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A compound was proposed by error but then showed very good inhibitory properties. How to argue this in a manuscript?

A compound was proposed by error but then showed very good inhibitory properties. How to argue this in a manuscript?



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In a project, we performed some virtual screening calculations in order to predict inhibitors, and then purchased the compounds and tested them in the lab. One of them was a very good TK inhibitor (IC50 nM).

However, when we processed the results we realized that the presence of this particular compound in the original list of virtual screening hits was due to errors in the original calculations (it was added to the list by random factors, not by docking score).

So now we are writing the paper and we wonder how to argue this. We can not just tell the story like in this post (it was discovered by randomness or by our error). How would you argue this?


Be honest (and possibly split the papers).

If the compound was identified as an error and you want to include in a paper describing the process of your simulation and the effectiveness of the predicted inhibitors, you'd have to do so as a sort of negative control for your calculations (unless there is some way to reproduce/rationalise why it showed up randomly). Since this is the only good inhibitor you have, you'd have to conclude that your calculation isn't much better than chance, or explain why it is better even though you found a better inhibitor by chance. If you have reason to argument that your calculations are good and make sense then exclude the 'random' inhibitor from that paper. You can the write an individual paper about a good inhibitor, the fact that you found it by chance isn't that important anymore.


Antibiotic-Loaded Cement in Orthopedic Surgery: A Review

Infections in orthopaedic surgery are a serious issue. Antibiotic-loaded bone cement was developed for the treatment of infected joint arthroplasties and for prophylaxes in total joint replacement in selected cases. Despite the widespread use of the antibiotic-loaded bone cement in orthopedics, many issues are still unclear or controversial: bacterial adhesion and antibiotic resistance, modification of mechanical properties which follows the addition of the antibiotic, factors influencing the release of the antibiotic from the cement and the role of the surface, the method for mixing the cement and the antibiotic, the choice and the effectiveness of the antibiotic, the combination of two or more antibiotics, and the toxicity. This review discusses all these topics, focusing on properties, merits, and defects of the antibiotic loaded cement. The final objective is to provide the orthopaedic surgeons clear and concise information for the correct choice of cement in their clinical practice.

1. Introduction

The purpose of this review is to analyze the main issues of antibiotic loaded bone cements and to comment their basic properties, main characteristics, merits, and defects. The final goal is to provide the orthopaedic surgeons of clear and concise information for a correct choice of antibiotic loaded cement in the clinical practice.

1.1. Biomaterials, Infections, and Orthopedics

The presence of biomaterials in orthopedic surgery involves a high risk of developing deep infections [1]. One of the main factors is the phenomenon of the adhesion of the bacteria to the biomaterials and the production of a biofilm from the bacterial strains [2–5]. In fact, it was demonstrated that bacteria have the ability to bind to the surface of biomaterials, due to specific physical and chemical properties [2, 6, 7]. Infections around joint arthroplasties are among the most difficult to manage and to heal. Until decades ago, the antibiotics available for the prevention and the treatment of the orthopedic infections were only a few and these antibiotics could have been ineffective against certain bacteria like staphylococci and gram-negative. With the spread of the prosthetic joint replacement in the seventies, the problem increased [1, 8–10]. Actually, the mainstay of treatment of an infected joint prosthesis is based on the removing of the implant and on the accurate toiletries around the surrounding necrotic soft and bone tissue, either in on- or two- stage technique [11–13].

The positioning into the surgical site of cement loaded with antibiotics may be useful to maintain at local level a high concentration of drug, which could not be reached by the venous administering without general complications and toxicity [14, 15]. Nevertheless, the real effectiveness of the antibiotic loaded cement is currently under debate [16]. After the surgical treatment and cleaning, the systemic administering of antibiotics for prolonged time is anyway mandatory, being the toilet alone not enough. After years of scientific debate, there still are many doubts and conflicting opinions on several aspects of the use of antibiotic cement: the method of preparation, the choice of the antibiotic, the effective release and diffusion of the antibiotic in the surrounding tissues, and the mechanical properties of the antibiotic loaded cement.

2. Cement Development and Joint Infections

The subject of this review, the so called bone cement is a polymer-based material composed of poly-methyl-methacrylate (PMMA) or copolymers, is a polymeric material commonly used for the fixation of the joint implants to the bone.

In recent years, thanks to the improved surgical techniques, to the adoption of stringent and efficient antiseptic pre-operative and intra-operative procedures and, above all, thanks to the optimization of the peri-operative systemic antibiotic prophylaxis a significant reduction in the number of deep infections and subsequent revisions occurred [17–20]. It was estimated that the rate of the infections was reduced from 5–10% to approximately 1-2% during the last twenty years. Among the many procedures to fight periprosthetic infection, the use of antibiotic enriched bone cement is widely used [21], particularly in case of revision and septic failure of the arthroplasties. Over 30 years ago, Bulchoz and Engelbrecht reported that penicillin, erythromycin, gentamicyn introduced into the cement used to stabilize the hip, spread into the surrounding tissues for months, bringing as a result a prolonged local concentration of antibiotic [22]. After these findings, the interest in the application of cement impregnated with antibiotic in the treatment of osteomyelitis grew. In 1979, as an alternative to the introduction of large deposits of antibiotic cement in the site of chronic osteomyelitis, Klemm introduced gentamicyn in cement beads and used them as temporary filler for the gap that was created after the removal of necrotic tissue. The cement impregnated with antibiotic (ALBC: antibiotic loaded bone cement), since the late 90ties, was increasingly used for the prevention of the arthroplasty-related infections. Also, over the time, this choice has undergone significant changes and improvements concerning the chemical formulation, the techniques of preparation and clinical applications [22].

3. Bacterial Adhesion and Antibiotic Resistance

The addition of antibiotics to the cement must be considered as a support strategy in preventing the onset of infections and not the solution: the key point is still represented by the sterility in the operating room and by the antiseptic surgical procedures. Nevertheless, any procedure which can potentially reduce the adhesion and the bacterial colonization is welcome in orthopedics. Hypotheses were formulated about the effectiveness of the addition of antibiotics in terms of reduction of the bacterial biofilm on the different types of cement and it appeared as a multi-factorial process, not related only to the kinetics of release of the antibiotic. Some sustained that the production from the bacteria of a kind of glycocalyx (extracellular structure that covers the external surface of tissues with a “sheath” that is found mainly in epithelia), which adheres to the biomaterial, causes physiological changes on bacteria themselves and confers antibiotic resistance [16, 23]. Others proposed that the main factor can be the hydrophobicity of the implanted material, the electrostatic interactions and/or the roughness of the surface [24, 25]. Emerging evidences showed that the bacterial adhesion to a biomaterial is the result of a development of the antibiotic resistance [22]. It was hypothesized that the bacterial growth is privileged on certain biomaterials: for example, coagulase negative staphylococci would prefer to join the bone cement, while S. Aureus would show preferential adhesion to metallic biomaterials [26]. Prolonged exposure to antibiotic at a dose concentration below the inhibitory one, allows the development of mutational resistance in bacteria. Therefore, the wide clinical use of ALBC with preventive purposes must be carefully considered [16, 22]. The use of cement added with gentamicin for first implants was associated with the development of coagulase-negative staphylococci resistant to this drug [27]. Also, bacterial strains resistant to gentamicin were found in the 88% of the cases of infection in arthroplasty where cement was loaded with antibiotic, compared to the 16% found after those where the common cement was used [28, 29]. Another important factor for bacteria adhesion is the roughness of the surface: in general the higher the roughness, the higher the adhesion and the PMMA is characterized by a rough surface [11].

4. The Use of Antibiotic-Loaded Bone Cement: General Principles

The PMMA enters the operating room packaged in monomer (liquid) and polymer (powder) separately. At the time of the preparation, when mixed, it becomes a viscous material paste, which solidifies in few minutes by an exothermic reaction. It acts as a fixation between the prosthetic components and the cancellous bone. During the mixing, pores of different sizes are produced as consequence of the chemical reaction and volume variations. These microholes may represent the start point of cracks and thus can be responsible of the premature failure of the cement. To avoid the formation of these pores, it is possible to prepare the cement under vacuum conditions. Nevertheless, the preparation under vacuum leads to a greater reduction in volume during the polymerization, thus resulting in higher shrinkage and worse adhesion on the bone-prosthesis interface compared to the nonvacuum mixed cement. It was demonstrated that among the compounds prepared in the operating room, those made under vacuum conditions present improved mechanical properties [30–32]. However, it is not the purpose of this work to discuss the method of preparation of cement and the relative advantages or disadvantages of the vacuum preparation on the biomechanical properties.

Some authors do not recommend the use of antibiotic cement in primary arthroplasty [30], first of all for the reduction of mechanical properties and secondly because its spread use might lead to the selection of antibiotic resistant bacteria. The use of the antibiotic loaded cement in primary implants is indicated in patients with hag surgical risk, in elder patients, in patients with general health problems like immuno-depression, diabetes, history of previous prosthetic and periprosthetic infections, and particular diseases such as rheumatoid arthritis and SLE, or in conditions of malnutrition [33, 34]. In contrast, the use of the antibiotic loaded cement is recommended by most authors for joint arthroplasty revisions, which are at higher risk of infection compared with the first implants [33, 34]. The use of the antibiotic-loaded cement is particularly indicated for septical revisions [30]. The revision of an infected joint arthroplasty can be performed in a single surgical operation (one-stage), where the removal of the implant and of the infected and necrotic tissue is followed by an accurate cleaning of the area and by the implant of the new prosthesis. On the contrary, the revision can be performed with the so-called “two-stage” technique, where the first step is the implant removal and the surgical toilette, then a temporary spacer is implanted, and finally the new prosthesis is implanted at a distance of 6–8 weeks after surgery with a new operation [12, 13, 35]. During the period of time between the two surgeries, the area of the joint cleaned up can be left empty or most commonly is filled with a spacer. Antibiotic loaded cement is the most used spacer, due to its plasticity and to the capability to release the antibiotic in situ [27, 30, 36]. In this case a therapeutic local effect is added to the primary function of the spacer: to avoid the retraction of the tissues and to maintain the joint space, thus facilitating the revision surgery [37].

5. Method for Mixing the Cement and the Antibiotic

The method of mixing is considered one of the most important factors that affect the release of the antibiotics and the mechanical properties of cement. The preparation should be as porous as possible in order to increase the spread of the antibiotic, but not excessively porous to weaken the structure of the cement itself. A fundamental distinction regards the method of addition of the antibiotic to the cement: manually mixing at the time of implantation or industrial mixing by the several companies which provide premixed antibiotic loaded bone cement [31, 38, 39].

The antibiotic must be a powder preparation for a better integration with the cement and a reduced interference with the mechanical properties of the cement [39]. Until now, no studies were conducted to correlate the changes in the release of the antibiotic with the temperature. It must be considered, however, that the process of polymerization of the cement is an exothermic reaction with temperatures up to 60°–80°C. Therefore, the antibiotics destined to be mixed with the cement must be chemically and thermally stable [40]. The manual preparation, according to a study conducted on the Simplex-P spiked with tobramycin, reduces the strength of cement of 36% compared to the ALBC prepared industrially [30]. The improvement of mechanical properties due to the greater compactness of the structure of the cement, however, could lead to a decrease in the rate of diffusion of the antibiotic [32]. This difference however is not considered significant by most surgeons.

6. The Choice of the Antibiotic

The choice of the antibiotic is a fundamental issue. The antibiotic must have a broad antibacterial spectrum (including gram positive and gram negative bacteria) and a low percentage of resistant species. The most commonly mixed antibiotics are gentamicin and tobramycin (aminoglycosides with particular effectiveness against gram-negative bacteria) and vancomycin (glycopeptide active mainly on gram-positive like, e.g., Staphylococcus aureus). In addition, the antibiotic must provide a local concentration able to overcome the “break point sensitivity limit” of pathogens. This is generally defined as the antibiotic concentration that marks the transition from bacterial sensitivity to induction to resistance to antibiotics for at least three or four weeks. The final aim is to reach appropriate antibiotic concentrations in the tissues and bone avoiding the toxicity of the concentration of the drugs [22].

A study showed that coagulase negative staphylococci are found in the 88% of the infections in patients undergoing a primary arthroplasty where cement loaded with gentamicin was used [41]. A study [42] in vitro analyzed the behaviour of Staphylococcus aureus in function of the kinetics of gentamicin release in different cements: none of the antibiotic loaded cements was able to immediately reduce the growth of bacteria, but everyone led to a significant decrease in bacterial growth if compared with non-antibiotic cements. Apparently, the CMW3 cement showed the ability to reduce bacterial colonization for a longer period (24–72 hours) compared to other cements. It was also noted that the gentamicin may act differently when added to different cements, although the mechanism of bacterial adhesion is always the same. For example, it was demonstrated that the release of gentamicin is much more effective in Palacos than in Simplex [14]. Similarly, changes in elution related to the type of cement were found also for the vancomycin. The release of this antibiotic was compared in three different types of cement (CMW1, Palacos-R, and Simplex-P) and the first showed an increased release compared with the other two [43]. Other studies on various cements (Cemex, Palacos, and Simplex) demonstrated that vancomycin alone had a minor and less effective release compared with gentamicin [44]. This lower diffusion of the vancomycin would be related to several factors such as physical-chemical properties of the antibiotic, the molecular weight, the stability of the molecules in presence of biological fluids, the temperature, as well as the different morphology of the cement itself (porosity, roughness, surface, etc.). The type of cement and the method of preparation may modify the elution of the antibiotic, although other studies affirm the opposite and argue that the spread of vancomycin and tobramycin would not depend on the type of cement used [45].

One study [40] showed that the tobramycin added to the Simplex cement has good activity against 98% of the bacterial tested, using a wide spectrum of pathogens clinically relevant in orthopaedic infections (aerobic Gram-positive and Gram-negative bacteria, anaerobic ones, and mycobacterium). Tobramycin resistant bacteria at the usual systemic concentrations, such as Enterococcus faecalis, methicillin-resistant staphylococci, and Staphylococcus epidermidis, exhibited a limited sensitivity also to the antibiotic released from the cement. The study confirmed the effectiveness of the tobramycin bounded to PMMA for the prevention and reduction of infections caused by a wide spectrum of micro-organisms: tobramycin is stable during the exothermic cement polymerization and its release on the surface of PMMA occurs at concentrations that usually inhibits the growth of the majority of the examined bacteria. Another study from the same authors [46] compared the two principal aminoglycosides used in prosthetic surgery, gentamicin and tobramycin, respectively, added to Palacos and Simplex. The results showed that Simplex-tobramycin has antibacterial activity against 98% of the tested strains of P. Aeruginosa, while Palacos-gentamicin contrasts the 93% of the same bacteria. These results would suggest an antibacterial activity of the tobramycin from 2 to 8 times better than gentamicin. Aminoglycosides act through a mechanism directly correlated to the concentration therefore increasing the dose of the antibiotics corresponds to an increased antibacterial efficacy. In addition, the release of antibiotic is positively correlated to the quantity added to the cement [47].

7. Dose of Antibiotic

The dosage of the antibiotic varies according to the use for which the cement is destined. Many authors argue that in case of acute infections high doses of antibiotics should be used: more than 2 g each 40 g of cement, usually from 6 to 8 g each 40 g, for a prolonged and effective release against pathogens [16, 22, 48]. Whereas if the ALBC is used for prophylaxis in first implants, where the first function of the cement is to fix the implant, the antibiotic can be mixed at low doses: less than 2 g each 40 g of antibiotic cement. An inadequate dose may be seen as the cause of failure of the prosthesis, as it may generate the emergence of resistant bacteria [16, 22, 48].

8. Association of More Than One Antibiotic into the Bone Cement

The activity of the release of two or more antibiotics from the bone cement was studied. The idea to add more than one single antibiotic arose after the emergence of resistant bacteria and after the possible synergistic combination of two antibiotics has become an increasingly common practice in infectivology (usually, vancomycin and aminoglycosides are often combined for their synergic potential effect in the treatment of serious infections caused by the S. aureus). Since 1970, it has been documented that β-lactamic antibiotics can be combined with most of aminoglycosides, when there is a high concentration of both substances, or when their excretion is delayed. The combination of a molecule of a β-lactam with an aminoglycoside molecule can inactivate equimolar amounts of both antibiotics. When high doses of both substances are combined with cement, the inactivation of these can affect the properties of the combination, but this phenomenon has not been studied yet [41].

A study conducted on 20 patients with infections due to S. aureus, S. epidermidis, E. coli and P. aeruginosa, showed a superior effectiveness of spacers loaded with a combination of gentamicin and vancomycin compared to spacers loaded with gentamicin alone [36]. The emerging capacity of staphylococcal survival on prosthetic materials and the in vitro effects of gentamicin and vancomycin-loaded polymethylmethacrylate (PMMA) were studied on hospital acquired staphylococcal strains systematically inoculated on four orthopedic materials: Ultra-High Molecular Weight Polyethylene (UHMPWE), Palamed cement without antibiotics, Palamed-G cement, and Palamed-G cement loaded with vancomycin (1 g of antibiotic each 40 g of cement) [49]. The sample with the association of vancomycin-gentamicin was the most effectively protected from bacterial colonization. The result is coherent with other similar tests carried out by other authors on other cements (Palamed and Palacos) and various antibiotics on other strains of bacteria [50, 51].

The additive or synergistic effect of tobramycin on vancomycin released from acrylic cement was demonstrated [52]. This phenomenon was called “passive opportunism” because the second antibiotic appears to act simply as a soluble passive additive. The elution of tobramycin and of vancomycin alone and combined from the disks of acrylic cement was studied: it was demonstrated that combining two antibiotics in bone-cement improves elution of both antibiotics in vitro and may translate into enhanced elution in vivo [53].

The characteristics of elution of vancomycin and tobramycin alone and together were compared in two types of cement, Palacos and Simplex [15], divided into three groups: a first group (low) contained 1.2 g of tobramycin and 1 g of vancomycin, a second group (medium) 2.4 g of tobramycin and 2 g of vancomycin, and a third group (high) 3.6 g of tobramycin and 3 g of vancomycin. At low dose both antibiotics showed very low elution as well as Simplex in the medium-dose group. Palacos resulted in a greater release than Simplex in medium- and high- dose groups. In particular, Palacos with high concentration of antibiotics showed a level of activity that passed for more than eighty days the level of minimum inhibitory concentration (MIC) of the most common pathogens [15]. Also, the amount of tobramycin released from Palacos was higher than that of Simplex (10 days). Considering the vancomycin, the kinetics of elution was inadequate in all three groups for both cements (taking as limit the detection of 25 μg/mL). Nevertheless, vancomycin resulted active for the first day. Considering the tobramycin, for groups with low and medium dose an inadequacy in the kinetics of release was observed again, but for the high-dose group (especially for Palacos) the duration of release was high. In general, Palacos has a much higher release level (above the MIC for most common pathogens encountered) and for a longer period of time compared to Simplex and tobramycin showed an improved efficacy profile compared to vancomycin [53, 54]. However, the combination of the two antibiotics greatly increases the release and, probability, the efficacy [36]. In another study about the combination of antibiotics [38] the release of antibiotics from a spacer in vitro was measured with the purpose to establish the best pairing cement-antibiotic against specific bacteria (S. aureus, S. epidermidis, Enterococcus faecalis, and MRSA). Palacos-R and three different antibiotics (gentamicin, vancomycin, and teicoplanin) were used alone or in combination (gentamicin plus vancomycin or teicoplanin plus gentamicin). The study showed that the combination of two antibiotics in a spacer has a bactericidal activity more prolonged than a spacer loaded with a single antibiotic. Also, the synergistic action of gentamicin and teicoplanin had superior bactericidal activity compared to gentamicin and vancomycin and the coupling of a glycopeptide with an aminoglycoside covers both Gram-negative and Gram-positive bacteria.

9. Factors Influencing the Release of Antibiotic from the Cement and the Role of Surface

According to some authors the release of the antibiotic can last for many days [15], while for the majority of the authors the process occurs for the first days only [55]. Others sustain that it is a process of the duration of few hours [56]. The amount and the duration of the release of the antibiotic from the cement is a debated issue which still has not been completely understood [3, 22, 30, 55, 56].

The release of the antibiotic from the cement is influenced by the type (viscosity) of the cement, by the surface of contact/exchange, by the conditions of the compound, and the type and amount of antibiotic. The antibiotic is released from the surface of the cement and from cracks and voids in the cement itself [57]. The nature of the polymer allows the passage of fluids, allowing the release of the incorporated antibiotic. Nevertheless, while the hydrophobicity of the cement limits this release at less than the 10%, the most of the antibiotic is released in the first hours and days after surgery [58]. In addition, a significant amount may still be trapped in cement for long time [58]. According to a study [59], the Palamed, given the same procedure of preparation, is the cement that permits the biggest release of antibiotic over time (17%), compared with Palacos (8.4%) and CMW (4–5.3%). Many authors interpreted this release as a phenomenon of surface, while others argue what occurs throughout the polymeric matrix. It was shown that the initial release is directly proportional to the roughness of the surface: the higher the roughness, the wider the area of release [36, 48]. Also, a linear correspondence after a week between the porosity of the cement and the release of the antibiotic was demonstrated: the continuous release after several days would depend on the deep penetration of the antibiotic in the cement previously determined by the porosity [36, 48].

The effect of the direct contact with the surface of biomaterials, such as PMMA, on the characteristics of the bacteria and a consequent possible change in the population and bacterial resistance were studied [60]. Also, different types of antibiotics were evaluated for this subject: β-lactams, aminoglycosides, macrolides, and others investigating the susceptibility to antibiotics both from bacteria adherent to the cement and from nonadherent bacteria. The contact with the material gave significant differences in terms of growth for all tested antibiotics, with the exception of clindamycin. These data suggest that the characteristics of the surface of the material could be important in the interaction with bacteria and that the bone cement can lead to changes in bacterial adhesion to the biomaterial modifying the antibiotic resistance [60, 61].

10. Mechanical Properties of Antibiotic Cement

It was suggested, as mentioned above, that the addition of antibiotics may play a role in weakening the structure and the mechanical properties of the cement. Various cements and antibiotics were compared, to determine which might be the more resistant over the time: studies conducted on Palacos-R, CMW1, and CMW3 with and without the addition of gentamicin or Simplex-P with erythromycin, colistin, or tobramycin did not show significant effects on fatigue resistance in comparison to the respective simple cements [33]. It must be noted that the majority of the studies which demonstrated a theoretical disadvantage of the cement loaded with antibiotics are in vitro studies [47]. On the contrary, the majority of the clinical studies reported an increased rate of mechanical failures when high dosages were used in comparison with the ALBC loaded at low dose [47, 49].

11. Toxicity

At our knowledge, there are no reports in literature of systemic toxicity related to the use of ALBC. Various researches focused about local toxicity, with particular interest to the function of osteoblasts and osteocytes: event though there are no reports of clinical adverse effects on these cells, some in vitro studies raised doubts about this subject. In addition, the concerns are more consistent in case of cement loaded at high doses, where the local levels of antibiotics may exceed 200 μg/mL. In particular, when osteoblasts derived from trabecular bone were exposed to materials containing various concentrations of gentamicin (0 to 100 μg/mL), the activity of alkaline phosphates decreased significantly in all the cultures with gentamicin concentration >100 μg/mL, the incorporation of 3H-thymidine decreases at the same concentration of antibiotic, and the total DNA decreases for concentrations ≥700 μg/mL [62, 63]. A study about the effect of tobramycin (concentrations between 0 and 10,000 μg/mL) on osteoblasts showed that local levels <200 μg/mL have no effect on replication of these cells, whereas at concentrations >400 μg/mL replication decreases, and with 10,000 μg/mL cell death occurs. Also the effects of vancomycin on osteoblasts were studied for concentrations ranging between 0 and 10,000 μg/mL: levels of vancomycin <1.000 μg/mL had little or no effect on replication, but concentrations of 10,000 μg/mL caused the death of the osteoblasts [64]. Vancomycin seems to be less toxic than aminoglycosides at high concentrations and Gentamicin has lower critical concentrations than those of tobramycin, despite they are both aminoglycosides.

12. Conclusions

The majority of the studies demonstrated an antibacterial effectiveness of cements loaded with antibiotics in the treatment of deep infections following hip and knee arthroplasty (onestage and two stages).

The main problem of these analyses is the minimus time of treatment that allows the antibiotic effects without developing bacterial-resistance. Recent studies in vitro showed that the highest concentration of antibiotic released is found in the first two days in contrast, studies in vivo did not reach statistically significant evidence.

The literature demonstrated that the best results are obtained with the association of antibiotic-loaded cement and the systemic antibiotic administration, if possible with targeted testing.

Finally, a significant difference between the intravenous administration of antibiotics and the use of the antibiotics into the cement for prophylactic use in patients with standard risk was not found so it is not advisable to use antibiotic loaded cement for routine as prophylaxis.

In conclusion, it is recommended not to trust excessively in the role of antibiotic loaded bone cement and not to give it therapeutic properties that it does not posses. It is clear how ALBCs are more effective than simple cements, but undoubtedly the “window of effectiveness” cannot be attributed only to antibiotics. Other properties related to the cement itself such as roughness, porosity, technique of preparation, and many patient-related features must be reminded.

However, it is necessary to underline that ALBC, especially if targeted by a specific antibiogram or integrated with an association of molecules more than a single one, is an important aid in the prevention and in the treatment of prosthetic infections.

Disclosure Policy and Conflict of Interests

The authors have no conflicts of interest in this submitted manuscript and did not receive grant, funds, and financial support. This research was not influenced by a secondary interest, such as financial gain. All authors read and agreed to all Hindawi Copyright and Licence Agreement terms.

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Copyright

Copyright © 2011 Alessandro Bistolfi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Introduction

Bacteria have developed resistance to most of the currently used antibiotics [1], thus, making bacterial infections a major cause of mortality in the health care system [2]. There is evidence to suggest that bacterial infections are becoming difficult to treat because the pathogens are capable of developing biofilm which aids in host establishment, population expansion and in disease proliferation [3]. More than 60% of all the human bacterial infections have been attributed to the persistence of biofilm formation by the respective bacteria [4]. When bacteria form biofilms, the biofilm structure facilitates the survival of disease-causing pathogens even in hostile environmental conditions. It has been shown that the nature of biofilm structure and physiological attributes of biofilm forming organisms confer an inherent resistance to hostile conditions including antimicrobial agents such as antibiotics [5]. Hence, there is need to develop new antibacterial agents which can inhibit formation or destroy the mature biofilms and thus, increasing susceptibility of microbes to antibiotics.

There is an increasing interest in the use of medicinal plant-derived compounds as alternative antibacterial agents [6]. Plants have formed the basis of traditional medicinal systems that have been in existence for thousands of years, and continue to provide humanity with new remedies [7]. Some of the drugs which are widely used in clinical practice have been obtained from plants [8]. Vernonia adoensis is a plant which is commonly used in African ethnomedicine [9]. It is a herbaceous plant from the Vernonia genus belonging to the family Asteraceae which is the largest genus with close to 1000 species [10]. In East Africa the decoction of the roots is mixed with other trees for the treatment of heart and kidney problems [11]. In the Rift valley and Western part of Kenya, V. adoensis is used traditionally to treat symptoms of sexually transmitted diseases such as gonorrhea [11]. The leaves of the plant are also used in the treatment of the symptoms of malaria [12]. In Tanzania this plant has been traditionally used in African ethnomedicine for the treatment of fever and upper respiratory tract infections [9]. V. adoensis is native and commonly distributed throughout Zimbabwe where it is known in vernacular Shona language as Musikavakadzi [13].

Several studies have been carried out to evaluate the antimicrobial potential of V. adoensis with the ultimate goal of justifying the traditional use of the plant species or discovering drugs [14, 15]. The plant extracts have shown antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus and Bacillus subtilis in-vitro [16]. The plant leaves have been reported to have very high anti-plasmodial activity against Plasmodium falciparum [17]. Chimponda and Mukanganyama [18], reported that the leaves of V. adoensis had inhibitory activity against Mycobacterium aurum and Corynebacterium glutamicum. There are studies which have been done to isolate phytochemicals from some Vernonia species and the compounds isolated were shown to have biological activities [19,20,21]. V. adoensis has been shown to possess some important pharmacological phytochemicals [21] but there is limited information on the biological activity of the specific compounds isolated from the plant. Most of the biological activity tests from the various studies were conducted using crude extracts obtained from different parts of the plant. Hence, the need to isolate phytochemicals from V. adoensis as they may yield lead compounds that have significant antimicrobial potential against selected strains of bacteria. Studies have shown that bacteria such as P. aeruginosa, S. aureus and K. pneumoniae are among the major causes of nosocomial infections that have produced a poor prognosis in the hospital [22]. From the advent of antimicrobial application in treatment of bacterial diseases, pathogenic bacteria have responded by developing varied forms of resistance [23]. Klebsiella pneumoniae has become resistant to carbapenem antibiotics which are often the last line of defense against Gram-negative infections [24]. Staphylococcus aureus strains are now resistant to penicillin [25]. In most Asian countries, 70–80% of the same strain have become methicillin-resistant [25]. Pseudomonas aeruginosa infections are now responsible for most nosocomial infections in hospitals and healthcare centers because there are no effective antimicrobial agents against it [26]. Also infection by P. aeruginosa through biofilm in lungs of cystic fibrosis (CF) patients is causing high morbidity and functional failure of this organ [27].

There is need for new antibacterial agents as bacteria have developed resistance to most of the currently used antibiotics [1]. In addition, current antibiotics have considerable limitations in terms of antimicrobial spectrum and side effects [28]. The indiscriminate use and misuse of antibiotics has led to increasing clinical resistance of previously sensitive microorganisms and to the occurrence of uncommon infections. Plant-based natural products have been found to be a rich source of antimicrobial agents [29]. Traditional medicinal plants have received significant attention as a source of new chemical entities because their phytochemicals may lead to new leads in drug discovery [30]. The aim of this study was to isolate the phytochemicals found in V. adoensis and evaluate their antimicrobial activity since previously the crude extracts were shown to have high antibacterial activities [31].


Endophytic Penicillium species and their agricultural, biotechnological, and pharmaceutical applications

Penicillium genus constituted by over 200 species is one of the largest and fascinating groups of fungi, particularly well established as a source of antibiotics. Endophytic Penicillium has been reported to colonize their ecological niches and protect their host plant against multiples stresses by exhibiting diverse biological functions that can be exploited for countless applications including agricultural, biotechnological, and pharmaceutical. Over the past 2 decades, endophytic Penicillium species have been investigated beyond their antibiotic potential and numerous applications have been reported. We comprehensively summarized in this review available data (2000–2019) regarding bioactive compounds isolated from endophytic Penicillium species as well as the application of these fungi in multiple agricultural and biotechnological processes. This review has shown that a very large number (131) of endophytes from this genus have been investigated so far and more than 280 compounds exhibiting antimicrobial, anticancer, antiviral, antioxidants, anti-inflammatory, antiparasitics, immunosuppressants, antidiabetic, anti-obesity, antifibrotic, neuroprotective effects, and insecticidal and biocontrol activities have been reported. Moreover, several endophytic Penicillium spp. have been characterized as biocatalysts, plant growth promoters, phytoremediators, and enzyme producers. We hope that this review summarizes the status of research on this genus and will stimulate further investigations.

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Screening of Compounds

Experimental procedure and screening of potential inhibitors

All inhibitory assays of the phosphorylation activity of Yes were performed in accordance with the Promega Technical Manual for the ADP-Glo™ Kinase Assay (Fitchburg, WI, USA. Catalog number: V9102). The human recombinant Yes [a.a. 2–543 (end)] was purchased from BPS Bioscience (catalog number: 40488). The details of the assay protocol and reagent information are given elsewhere 10 . Here, we briefly describe the screening of the compounds based on inhibition activity and results.

The screening was conducted in three steps consisting of the first screening, the second screening, and the IC50 determination, as can be illustrated in Fig. 1b. First, we determined the inhibition rates of the 1,991 compounds. Each compound was placed in 4 wells of a 384-well plate. In total, 80 compounds were assayed on one plate and the other wells were used for positive and negative controls. The compounds were randomly placed on plates so that compounds proposed by one group were not placed on a plate together. A mean of four inhibition rates for each compound was compared to criteria for the first screening. These criteria included that the inhibition rate was greater than 25% and the inhibition rate was greater than the mean plus three-fold of the standard deviation of the plate on which the compound was assayed, where observed inhibition rates of positive and negative controls were not taken into consideration. As a results, 68 compounds passed the screening. Information of the dropped compounds is given in Table S3 of the Supporting Information. Second, the inhibition rates of the screened compounds were determined on one plate using the same procedure of the first screening, where compounds were dissolved from fresh powder. As a result, 16 compounds showed inhibition rates greater than the threshold of the second screening (i.e., approximately 50%). Information for screened and dropped compounds is given in Table S4 of the Supporting Information. Screened compounds were then evaluated for their IC50 values. The chemical structure and assay results of these compounds are given in Table 2.

Among the 16 compounds, 10 compounds showed an IC50 less than our hit criterion, which was an IC50 less than 10 μmol L −1 , as shown in Table 2. These compounds showed a clear dose-response relationship (DRR) as can be seen in Figure S1 of the Supporting Information. As for Z1252403274 and Z275023406, which showed a good DRR, they were not defined as hit compounds because of insufficient potency. The other four compounds, Z50080378, Z57745307, Z50080181, and Z1283491630, did not reveal a DRR, having “inhibition activity” around 50% in the whole range of concentrations, which may be due to their non-specific interactions with the target (promiscuous protein binding, protein aggregation) or solubility-related issues. For these reasons, these compounds were excluded from consideration. Note that we confirmed that the threshold used for the second screening was reasonable as can be seen in Figure S2 of the Supporting Information.

The 10 hit compounds were compared to the pan-assay interference compounds (PAINS) filters, filters A, B, and C described in the literature 35 , which suggests potential functional groups of frequent hitters extracted from HTS assays. We found that the 10 compounds do not have these potential functional groups. This means that all the hit compounds are promising for further investigation. It should be noted that some hit compounds have “questionable” chemotypes from a medicinal chemistry point of view, i.e., hydrazones (Z49895016, Z57745314, Z57745304, Z295464022) and a potential Michael acceptor (Z449737600), which may present a reactivity/toxicity liability. In the present study, we did not exclude them because only the biochemical assays, not cell-based, were used for screening in this study. This strongly decreases the chance of getting false positives with these compounds during the primary screen. The emphasis was also placed on avoiding the potential loss of any active scaffolds identified by the competing computational groups, rather than on the early elimination of less desirable chemical series. Substituting hydrazones with their non-reactive isosteres during hit-to-lead optimization is a feasible medicinal chemistry endeavor as is illustrated by some research publications 36,37,38 . We will discuss these hydrazone-containing compounds in more details in the following section. The Michael acceptor could be substituted by an amide group between an acid and a cyclical secondary amine, to retain molecular rigidity.


4 Computational Analysis of the Physicochemical and Structural Properties of Natural Products

Cheminformatics has been playing a key role in the characterization of NPs by their physicochemical and structural properties, and in the comparison of NPs with small-molecule drugs, drug-like compounds and other types of (organic) molecules. NPs cover a much broader chemical space than synthetic compounds and they populate also areas in chemical space that are generally not (or only with great difficulties) synthetically accessible. 6, 8, 19, 57, 58 The structural uniqueness (and complexity) of some NPs could allow them to target macromolecules that are otherwise undruggable. 16

NPs are on average heavier and more hydrophobic than synthetic drugs and synthetic, drug-like compounds. 59 Their structural complexity is also often higher, in particular with regard to stereochemistry (commonly quantified by the number of chiral centers, 57, 59-66 the number of fraction of Csp 3 atoms, 6, 8 and/or the number of bridgehead atoms in ring systems 67 ) and 3D molecular shape. 8, 68

NPs show an enormous diversity of ring systems, in particular of aliphatic systems. 6, 8, 57, 63, 65 One study showed that 83 % of core ring scaffolds of NPs are absent in commercially available screening databases. 69 With regard to atom composition, two of the most discriminative features of NPs over synthetic compounds are the (on average) low number of nitrogen atoms and high number of oxygen atoms. 57, 59, 62-64 Nevertheless, a clear majority of the known NPs, and even more so in physical NP libraries, are drug-like. 6

NPs from different kingdoms have distinct physicochemical and structural properties. 66, 70-76 For example, NPs with macrocycles or long aliphatic chains are more commonly to marine species than terrestrial species. 74 Also bacteria produce many macrocyclic NPs. 75 Their NPs are characterized by a high proportion of heteroatoms and, related to this, a high diversity of functional groups. 76


Acknowledgements

We thank Per Anders Enquist (Chemical Biology Consortium Sweden Umeå), Christa Testerink and Iko Koevoets (Wageningen University) for advice. Kamaleddin Hajmohammadebrahimtehrani, Colin Snoeker, Jan Orsel and Lennert Zorn (Utrecht University) are thanked for technical assistance. This work was supported by Graduateschool Uitgangsmaterialen grant NWO# 831.13.002 to LvdW and MvZ by the Netherlands Organisation for Scientific research (NWO), a Facility access and support grant for chemical genomics projects to LvdW and MvZ from the Laboratories for Chemical Biology (Chemical Biology Consortium Sweden Umeå), Umeå, Sweden, an Erasmus Placement grant to LvdW, an European Research Council (ERC) starting grant No. 258413 to MK, Deutsche Forschungsgemeinschaft (DFG) grant INST 20876/127-1 FUGG to MK and ERC consolidator grant 616449 and BBSRC grants BB/R017913/1 and BB/S003193/1 to RH. KL acknowledges support from the Swedish Foundation for Strategic Research (VINNOVA), the Swedish Research Council (VR), the Swedish Metabolomics Centre for the Use of Instrumentation and the Knut and Alice Wallenberg Foundation (KAW). ON was financially supported by the Ministry of Education Youth and Sports of the Czech Republic through the European Regional Development Fund-Project ‘Plants as a tool for sustainable global development’ (CZ.02.1.01/0.0/0.0/16_019/0000827).


Discussion

Successful control and treatment strategies for malaria currently rely on the availability of effective antimalarial drugs. The emergence of resistance to the critically important artemisinin family of drugs [7–9] highlights the need for new drugs to partner and replace current therapies, and new drug development strategies. Drugs that target merozoite invasion have been proposed as a novel strategy for antimalarial development [11–14]. We have applied robust methods for the purification of viable P. falciparum merozoites and invasion assays [13, 18] to screen for inhibitors of merozoite invasion. The antibiotic azithromycin and related compounds were identified as rapid inhibitors of P. falciparum merozoite invasion into erythrocytes. This is the first time that an antimalarial in clinical use has been linked to inhibition of merozoite invasion in vitro, and the first clear identification of antimalarial compounds with dual mechanisms of action against merozoite invasion and intra-erythrocytic parasite development. Live video microscopy revealed that azithromycin acts to prevent the essential step of tight junction formation during invasion. Furthermore, the inhibitory activity of azithromycin was largely ablated by selective removal of glycan groups, and we identified modified macrolides with increased potency against merozoite invasion.

Azithromycin is a macrolide antibiotic with a 15-membered macrolactone ring that is well tolerated and safe for clinical use by children and pregnant women [28]. Azithromycin and related macrolides are known to inhibit ribosomal protein synthesis in the asexual-stage parasite apicoplast by binding to the 50S subunit of the apicoplast (70S) ribosomal complex [49, 50]. Typically, clinical concentrations of azithromycin based on current dosing regimens result in ‘delayed death’ of the parasite in in vitro assays. The progeny of azithromycin-treated parasites fail to form functional apicoplasts in the daughter merozoites, thereby causing the death of the second generation of parasites after treatment [31, 32]. Azithromycin has been considered a candidate for inclusion in artemisinin combination therapies (ACT) and for the prevention of malaria in pregnancy [51] due to its safety and its long half-life (over 50 hours) [25, 27, 29].

In addition to its ‘delayed death’ effect on parasite growth, azithromycin has been reported to kill parasites within the first cycle of in vitro parasite growth at a drug concentration just above reported peak plasma concentrations [29, 32]. It has been speculated that inhibition of parasite growth during the first cycle of treatment is a result of azithromycin having a secondary target in addition to the apicoplast ribosome [32, 41]. Given growth inhibition via apicoplast-targeting ‘delayed death’ is approximately 150-fold more potent than that achieved for 1 cycle growth inhibition in vitro, azithromycin is thought to act predominantly through targeting the apicoplast when used clinically. Because of azithromycin’s safety profile, several studies have investigated modifications to the drug with a view to lowering the IC50 of short-term treatment (1 cycle of growth) to the low nanomolar range [42–44]. Yet despite this interest in azithromycin, the target of drug inhibition during the first cycle of growth is unknown, and the potential of this novel mechanism of action as a clinical treatment remains unclear.

Several lines of evidence indicate that azithromycin has a secondary mechanism and target(s) of action to inhibit invasion, in addition to binding to the 50S apicoplast ribosomal subunit as described for the ‘delayed death’ phenotype [31, 32]. Clindamycin, a smaller and structurally unrelated antibiotic that has overlapping binding sites to azithromycin on the apicoplast ribosome [39], as well as an identical mode of action, had very little invasion inhibitory activity. Selection of a D10 line for resistance (57-fold reduced sensitivity) to azithromycin in the 2 cycle ‘delayed death’ assays did not result in a similar increase in resistance to azithromycin invasion inhibition assays when compared to a parental D10 (‘delayed death’ sensitive) line. Comparison of azithromycin and its modified analogues (1j, 12e) showed that modifications increasing the potency of these analogues over azithromycin in invasion inhibition, in cycle (40 hour) and 1 cycle (90 hour) assays did not appreciably increase the potency of the compounds over 2 cycle ‘delayed death’ assays, suggesting the mechanism of enhanced inhibitory activity was independent of anti-ribosomal activity.

The near instantaneous inhibition of merozoite invasion caused by azithromycin also suggests that this drug can rapidly kill parasites through a secondary mechanism of action. Typical ‘delayed death’ inhibition by macrolide antibiotics results in the loss of apicoplast functionality [31, 32], with this then leading to the loss of isoprenoid precursor biosynthesis, the only essential function of the apicoplast during blood-stage development [33]. The long timeframes required for apicoplast translation inhibition and the resulting ‘delayed death’ caused by macrolide antibiotics is in striking contrast to the very rapid inhibition of merozoite invasion at higher drug concentrations. Furthermore, removal of the apicoplast in the experiments by Yeh et al. [33] was not reported to result in an invasion defect. Combined, the available evidence suggests it is very unlikely that the apicoplast plays some as yet undefined role in merozoite invasion and implicates a secondary mechanism of action of azithromycin against invading merozoites. Whether the mechanism of invasion inhibition and intracellular asexual-stage parasite growth inhibition (within 1 cycle in the absence of invasion inhibition) is the same remains to be elucidated. However, the similar IC50 values seen for both inhibitory phenotypes raises the intriguing possibility that this secondary mechanism of action could be targeted throughout the disease causing blood-stage of P. falciparum malaria.

The ability of azithromycin to rapidly inhibit merozoite invasion, in addition to the apicoplast-targeting ‘delayed death’ mechanism of action, is an exciting prospect for the development of macrolide-based drugs in combination therapies. This dual modality could see azithromycin assist in rapid clearance of parasites as well as offering longer-term treatment of the remaining low-level blood and liver-stage parasites through its activity against the apicoplast. Such a drug treatment strategy has the potential to increase drug efficacy, while reducing the chances for the development of resistance.

Although the in vitro merozoite invasion inhibitory activity of azithromycin required drug concentrations at the upper end of what is achieved clinically [29, 32], we demonstrate that modification of the macrolides can lead to a substantial improvement in invasion inhibitory activity this suggests that invasion inhibitory macrolides could be developed as therapeutics. Addition of an L-megosamine sugar to erythromycin A through an in vivo fermentation process [41] produced a compound with 32-fold greater invasion inhibitory activity than parent erythromycin A. Furthermore, a selection of azithromycin analogues [42–44] lowered the invasion inhibitory IC50 by up to 5-fold. The azithromycin derivatives were tested only when solubilized in DMSO due to limitations in the amount of available material. Importantly, the use of DMSO as vehicle for solubilization reduced the potency of the parent compound azithromycin 4-fold relative to ethanol as vehicle. If this observation were to hold true for the derivatives, the invasion inhibitory IC50 of the compound 1j (5) [44] would be in the range of 1–2 μM.

The invasion inhibitory IC50 of azithromycin (10 μM) and analogues (1j, 7 μM 12e, 15 μM) is comparable to that achieved for inhibitors that specifically target the function of essential merozoite proteins. Two recent proof-of-concept studies identified small molecule invasion inhibitory compounds targeting interactions between essential invasion ligands RON4-AMA1 (inhibitory range 30–6 μM) and MSP1-19 (replication inhibition 21.7 μM) [12, 14], and reported invasion inhibitory IC50 values in a very similar range to azithromycin and analogues. Another highly selective invasion inhibitor, the 3D7-AMA1-specific peptide inhibitor R1 [52], has an invasion inhibitory IC50 of 2.5 μM in assays using purified merozoites [18]. Azithromycin, with minimal modification, can rapidly inhibit merozoite invasion at concentrations comparable to some of the best and most selective invasion inhibitors available for drug development and research. The encouraging improvements evident in the invasion inhibitory activity of azithromycin from only a small panel of derivatives, combined with the proven safety and efficacy of macrolides, support the potential for developing such compounds as therapeutics.

Removal of azithromycin’s glycan groups provided insight into the structural requirements for invasion inhibitory activity. Removal of the cladinosyl sugar resulted in a 5-fold reduction in invasion inhibitory activity. Strikingly, removal of both sugars resulted in complete loss of invasion inhibitory activity, strongly suggesting that the desosamine sugar, rather than the cladinosyl, is critical for invasion inhibitory activity. Interestingly, the desosamine sugar is also considered the critical glycosylated group in binding of macrolides to microbial ribosomes [39] with clinically used macrolide antibiotics such as telithromycin having dispensed with the cladinosyl group altogether. Screening of a larger number of macrolides and modified analogues will help identify others with greater potency, and allow assessment of which modifications and groups are most important for activity.

The fact that azithromycin inhibits host invasion by Plasmodium merozoites (from human and rodent malaria) and T. gondii tachyzoites parasites suggests that the target of inhibition is shared amongst some apicomplexan invasive stages. The fact that the potency of azithromycin against P. falciparum (human) and P. berghei (rodent) malarias was very similar raises the possibility that targeting this pathway could be an effective strategy to treat other malaria species. The results of the T. gondii invasion assays suggest that host cell invasion of this more distantly related apicomplexan parasite can also be targeted by azithromycin, albeit at higher concentrations. Importantly, the analogue 12e had equal or better invasion inhibitory potency than azithromycin (in ethanol or DMSO, respectively) when tested at a 6-fold lower concentration. This strongly suggests that analogues of azithromycin could be developed with superior inhibitory activity against tachyzoite invasion and confirms that the invasion inhibitory activity of azithromycin and analogues is shared across distantly related apicomplexan parasites.

Macrolide antibiotics have been linked to mechanisms of action other than binding to microbial ribosomes, including interference with intracellular signalling mechanisms such as Ca 2+ and MAPK through an as yet undefined mechanism (reviewed in [53]). Intracellular Ca 2+ signalling is thought to play an important role in invasion (reviewed in [54]) so interference with this mechanism by macrolide antibiotics could contribute to inhibition of invasion. Macrolide antibiotics have also been shown to bind to negatively charged phospholipid bilayers and interfere with normal membrane function [55, 56]. Intriguingly, the relative activity of the macrolides appeared to be in part related to the number of cationic groups present on the macrolide.


Background

Maize, one of the three major food crops in the world, is consumed vast. However, the usable area for maize cultivation is gradually decreased every year. Facing the conflict, the major solution is to improve the yield per unit area, which can be realized by cultivating new varieties, increasing planting density and improving the farming condition. Among these approaches, the easiest way is to increase the planting density, but it brings the problem of lodging. Moderately reducing plant height is an effective strategy for improving lodging resistance in maize grown at high density. Meanwhile, the heavy rain and the strong winds, lead directly to the maize plants becoming flooded or lodged. Lodging leads to multiple adverse impacts, including the reduce of yield by 15–18%, later maturing, quality reduction, harvest difficulty, aggravation in diseases, pests and rats [66, 106].

The plant height is generally positively correlated with lodging rate. Study the growth mechanism and plant height regulation of maize has great significance for improving lodging tolerance [107]. The plant height of maize is mainly determined by internode number and length particularly the 7th to 9th internodes, which are the usual occurrence of the stalk lodging for maize [68]. The internode can be divided into three parts during the elongation stage, including meristem region, elongation region, and fixed region [120].

The lower end of the elongating internode is the meristem, the region with active cell division. The elongation region is located above the meristem, where the cell expansion and primary cell walls formed [51, 95]. The fixed region, also known as the maturation region, is located at the upper end of the internode, in which the extended growth of cells is stopped and the deposition of secondary wall is main process [51]. Gene activity is closely associated with specific biological processes in these three type regions. It has been showed that the genes, which were related to gibberellin (GA) and auxin, mainly expressed in internode elongation region, such as dwarf1, dwarf3 and brachytic2 [12, 54, 102]. And NACs and CAD genes which involve in regulating the secondary cell wall synthesis are mainly expressed in fixed regions [15, 39]. Nevertheless, information on the transcriptional differences between meristem, elongation and fixed regions is far from clear.

The genes related to many hormones have been showed to be involved in the plant height, such as genes participated in biosynthesis, transport and signaling pathways of GA and jasmonic acid (JA) [16, 55, 97]. Some genes affect plant height by regulating GA synthesis and transduction, such as dwarf1, dwarf3, GA20oxs, GA3oxs, CPS, dwarf plant 8 and dwarf plant 9 [7, 73, 81, 83, 101, 102, 108]. JA affects plant height mainly via complex phytohormone crosstalk with GA and auxin. Studies showed that JA can affect the formation and distribution of auxin by inducing the ASA1 expression and regulating the PINs and PLETHORA [98], thereby affecting cell elongation. In addition, DELLAs, GA signal reverse regulation factor, can interact with the JA pathway to coordinate normal growth and defense to biotic stresses [110]. Therefore, phytohormones are of great significance to control internode development. However, the high production cost and the instability of molecular structure in the vitro environment make direct application of phytohormones very difficult in yield. Plant growth regulators, which are compounds with similar effects to phytohormones, overcome these difficulties [105, 112]. Currently, the main component of plant growth regulators used in agriculture is 1,1-dimethyl-piperidinium chloride (DPC) or ethephon [70, 121]. However, maize is not sensitive to DPC and the ethephon decreases grain yield of maize [52, 71]. With the increase of planting density and mechanization level, a more efficient and safe new plant growth regulator is urgently needed.

Coronatine (COR), secreted by Pseudomonas syingae pathovars, is a phytotoxin [46, 50, 76], with similar function as JA [36, 115]. It has been showed that the COR is an analog of JA [100], and is 1000 times more active than JAs [93]. The COR can lead to adverse effects for plants, such as leaf chlorosis and disease symptoms [94]. However, COR of low concentrations can increase the abiotic stress resistance [40, 104, 124]. At present, COR can be produced by microbial fermentation, and has the advantages of lower environmental pollution and chemical residues. Therefore, as a new environmentally friendly plant growth regulator, COR is expected to be widely used in agriculture. Previous researches have shown that COR can inhibit the elongation of maize root, hypocotyl and mesocotyls [62]. Our previous studies have showed that COR had certain effect on reducing plant height [85, 99], while the molecular mechanism of COR in reducing plant height of maize is not well known.

In our study, the plant height of ZD958 and XY335, two wildly cultivated maize hybrids, could be significantly decreased under COR treatment via reducing internode length and thus improve lodging resistance. To research the underlying gene different expression that drive the responses of internode to COR, spatio-temporal transcriptome of inbred B73 internode were produced under control and COR treatment, containing the maturation, meristem and elongation regions of internode. The differences in transcription levels of the three regions at normal condition were displayed and then were compared with that upon COR treatment. In total, 8605 COR-responsive genes (COR-RGs) were reported, and internode specific genes accounted for 9.3% (802 genes). For these COR-RGs, 614, 870, 2123 of which showed expression changes in only fixed, meristem and elongation region, respectively. Gene ontology enrichment analysis indicated that different genes in the three regions control their growth. Moreover, we found that 84% of GA related gens and 80% of JA related genes were significant affected under COR treatment. In summary, the differential expression map of gene expression response in internode to COR provides a theoretical support for future study of the molecular mechanism of plant height decreased by COR.


Acknowledgments

We would like to thank our many colleagues and collaborators who have worked with us on PI3K inhibitors. We thank Dr. Swen Hoelder for suggesting the Rubik's cube analogy and Professor Keith Jones for help with Figures 1 and 2. We are grateful to Val Cornwell for help with preparation of the manuscript and Ann Ford for administrative support. We apologize to the authors of many excellent reports that could not be cited because of space restrictions.

Grant Support: Cancer Research UK program grant number C309/A8274 and National Health Service funding to the National Institute for Health Biomedical Research Centre. Paul Workman is a Cancer Research UK Life Fellow.


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