The mean P-R interval in 22 patients taking placebo and immediate-release verapamil was 0.18 second. The borderline first-degree atrioventricular block of three patients did not change during treatment. Plasma verapamil concentrations of patients with a P-R interval longer than 0.20 second and of those with a P-R interval of 0.20 second or less were 169 +/- 73 ng/mL and 63 +/- 8 ng/mL, respectively. Six patients taking sustained-release verapamil had a maximal mean P-R interval of 0.19 +/- 0.01 second during 24-hour ambulatory electrocardiographic monitoring. P-R intervals were 0.22 second or more in two patients, but they returned to normal by hour 7 for one and by hour 20 for the other patient. In summary, transient P-R prolongation occurred with oral verapamil therapy, but no patient, regardless of baseline P-R interval, developed high-grade atrioventricular block.

Antihypertensive effects of a new sustained-release formulation of nifedipine.

The blood pressure response to a new sustained-release formulation of nifedipine was evaluated in an 8-week, double-blind, placebo-controlled study. Twenty-nine patients with mild essential hypertension were randomized to receive placebo (N = 9), 30 mg nifedipine (N = 10), or 60 mg nifedipine (N = 10). During treatment, 30-mg and 60-mg doses of nifedipine administered once daily decreased office blood pressures from 137/98 +/- 8/2 mm Hg and 141/98 +/- 15/2 mm Hg at baseline, respectively, to 126/89 +/- 9/7 mm Hg and 126/86 +/- 6/7 mm Hg (P less than .005). Noninvasive automatic ambulatory blood pressure monitoring demonstrated a marginally significant (P less than .10) reduction in the mean 24-hour blood pressure of 2/6 +/- 8/8 mm Hg and 5/6 +/- 9/9 mm Hg for patients taking 30 mg and 60 mg nifedipine once daily, respectively. Diastolic blood pressure load (the percentage of ambulatory diastolic blood pressure readings greater than 90 mm Hg) during 24 hours was decreased by 41% and 35%, with 30 mg and 60 mg nifedipine administered once daily, respectively. No significant dose response to nifedipine at these dose levels was observed. Although the once-daily formulation of nifedipine achieved effective control of office blood pressure, similar control was not observed in awake and 24-hour periods in all patients.

Anterior cruciate ligament injuries. Evaluation, arthroscopic reconstruction, and rehabilitation.

The advantages of arthroscopic reconstruction of the anterior cruciate ligament tear over arthrotomy are quite obvious: reduced pain and morbidity. Some arthroscopists are performing these procedures on an outpatient basis. The physician can choose from several graft substitutes for anterior cruciate ligament replacement. Autografts consisting of the iliotibial band, semitendinosus, gracilis, and meniscus have been used as grafts. The most common autograft is the bone-patellar tendon-bone, which has been used since 1930 and has been shown to have a tensile strength near that of the anterior cruciate ligament. The state of the art in surgical alternatives for anterior cruciate ligament tears is arthroscopic reconstruction using the midthird of the patellar tendon. Treatment of anterior cruciate ligament injuries requires prompt and adequate evaluation of the laxity of the ligament as well as other structures in the knee, appropriate treatment options offered to the patient with complete descriptions of knee function after each treatment option, and comprehensive rehabilitation program. Patient compliance is an integral part of the success of this procedure. The nurse must include a description of the injury, preoperative testing, surgical intervention, and rehabilitation program when educating the patient. The successful postoperative anterior cruciate ligament rehabilitation program is multifaceted. In general, there must be specific guidelines applied by a physical therapist who has knowledge of the surgical procedure, understands principles of ligament healing, and has the ability to individualize the program as needed. For any level of athlete or active person, there must be achievement of all goals per phase to a high performance level. In addition, there must always be objective measurements to document progress to the physical therapist and physician but, perhaps most importantly, to reassure the patient that normalcy is being restored.

The physiological genetics of denitrifying bacteria.

The genetics of denitrification is a relatively unexplored area that has great promise. Species of Pseudomonas are probably best suited for study because they are widely found among natural denitrifying populations and are quite readily amenable to genetic analysis. The techniques for mutagenesis and for the exchange of chromosomal genes to characterize mutant strains have been well-developed in P. aeruginosa and are being developed in P. stutzeri. Mutants defective in the denitrification of nitrate, nitrite, and nitrous oxide are now available and will aid in describing the catalytic and regulatory elements of the denitrification pathway.

The biology of natural transformation.

Natural transformation is widely distributed among bacteria. Its variations, in terms of specific mechanisms, may in part reflect responses to different selective pressures in different bacteria. We have suggested that both gene transfer and acquisition of carbon, nitrogen, and energy represent physiological needs that may have contributed to the evolution of natural transformation. While natural transformation was the first mechanism of genetic exchange to be detected, it is perhaps the least understood. Our understanding of the mechanism for uptake and incorporation of soluble DNA has increased significantly in the last two decades, but the overall picture of transformation as a biologically significant function is still unfolding. The mechanism by which DNA is released for transformation, the control of genes involved in DNA release and uptake, the potential for transformation in the natural environment, and the potential of natural transformation as a tool for other microbiological studies are but a few of the important issues that remain.

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.

Involvement of arginine residues in the allosteric activation of Escherichia coli ADP-glucose synthetase.

Inactivation of Escherichia coli ADP-glucose synthetase (EC 2.7.2.27) by the arginine-specific reagents cyclohexanedione and phenylglyoxal resulted primarily from interference with normal allosteric activation. Partial modification by phenylglyoxal resulted in a lessened ability of fructose 1,6-bisphosphate (fructose-P2) to stimulate and of 5'-AMP (5'-adenylate) to inhibit enzymic activity. The apparent affinity for fructose-P2 and the Vmax at saturating fructose-P2 concentrations were decreased by the arginine modification. Fructose-P2, 5'-adenylate, and several other allosteric effectors were able to partially protect the enzyme from inactivation. However, catalytic activity was not decreased by arginine modification under conditions where the enzyme was assayed in the absence of fructose-P2. The two arginine-modifying reagents differed markedly in their reactivity with the enzyme. Cyclohexanedione inactivated the enzyme quite slowly and eventually reacted with at least 14 of the 32 arginines present per subunit. Phenylglyoxal was some 50-fold more effective in inactivation, but it modified only one arginine residue per subunit.

Modification of the allosteric activator site of Escherichia coli ADP-glucose synthetase by trinitrobenzenesulfonate.

Limited modification of Escherichia coli B ADP-glucose synthetase (EC 2.7.7.27) by trinitrobenzenesulfonate (TNBS) appeared to affect primarily the allosteric properties of the enzyme. There was little loss of the catalytic activity assayed in the absence of activator. However, the abilities of fructose 1,6-bisphosphate or hexanediol 1,6-bisphosphate to activate the enzyme, or of 5'-adenylate to inhibit the enzyme, were rapidly lost upon trinitrophenylation. Modification progressively decreased the affinity for activator, decreased the Vmax at saturating concentrations of activator, and decreased the cooperativity among activator binding sites. These effects could be completely prevented by the presence of allosteric effectors during reaction with TNBS, although a low amount of trinitrophenylation still occurred. Substrates partially protected the enzyme from reaction with TNBS. The lysyl epsilon-amino side chain was modified by trinitrophenylation, but the target was not primarily the same residue which could form a Schiff base with pyridoxal phosphate, another activator of the enzyme. A large peptide containing most of the trinitrophenyl residue was isolated after cleavage of the enzyme and was identified as part of the N-terminal amino acid sequence. The migration of the enzyme on polyacrylamide gel electrophoresis or on agarose column chromatography was unchanged by modification. However, the ability of fructose-1, 6-P2 to induce the oligomerization of a mutant form of the enzyme was completely prevented by trinitrophenylation. This effect could be protected against by the presence of activator or inhibitor during reaction with TNBS.

Inability of Pseudomonas stutzeri denitrification mutants with the phenotype of Pseudomonas aeruginosa to grow in nitrous oxide.

Pseudomonas aeruginosa PAO1 reduced nitrous oxide to dinitrogen but did not grow anaerobically in nitrous oxide. Two transposon insertion Nos- mutants of Pseudomonas stutzeri exhibited the P. aeruginosa phenotype. Growth yield studies demonstrated that nitrous oxide produced in vivo was productively respired, but nitrous oxide supplied exogenously was not. The defect may be in electron transport or in nitrous oxide uptake.

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.