Bacillus subtilis promotes plant phosphorus (P) acquisition through P solubilization and stimulation of root and root hair growth.

Bacteria can be applied as biofertilizers to improve crop growth in phosphorus (P)-limited conditions. However, their mode of action in a soil environment is still elusive. We used the strain ALC_02 as a case study to elucidate how Bacillus subtilis affects dwarf tomato cultivated in soil-filled rhizoboxes over time. ALC_02 improved plant P acquisition by increasing the size and P content of P-limited plants. We assessed three possible mechanisms, namely root growth stimulation, root hair elongation, and solubilization of soil P. ALC_02 produced auxin, and inoculation with ALC_02 promoted root growth. ALC_02 promoted root hair elongation as the earliest observed response and colonized root hairs specifically. Root and root hair growth stimulation was associated with a subsequent increase in plant P content, indicating that a better soil exploration by the root system improved plant P acquisition. Furthermore, ALC_02 affected the plant-available P content in sterilized soil differently over time and released P from native P pools in the soil. Collectively, ALC_02 exhibited all three mechanisms in a soil environment. To our knowledge, bacterial P biofertilizers have not been reported to colonize and elongate root hairs in the soil so far, and we propose that these traits contribute to the overall effect of ALC_02. The knowledge gained in this research can be applied in the future quest for bacterial P biofertilizers, where we recommend assessing all three parameters, not only root growth and P solubilization, but also root hair elongation. This will ultimately support the development of sustainable agricultural practices.

Differential influence of Bacillus subtilis strains on Arabidopsis root architecture through common and distinct plant hormonal pathways.

Plant growth-promoting microbes (PGPM) can enhance crop yield and health, but knowledge of their mode-of-action is limited. We studied the influence of two Bacillus subtilis strains, the natural isolate ALC_02 and the domesticated 168 Gö, on Arabidopsis and hypothesized that they modify the root architecture by modulating hormone transport or signaling. Both bacteria promoted increase of shoot and root surface area in vitro, but through different root anatomical traits. Mutant plants deficient in auxin transport or signaling responded less to the bacterial strains than the wild-type, and application of the auxin transport inhibitor NPA strongly reduced the influence of the strains. Both bacteria produced auxin and enhanced shoot auxin levels in DR5::GUS reporter plants. Accordingly, most of the beneficial effects of the strains were dependent on functional auxin transport and signaling, while only 168 Gö depended on functional ethylene signaling. As expected, only ALC_02 stimulated plant growth in soil, unlike 168 Gö that was previously reported to have reduced biofilms. Collectively, the results highlight that B. subtilis strains can have strikingly different plant growth-promoting properties, dependent on what experimental setup they are tested in, and the importance of choosing the right PGPM for a desired root phenotype.

Adaptation of Bacillus thuringiensis to Plant Colonization Affects Differentiation and Toxicity.

The Bacillus cereus group (Bacillus cereus sensu lato) has a diverse ecology, including various species that are vertebrate or invertebrate pathogens. Few isolates from the B. cereus group have however been demonstrated to benefit plant growth. Therefore, it is crucial to explore how bacterial development and pathogenesis evolve during plant colonization. Herein, we investigated Bacillus thuringiensis (Cry-) adaptation to the colonization of Arabidopsis thaliana roots and monitored changes in cellular differentiation in experimentally evolved isolates. Isolates from two populations displayed improved iterative ecesis on roots and increased virulence against insect larvae. Molecular dissection and recreation of a causative mutation revealed the importance of a nonsense mutation in the rho transcription terminator gene. Transcriptome analysis revealed how Rho impacts various B. thuringiensis genes involved in carbohydrate metabolism and virulence. Our work suggests that evolved multicellular aggregates have a fitness advantage over single cells when colonizing plants, creating a trade-off between swimming and multicellularity in evolved lineages, in addition to unrelated alterations in pathogenicity. IMPORTANCE Biologicals-based plant protection relies on the use of safe microbial strains. During application of biologicals to the rhizosphere, microbes adapt to the niche, including genetic mutations shaping the physiology of the cells. Here, the experimental evolution of Bacillus thuringiensis lacking the insecticide crystal toxins was examined on the plant root to reveal how adaptation shapes the differentiation of this bacterium. Interestingly, evolution of certain lineages led to increased hemolysis and insect larva pathogenesis in B. thuringiensis driven by transcriptional rewiring. Further, our detailed study reveals how inactivation of the transcription termination protein Rho promotes aggregation on the plant root in addition to altered differentiation and pathogenesis in B. thuringiensis.

Cost savings realized by use of the PhaSeal(®) closed-system transfer device for preparation of antineoplastic agents.

PURPOSE: Medication cost is a major factor associated with increasing health care costs in the United States. Expenditures for prescription drugs in 2013 are estimated to be $283.7 billion. Closed system transfer devices are widely used for preparation of hazardous drugs. Reports indicate the Phaseal(®) closed system transfer device maintains sterility in vials for 7 days, suggesting the unused portion of single-use vials could be salvaged. This study was done to determine whether using a closed system transfer device to extend the beyond-use date of single-use vials of antineoplastic medications would result in a measurable cost saving. METHODS: A list of 25 drugs available in single-use vials, with a chemical stability of at least 48 hours, was compiled. Use of these agents was recorded during a 50-day period in April through June 2012. Use from a total of 296 vials of 21 antineoplastic agents was recorded. After allowing for the initial use of each vial, the mean potential percentage of drug waste was calculated to be 57.03%. RESULTS: Actual savings during the study period was $96,348.70. The pharmacy avoided nearly half of the potential waste and saved a mean of 29% of each vial. The cost-saving during the study period represents a $703,047.67 annual saving; which more than offsets the $106,556.55 the pharmacy spent for the Phaseal(®) system in 2012. CONCLUSION: In addition to being a protective measure to reduce exposure to hazardous agents, use of the Phaseal(®) system results in a reduction in drug waste, and a noticeable cost saving for antineoplastic agents.

Job satisfaction and the association with involvement in clinical activities among hospital pharmacists in Hong Kong.

OBJECTIVES: To describe the relationship between job satisfaction of hospital pharmacists and the extent of their involvement in clinical pharmacy activities, and to examine if demographics and practice characteristics are associated with the extent of involvement in clinical pharmacy activities and job satisfaction. METHODS: A cross-sectional study was conducted by surveying with a self-administered questionnaire mailed to all full-time pharmacists employed by the Hospital Authority, Hong Kong. KEY FINDINGS: Respondents reporting job and career satisfaction averaged near the neutral point. The results indicated an unmet expectation of work balance between clinical activities and drug distribution, with the majority of responding pharmacists desiring a shift of work balance from more drug distributive roles towards more clinical activities. The results also suggested that an unmet expectation in work balance affects job and career satisfaction, particularly in younger, frontline pharmacists. CONCLUSIONS: Younger, frontline pharmacists reported lower job satisfaction and a greater gap of unmet expectations in their work balance. This study highlights the importance of pharmacists' involvement in clinical activities, as job enrichment would improve job satisfaction and maximise benefits towards patients and healthcare organisations.

Use of antiplatelet drugs in the stroke unit of a Hong Kong hospital.

OBJECTIVE: Antiplatelet drugs are widely used for secondary prevention of thrombotic cerebrovascular disease. The antiplatelet prescribing patterns has been evaluated in several studies but data about Hong Kong patients are lacking. This study is to investigate the prescribing patterns of antiplatelet agents in a Hong Kong hospital. METHOD: All patients over 18 years, who attended the stroke unit between 1 October 2002 and 31 December 2002 and were on antiplatelet therapy were included in the study. Data were collected through retrospective chart review and recorded on a standardized data collection form. Continuous and categorical data were expressed as mean and counts respectively. Factors that determine frequency and pattern of antiplatelet therapy were assessed in multiple logistic regression models. RESULTS: Three hundred and nineteen patients were included in the study. Aspirin and clopidogrel accounted for 82.1 and 16.3 of all prescriptions respectively and remaining patients were on aspirin/dipyridamole. Patients with no history of aspirin use were less likely to be treated with clopidogrel compared with those experienced aspirin intolerance (OR=0.004, 95 CI 0.000-0.051). Patients with history of gastrointestinal (GI) diseases were more likely to receive clopidogrel than those had no history of GI disorders (OR=154.86, 95 CI 33.76-710.38). Atrial fibrillation (AF) was positively associated with clopidogrel prescription (OR=11.83, 95 CI 1.21-115.85). In addition, patients with concomitant gastroprotective drugs received clopidogrel significantly less often than those without gastroprotective agents (OR=0.06, 95 CI 0.01-0.29). CONCLUSION: Use of antiplatelet agents in patients receiving antiplatelet therapy of the stroke unit has complied with existing evidences. Several factors that determine choice between aspirin and clopidogrel were identified which included history of aspirin use and GI disorders, AF and co-prescribed gastroprotective drugs.

A phase I pharmacologic and pharmacogenetic trial of sequential 24-hour infusion of irinotecan followed by leucovorin and a 48-hour infusion of fluorouracil in adult patients with solid tumors.

PURPOSE: In preclinical studies, sequential exposure to irinotecan (CPT-11) then fluorouracil (5-FU) is superior to concurrent exposure or the reverse sequence; a 24-hour infusion of CPT-11 may be better tolerated than shorter infusions. EXPERIMENTAL DESIGN: CPT-11 was first given at four levels (70-140 mg/m(2)/24 hours), followed by leucovorin 500 mg/m(2)/0.5 hours and 5-FU 2,000 mg/m(2)/48 hours on days 1 and 15 of a 4-week cycle. 5-FU was then increased in three cohorts up to 3,900 mg/m(2)/48 hours. RESULTS: Two patients had dose-limiting toxicity during cycle 1 at 140/3,900 of CPT-11/5-FU (2-week delay for neutrophil recovery; grade 3 nausea despite antiemetics); one of six patients at 140/3,120 had dose-limiting toxicity (grade 3 diarrhea, grade 4 neutropenia). Four of 22 patients with colorectal cancer had partial responses, two of which had prior bolus CPT-11/5-FU. The mean 5-FU plasma concentration was 5.1 micromol/L at 3,900 mg/m(2)/48 hours. The end of infusion CPT-11 plasma concentration averaged 519 nmol/L at 140 mg/m(2)/24 hours. Patients with UDP-glucuronosyltransferase (UGT1A1; TA)6/6 promoter genotype had a lower ratio of free to glucuronide form of SN-38 than in patients with >/=1 (TA)7 allele. Thymidylate synthase genotypes for the 28-base promoter repeat were 2/2 (13%), 2/3 (74%), 3/3 (13%); all four responders had a 2/3 genotype. CONCLUSIONS: Doses (mg/m(2)) of CPT-11 140/24 hours, leucovorin 500/0.5 hours and 5-FU 3,120/48 hours were well tolerated.

A phase I and pharmacologic study of weekly gemcitabine in combination with infusional 5-fluorodeoxyuridine and oral calcium leucovorin.

PURPOSE: Since preclinical studies have shown more than additive cytotoxicity and DNA damage with the combination of gemcitabine and 5-fluoro-2'-deoxyuridine (FUDR), we studied this combination in a phase I trial. METHODS: Gemcitabine alone was given in cycle 1 as a 24-h, 2-h or 1-h i.v. infusion weekly for 3 of 4 weeks; if tolerated, a 24-h i.v. infusion of FUDR was added with oral leucovorin. The cycle was aborted for grade 3 thrombocytopenia, grade 4 neutropenia, and grade 2 or worse nonhematologic toxicity. RESULTS: During cycle 1, six of eight patients who received 150 or 100 mg/m2 over 24 h had dose-limiting neutropenia, thrombocytopenia, fatigue or mucositis. Six of seven patients treated with 1000 mg/m2 over 2 h required a gemcitabine dose reduction for cycle 2 (thrombocytopenia, neutropenia, fatigue). Of 25 assessable patients who received gemcitabine 1000 mg/m2 over 1 h, 7 did not complete cycle 1 due to thrombocytopenia (n=6) or diarrhea (n=1). Of 42 patients entered, 27 received at least one course of gemcitabine/FUDR (5-19.5 mg/m2 over 24 h) without appreciable toxicity. Due to a shortage of FUDR, the protocol was closed early. Gemcitabine plasma concentrations averaged 0.061 micro M (24 h), 16.3 micro M (2 h), and 31.9 micro M (1 h). In 21 paired bone marrow mononuclear cell samples obtained before treatment and during FUDR infusion, thymidylate synthase ternary complex was only seen during FUDR infusion. CONCLUSIONS: Gemcitabine 100-150 mg/m2 over 24 h was poorly tolerated, whereas toxicity was acceptable with 800-1000 mg/m2 over 1 h. Inhibition of the target enzyme was demonstrated at all FUDR doses.

A phase I study of 9-aminocamptothecin as a colloidal dispersion formulation given as a fortnightly 72-h infusion.

PURPOSE: A phase I pharmacologic study was undertaken to determine the maximum tolerated dose (MTD), to characterize the pharmacokinetic profile, and to evaluate all toxicities of the aqueous colloidal dispersion formulation of 9-aminocampothecin (9-AC). METHODS: 9-AC was administered as a constant 72-h i.v. infusion every 2 weeks to adult cancer patients at dose rates ranging from 25 to 59 microg/m2 per hour. RESULTS: Twenty patients with refractory solid tumors received a total of 86 courses of 9-AC at four dose levels. Myelosuppression, particularly granulocytopenia, was the most common toxicity. Two of six assessable patients entered at 59 microg/m2 per hour had dose-limiting toxicity (grade 3 diarrhea or need for a 2-week treatment delay to permit granulocyte recovery), whereas lower doses were well tolerated. At the recommended dose, 47 microg/m2 per hour, the average steady-state plasma levels (Cpss) and area under the curve (AUC) of 9-AC lactone and total drug were 15 and 75 nM, and 1034 and 4220 nM.h, respectively. A moderate correlation was seen between 9-AC lactone AUC and the percentage decrease in granulocytes. CONCLUSIONS: The recommended phase II dose of 9-AC colloidal dispersion as a 72-h infusion every 14 days is 47 microg/m2 per hour (1.13 mg/m2 per day). The Cpss of 9-AC lactone at this dose exceeded the 10 nM threshold level for preclinical activity.

A phase I pharmacologic and pharmacodynamic study of pyrazoloacridine given as a weekly 24-hour continuous intravenous infusion in adult cancer patients.

PURPOSE: Pyrazoloacridine (PZA) is an investigational nucleic acid binding agent that inhibits the activity of topoisomerases I and II through a mechanism distinct from other topoisomerase poisons. PZA shows schedule-independent cytotoxicity against tumor cells, whereas host toxicity is greater with shorter infusions. We assessed the clinical toxicities and pharmacologic effects of PZA given as a 24-h i.v. infusion weekly for 3 of 4 weeks. EXPERIMENTAL DESIGN: Thirty-two adult patients with solid tumors received PZA at five dose levels (100-351 mg/m(2)). Plasma samples were obtained at the end of the PZA infusion at all of the dose levels, with extended sampling in a cohort treated at the recommended dose. RESULTS: Dose-limiting granulocytopenia and mucositis occurred in 2 of 6 patients at 351 mg/m(2), but lower doses were well tolerated. No responses were seen, but 28% had stable disease for > or =3 months. Plasma levels strongly correlated with the degree of granulocytopenia. Extended pharmacokinetics in 7 patients treated with 281 mg/m(2) indicated the following averages: maximum plasma level, 1.6 microM; area under the plasma concentration-time curve, 56 microM.h; terminal half-life, 27 h; urinary recovery, 17% over 72 h. DNA fragmentation in post-PZA bone marrow mononuclear cells was seen in 9 of 28 samples (all at > or =281 mg/m(2)). CONCLUSIONS: Unlike other schedules of PZA, neurotoxicity and thrombocytopenia were not problematic with a weekly 24-h infusion of PZA. The recommended Phase II dose is 281 mg/m(2), which was well tolerated. Both end of infusion plasma levels and presence of DNA damage correlated with granulocyte toxicity.