scholarly journals High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

2013 ◽  
Vol 80 (2) ◽  
pp. 462-469 ◽  
Author(s):  
Soumya Palliyil ◽  
Christina Downham ◽  
Ian Broadbent ◽  
Keith Charlton ◽  
Andrew J. Porter
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Monoclonal Antibodies ◽  
Bacterial Infections ◽  
Recombinant Antibody ◽  
Cell Communication ◽  
High Sensitivity ◽  
Therapeutic Benefit ◽  
Cross Reactivity ◽  
Content Type ◽  
Homoserine Lactones

ABSTRACTA number of bacteria, including pathogens likePseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role inP. aeruginosabiology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to preventP. aeruginosainfections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs inP. aeruginosacultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model ofPseudomonasinfection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

scholarly journals Light-Mediated Decreases in Cyclic di-GMP Levels Inhibit Structure Formation in Pseudomonas aeruginosa Biofilms

2020 ◽  
Vol 202 (14) ◽  
Author(s):  
Lisa Juliane Kahl ◽  
Alexa Price-Whelan ◽  
Lars E. P. Dietrich
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Prolonged Exposure ◽  
Light Exposure ◽  
Research Attention ◽  
Content Type ◽  
Dependent Inhibition ◽  
Matrix Production ◽  
Biofilm Development ◽  
The Impact

ABSTRACT Light is known to trigger regulatory responses in diverse organisms, including slime molds, animals, plants, and phototrophic bacteria. However, light-dependent processes in nonphototrophic bacteria, and those of pathogens in particular, have received comparatively little research attention. In this study, we examined the impact of light on multicellular development in Pseudomonas aeruginosa, a leading cause of biofilm-based bacterial infections. We grew P. aeruginosa strain PA14 in a colony morphology assay and found that growth under prolonged exposure to low-intensity blue light inhibited biofilm matrix production and thereby the formation of vertical biofilm structures (i.e., “wrinkles”). Light-dependent inhibition of biofilm wrinkling was correlated with low levels of cyclic di-GMP (c-di-GMP), consistent with the role of this signal in stimulating matrix production. A screen of enzymes with the potential to catalyze c-di-GMP synthesis or degradation identified c-di-GMP phosphodiesterases that contribute to light-dependent inhibition of biofilm wrinkling. One of these, RmcA, was previously characterized by our group for its role in mediating the effect of redox-active P. aeruginosa metabolites called phenazines on biofilm wrinkle formation. Our results suggest that an RmcA sensory domain that is predicted to bind a flavin cofactor is involved in light-dependent inhibition of wrinkling. Together, these findings indicate that P. aeruginosa integrates information about light exposure and redox state in its regulation of biofilm development. IMPORTANCE Light exposure tunes circadian rhythms, which modulate the immune response and affect susceptibility to infection in plants and animals. Though molecular responses to light are defined for model plant and animal hosts, analogous pathways that function in bacterial pathogens are understudied. We examined the response to light exposure in biofilms (matrix-encased multicellular assemblages) of the nonphotosynthetic bacterium Pseudomonas aeruginosa. We found that light at intensities that are not harmful to human cells inhibited biofilm maturation via effects on cellular signals. Because biofilm formation is a critical factor in many types of P. aeruginosa infections, including burn wound infections that may be exposed to light, these effects could be relevant for pathogenicity.

scholarly journals Evolution of Pseudomonas aeruginosa Virulence as a Result of Phage Predation

2013 ◽  
Vol 79 (19) ◽  
pp. 6110-6116 ◽  
Author(s):  
Zeinab Hosseinidoust ◽  
Theo G. M. van de Ven ◽  
Nathalie Tufenkji
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mammalian Cells ◽  
Bacterial Infections ◽  
Membrane Damage ◽  
Human Keratinocytes ◽  
Host Population ◽  
Resistant Bacteria ◽  
Content Type ◽  
Metabolic Action

ABSTRACTThe rapid increase in the emergence of antibiotic-resistant bacteria has attracted attention to bacteriophages for treating and preventing bacterial infections. Bacteriophages can drive the diversification ofPseudomonas aeruginosa, giving rise to phage-resistant variants with different phenotypes from their ancestral hosts. In this study, we sought to investigate the effect of phage resistance on cytotoxicity of host populations toward cultured mammalian cells. The library of phage-resistantP. aeruginosaPAO1 variants used was developed previously via experimental evolution of an isogenic host population using phages PP7 and E79. Our results presented herein indicate that the phage-resistant variants developed in a heterogeneous phage environment exhibit a greater ability to impede metabolic action of cultured human keratinocytes and have a greater tendency to cause membrane damage even though they cannot invade the cells in large numbers. They also show a heightened resistance to phagocytosis by model murine macrophages. Furthermore, all isolates produced higher levels of at least one of the secreted virulence factors, namely, total proteases, elastase, phospholipase C, and hemolysins. Reverse transcription-quantitative PCR (RT-qPCR) revealed upregulation in the transcription of a number of genes associated with virulence ofP. aeruginosafor the phage-resistant variants. The results of this study indicate a significant change in thein vitrovirulence ofP. aeruginosafollowing phage predation and highlight the need for caution in the selection and design of phages and phage cocktails for therapeutic use.

scholarly journals Potentiation of Tobramycin by Silver Nanoparticles against Pseudomonas aeruginosa Biofilms

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Marc B. Habash ◽  
Mara C. Goodyear ◽  
Amber J. Park ◽  
Matthew D. Surette ◽  
Emily C. Vis ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Silver Nanoparticles ◽  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Bacterial Species ◽  
Acute Infection ◽  
Public Health Problem ◽  
Chronic Infections ◽  
Content Type

ABSTRACT Increasing antibiotic resistance among pathogenic bacterial species is a serious public health problem and has prompted research examining the antibacterial effects of alternative compounds and novel treatment strategies. Compounding this problem is the ability of many pathogenic bacteria to form biofilms during chronic infections. Importantly, these communities are often recalcitrant to antibiotic treatments that show effectiveness against acute infection. The antimicrobial properties of silver have been known for decades, but recently silver and silver-containing compounds have seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the ability of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the aminoglycoside antibiotic tobramycin, to inhibit established Pseudomonas aeruginosa biofilms. Our results demonstrate that smaller 10-nm and 20-nm AgNPs were more effective at synergistically potentiating the activity of tobramycin. Visualization of biofilms treated with combinations of 10-nm AgNPs and tobramycin reveals that the synergistic bactericidal effect may be caused by disrupting cellular membranes. Minimum biofilm eradication concentration (MBEC) assays using clinical P. aeruginosa isolates shows that small AgNPs are more effective than larger AgNPs at inhibiting biofilms, but that the synergy effect is likely a strain-dependent phenomenon. These data suggest that small AgNPs synergistically potentiate the activity of tobramycin against P. aeruginosa in vitro and may reveal a potential role for AgNP/antibiotic combinations in treating patients with chronic infections in a strain-specific manner.

scholarly journals Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

2016 ◽  
Vol 82 (8) ◽  
pp. 2300-2311 ◽  
Author(s):  
Eva J. Scharinger ◽  
Richard Dietrich ◽  
Ina Kleinsteuber ◽  
Erwin Märtlbauer ◽  
Kristina Schauer
Keyword(s):  
Monoclonal Antibodies ◽  
Rapid Detection ◽  
Foodborne Pathogen ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Powdered Infant Formula ◽  
Bacterial Cells ◽  
Cronobacter Sakazakii ◽  
Content Type ◽  
Cell Counts

ABSTRACTCronobacter sakazakiiis a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection ofC. sakazakiihave not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakiiof serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 103and 9 × 106CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains ofCronobacterspp. and otherEnterobacteriaceaewas observed, except for that withC. sakazakiiserotype O3 andCronobactermuytjensiiserotype O1. Moreover, the sandwich EIAs detectedC. sakazakiiin PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection ofC. sakazakiistrains but also enables simultaneous serotyping in a simple sandwich EIA method.

scholarly journals Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production

2016 ◽  
Vol 23 (8) ◽  
pp. 681-688 ◽  
Author(s):  
Jisheng Lin ◽  
Mark A. Smith ◽  
William H. Benjamin ◽  
Robert W. Kaminski ◽  
Heather Wenzel ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
High Sensitivity ◽  
Epidemiological Data ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Bacterial Surface ◽  
Bactericidal Assay

ABSTRACTThere is a significant need for an effective multivalentShigellavaccine that targets the most prevalent serotypes. MostShigellavaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS fromShigella flexneri2a,Shigella flexneri3a, andShigella sonnei. We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that theShigellaserotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) forShigella. Functional assays, such as thein vitrobactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. AnS. flexneri2a-specific monoclonal antibody killedS. flexneri2b isolates, suggesting thatS. flexneri2a LPS may induce cross-protection againstS. flexneri2b. Overall, theShigellaLPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency.

scholarly journals Method for the Selective Measurement of Amino-Terminal Variants of Procalcitonin

2009 ◽  
Vol 55 (9) ◽  
pp. 1672-1679 ◽  
Author(s):  
Joachim Struck ◽  
Martina Strebelow ◽  
Sonja Tietz ◽  
Christine Alonso ◽  
Nils G Morgenthaler ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bacterial Infections ◽  
Cross Reactivity ◽  
Functional Assay ◽  
Human Endotoxemia ◽  
Amino Terminal ◽  
Endotoxemia Model ◽  
Sandwich Assays ◽  
Kinetics Of

Abstract Background: Procalcitonin (PCT) is an established marker for diagnosing and monitoring bacterial infections. Full-length PCT [116 amino acids that make up procalcitonin (PCT1–116)] can be truncated, leading to des-Ala-Pro-PCT (des-Alanin-Prolin-Procalcitonin; PCT3–116). Current immunoassays for PCT (“total PCT”) use antibodies directed against internal epitopes and are unable to distinguish amino-terminal PCT variants. Here we describe the development of monoclonal antibodies recognizing the amino-termini of PCT1–116 and PCT3–116 and their use in the selective measurement of these PCT species. Methods: With newly developed monoclonal antibodies against the amino-termini of PCT1–116 and PCT3–116, and an antibody against the katacalcin moiety of PCT, we developed and characterized immunoluminometric assays for the 2 PCT peptides. We comparatively assessed the kinetics of PCT variants in a human endotoxemia model. Results: Monoclonal antibodies against the amino-termini of PCT1–116 and PCT3–116 showed <1% cross-reactivity with other PCT-related peptides. The sandwich assays for PCT1–116 and PCT3–116 had functional assay sensitivities of 5 and 1.2 pmol/L, respectively, and exhibited recoveries within 20% of expected values. Plasma PCT1–116 was stable for 6 h at 22 °C and 24 h at 4 °C, and PCT3–116 was stable for at least 24 h at both temperatures. During experimental endotoxemia in healthy people, both PCT1–116 and PCT3–116 increased early in parallel with total PCT, but further increases in PCT1–116 were significantly slower than for PCT3–116 (P = 0.0049) and total PCT (P = 0.0024). Conclusions: The new assays selectively measure PCT1–116 and PCT3–116. Both PCT species increase early during endotoxemia but differ in their kinetics thereafter. The selective measurement of PCT species with different in vivo kinetics may be useful in improving PCT-guided therapies.

scholarly journals Thermoregulation of Pseudomonas aeruginosa Biofilm Formation

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Suran Kim ◽  
Xi-Hui Li ◽  
Hyeon-Ji Hwang ◽  
Joon-Hee Lee
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Effect Of Temperature ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Protection Mechanism ◽  
Formation Pattern ◽  
Temperature Ranges ◽  
Different Strains

ABSTRACT We investigated the effect of temperature on the biofilm formation of Pseudomonas aeruginosa and revealed that the biofilm formation increased rapidly at temperatures lower than 25°C. P. aeruginosa formed the most robust biofilm of a conspicuous mushroom-like structure at 20°C. However, when the temperature increased to 25°C, the biofilm formation rapidly decreased. Above 25°C, as the temperature rose, the biofilm formation increased again little by little despite its less-structured form, indicating that 25°C is the low point of biofilm formation. The intracellular 3′,5′-cyclic diguanylate (c-di-GMP) levels also decreased rapidly as the temperature rose from 20 to 25°C. The expression levels of pelA, algD, and pslA encoding Pel, alginate, and Psl, respectively, were also dramatically affected by temperature, with pelA being regulated in a pattern similar to that of the intracellular c-di-GMP levels, and the pattern seen for algD regulation was the most similar to the actual biofilm formation pattern. Total exopolysaccharide production was thermoregulated and followed the regulation pattern of c-di-GMP. Interestingly, the thermoregulation patterns in biofilm formation were different depending on the strain of P. aeruginosa. Unlike PAO1, another strain, PA14, showed a gradual decrease in biofilm formation and c-di-GMP in the range of 20 to 37°C, and P. aeruginosa clinical isolates also showed slightly different patterns in biofilm formation in conjunction with temperature change, suggesting that different strains may sense different temperature ranges for biofilm formation. However, it is obvious that P. aeruginosa forms more biofilms at lower temperatures and that temperature is an important factor in determining the biofilm formation. IMPORTANCE Biofilm formation is an important protection mechanism used by most microorganisms and provides cells with many advantages, like high infectivity, antibiotic resistance, and strong survivability. Since most persistent bacterial infections are believed to be associated with biofilms, biofilm control is an important issue in medicine, environmental engineering, and industry. Biofilm formation is influenced by various environmental factors. Temperature is the most direct environmental cue encountered by microorganisms. Here, we investigated the effect of temperature on the biofilm formation of P. aeruginosa, a notorious pathogen, and found that temperature is an important factor determining the amount and structure of biofilms. Low temperatures greatly increase biofilm formation and give biofilms a highly conspicuous structure. Although thermoregulation of biofilm formation is mainly mediated by c-di-GMP, some c-di-GMP-independent regulations were also observed. This study shows how biofilms are formed at various temperatures and provides new insights to control biofilms using temperature.

scholarly journals Cinnamaldehyde Induces Expression of Efflux Pumps and Multidrug Resistance in Pseudomonas aeruginosa

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Alexandre Tetard ◽  
Andy Zedet ◽  
Corine Girard ◽  
Patrick Plésiat ◽  
Catherine Llanes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Content Adaptation ◽  
Toxic Metabolite ◽  
Regulatory Pathway ◽  
Content Type ◽  
Antagonistic Effects ◽  
Cinnamon Bark ◽  
Therapeutic Consequences

ABSTRACT Essential oils or their components are increasingly used to fight bacterial infections. Cinnamaldehyde (CNA), the main constituent of cinnamon bark oil, has demonstrated interesting properties in vitro against various pathogens, including Pseudomonas aeruginosa. In the present study, we investigated the mechanisms and possible therapeutic consequences of P. aeruginosa adaptation to CNA. Exposure of P. aeruginosa PA14 to subinhibitory concentrations of CNA caused a strong albeit transient increase in the expression of operons that encode the efflux systems MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY/OprM. This multipump activation enhanced from 2- to 8-fold the resistance (MIC) of PA14 to various antipseudomonal antibiotics, including meropenem, ceftazidime, tobramycin, and ciprofloxacin. CNA-induced production of pump MexAB-OprM was found to play a major role in the adaption of P. aeruginosa to the electrophilic biocide, through the NalC regulatory pathway. CNA was progressively transformed by bacteria into the less toxic metabolite cinnamic alcohol (CN-OH), via yet undetermined detoxifying mechanisms. In conclusion, the use of cinnamon bark oil or cinnamaldehyde as adjunctive therapy to treat P. aeruginosa infections may potentially have antagonistic effects if combined with antibiotics because of Mex pump activation.

scholarly journals Synergy of Silver Nanoparticles and Aztreonam against Pseudomonas aeruginosa PAO1 Biofilms

2014 ◽  
Vol 58 (10) ◽  
pp. 5818-5830 ◽  
Author(s):  
Marc B. Habash ◽  
Amber J. Park ◽  
Emily C. Vis ◽  
Robert J. Harris ◽  
Cezar M. Khursigara
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Silver Nanoparticles ◽  
Bacterial Infections ◽  
Antimicrobial Agents ◽  
Cell Envelope ◽  
Antimicrobial Properties ◽  
Planktonic Cell ◽  
Chronic Infections ◽  
Content Type

ABSTRACTPathogenic bacterial biofilms, such as those found in the lungs of patients with cystic fibrosis (CF), exhibit increased antimicrobial resistance, due in part to the inherent architecture of the biofilm community. The protection provided by the biofilm limits antimicrobial dispersion and penetration and reduces the efficacy of antibiotics that normally inhibit planktonic cell growth. Thus, alternative antimicrobial strategies are required to combat persistent infections. The antimicrobial properties of silver have been known for decades, but silver and silver-containing compounds have recently seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the efficacy of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the monobactam antibiotic aztreonam, to inhibitPseudomonas aeruginosaPAO1 biofilms. Among the different sizes of AgNPs examined, 10-nm nanoparticles were most effective in inhibiting the recovery ofP. aeruginosabiofilm cultures and showed synergy of inhibition when combined with sub-MIC levels of aztreonam. Visualization of biofilms treated with combinations of 10-nm AgNPs and aztreonam indicated that the synergistic bactericidal effects are likely caused by better penetration of the small AgNPs into the biofilm matrix, which enhances the deleterious effects of aztreonam against the cell envelope ofP. aeruginosawithin the biofilms. These data suggest that small AgNPs synergistically enhance the antimicrobial effects of aztreonam againstP. aeruginosain vitro, and they reveal a potential role for combinations of small AgNPs and antibiotics in treating patients with chronic infections.

scholarly journals The Combination of Fosfomycin plus Meropenem Is Synergistic for Pseudomonas aeruginosa PAO1 in a Hollow-Fiber Infection Model

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
G. L. Drusano ◽  
M. N. Neely ◽  
W. M. Yamada ◽  
Brandon Duncanson ◽  
David Brown ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Combination Therapy ◽  
Hollow Fiber ◽  
Bacterial Infections ◽  
Clinical Problem ◽  
The United States ◽  
Resistance Mechanisms ◽  
Infection Model ◽  
Bacterial Killing ◽  
Content Type

ABSTRACT Treating high-density bacterial infections is a challenging clinical problem. We have a paucity of new agents that can address this problem. Pseudomonas aeruginosa is a particularly difficult pathogen to treat effectively because of the plethora of resistance mechanisms it carries. Fosfomycin is an agent discovered circa 40 years ago. Recently, it has been resurrected in the United States and studied for intravenous therapy. We hypothesized that, to maximize its utility, it would require combination chemotherapy when used in a clinical circumstance in high-bacterial-burden infections. We chose to examine the combination of meropenem plus fosfomycin. These agents were studied in the hollow-fiber infection model. We utilized a fully factorial study design, looking at 2 doses of meropenem alone (1 and 2 g 8-hourly) and two doses of fosfomycin alone (6 and 8 g 8-hourly), as well as all possible combinations plus a no-treatment control. We used a high-dimensional model of 5 inhomogeneous differential equations with 5 system outputs to analyze all data simultaneously. Combination therapy outperformed all monotherapy regimens, with all combinations driving >6 log10 CFU/ml of bacterial killing. Combination therapy was able to counterselect resistance emergence (meropenem mutants being killed by the combination, as well as fosfomycin mutants being killed by the combination) in all regimens studied. The analysis demonstrated that the combination was significantly synergistic for bacterial cell killing and resistance suppression. Meropenem plus fosfomycin is a promising combination for therapy of high-burden Pseudomonas aeruginosa infections and requires further study.

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