The dual role of photodynamic therapy to treat cancer and microbial infection.

Photodynamic therapy (PDT) is a minimally invasive treatment showing promise against cancer and microbial infections. PDT targets tumor cells while sparing healthy tissue, reducing side effects. It induces immunogenic cell death, potentially stimulating antitumor immune responses and reducing cancer recurrence. In microbial treatment, PDT effectively combats bacteria, fungi and viruses. Combining PDT with chemotherapy, radiotherapy and immunotherapy enhances its efficacy. However, challenges such as tumor hypoxia, limited tissue penetration and phototoxicity necessitate ongoing research efforts to optimize PDT protocols and overcome limitations. Overall, PDT is versatile and continually advancing with refined protocols to improve its clinical utility against cancer and microbial infections.

It's all relative: analyzing microbiome compositions, its significance, pathogenesis and microbiota derived biofilms: Challenges and opportunities for disease intervention.

Concept of microorganisms has largely been perceived from their pathogenic view point. Nevertheless, it is being gradually revisited in terms of its significance to human health and now appears to be the most dominant force that shapes the immune system of the human body and also determines an individual's predisposition to diseases. Human inhabits bacterial diversity (which is predominant among all microbial communities in human body) occupying 0.3% of body mass, known as microbiota. On birth, a part of microbiota that child obtains is essentially a mother's legacy. So, the review was initiated with this critical topic of microbiotal inheritance. Since, each body site has distinct physiological specifications; therefore, they contain discrete microbiome composition that has been separately discussed along with dysbiosis-induced pathologies originating in different body organs. Factors affecting microbiome composition and may cause dysbiosis like antibiotics, delivery, feeding method etc. as well as the strategies that immune system adopts to prevent dysbiosis have been highlighted. We also tried to bring into attention the topic of dysbiosis induced biofilms, that enables cohort to survive stresses, evolve, disseminate and infection resurgence that is still in dormancy. Eventually, we put spotlight on microbiome significance in medical therapeutics. We didn't merely confine article to gut microbiota, that is being studied more extensively. Numerous community forms at diverse body sites are inter-related, and being exposed to awfully variable perturbations appear to be challenging to evaluate perturbation risks holistically. All aspects have been elaborately discussed to achieve a global depiction of human microbiota in order to meet urgent necessity for protocol standardisation. Demonstrates that environmental challenges (antibiotic use, alterations in diet, stress, smoking etc.) might cause dysbiosis i.e. transition of healthy microbiome composition to the one in which pathogenic microorganisms become more abundant, and eventually results in an infected state.

Orally fed EGCG coronate food released TiO2 and enhanced penetrability into body organs via gut.

Owing to unique nano-scale properties, TiO2-NPs (T-NPs) are employed as food-quality enhancers in >900 processed food products. Whereas, epigallocatechin-3-gallate (EGCG), a green tea polyphenol is consumed in traditional brewed tea, globally. Taken together, we aimed to investigate whether human gastric-acid digested T-NPs and complex tea catechins yield ionic species (Ti4+, Ti3+ etc.) and active EGCG forms to meet favourable conditions for in vivo bio-genesis of EGCG-coronated TiO2-NPs (ET-NPs) in human gut. Secondly, compared to bare-surface micro and nano-scale TiO2, i.e., T-MPs and T-NPs, respectively, how EGCG coronation on ET-NPs in the gut facilitates the modulation of intrinsic propensity of internalization of TiO2 species into bacteria, body-organs, and gut-microbiota (GM), and immune system. ET-NPs were synthesized in non-toxic aqueous solution at varied pH (3-10) and characterised by state-of-the-arts for crystallinity, surface-charge, EGCG-encapsulation, stability, size, composition and morphology. Besides, flow-cytometry (FCM), TEM, EDS, histopathology, RT-PCR, 16S-rRNA metagenomics and ELISA were also performed to assess the size and surface dependent activities of ET-NPs, T-NPs and T-MPs vis-a-vis planktonic bacteria, biofilm, GM bacterial communities and animal's organs. Electron-microscopic, NMR, FTIR, DLS, XRD and EDS confirmed the EGCG coronation, dispersity, size-stability of ET-NPs, crystallinity and elemental composition of ET-NPs-8 and T-NPs. Besides, FCM, RT-PCR, 16S-rRNA metagenomics, histopathology, SEM and EDS analyses exhibited that EGCG coronation in ET-NPs-8 enhanced the penetration into body organs (i.e., liver and kidney etc.) and metabolically active bacterial communities of GM.

Functional Amyloids in Pseudomonas aeruginosa Are Essential for the Proteome Modulation That Leads to Pathoadaptation in Pulmonary Niches.

Persistence and survival of Pseudomonas aeruginosa in chronic lung infections is closely linked to the biofilm lifestyle. One biofilm component, functional amyloid of P. aeruginosa (Fap), imparts structural adaptations for biofilms; however, the role of Fap in pathogenesis is still unclear. Conservation of the fap operon encoding Fap and P. aeruginosa being an opportunistic pathogen of lung infections prompted us to explore its role in lung infection. We found that Fap is essential for establishment of lung infection in rats, as its genetic exclusion led to mild focal infection with quick resolution. Moreover, without an underlying cystic fibrosis (CF) genetic disorder, overexpression of Fap reproduced the CF pathotype. The molecular basis of Fap-mediated pulmonary adaptation was explored through surface-associated proteomics in vitro. Differential proteomics positively associated Fap expression with activation of known proteins related to pulmonary pathoadaptation, attachment, and biofilm fitness. The aggregative bacterial phenotype in the pulmonary niche correlated with Fap-influenced activation of biofilm sustainability regulators and stress response regulators that favored persistence-mediated establishment of pulmonary infection. Fap overexpression upregulated proteins that are abundant in the proteome of P. aeruginosa in colonizing CF lungs. Planktonic lifestyle, defects in anaerobic pathway, and neutrophilic evasion were key factors in the absence of Fap that impaired establishment of infection. We concluded that Fap is essential for cellular equilibration to establish pulmonary infection. Amyloid-induced bacterial aggregation subverted the immune response, leading to chronic infection by collaterally damaging tissue and reinforcing bacterial persistence. IMPORTANCE Pseudomonas aeruginosa is inextricably linked with chronic lung infections. In this study, the well-conserved Fap operon was found to be essential for pathoadaptation in pulmonary infection in a rat lung model. Moreover, the presence of Fap increased pathogenesis and biofilm sustainability by modulating bacterial physiology. Hence, a pathoadaptive role of Fap in pulmonary infections can be exploited for clinical application by targeting amyloids. Furthermore, genetic conservation and extracellular exposure of Fap make it a commendable target for such interventions.

Chemically synthesised flavone and coumarin based isoxazole derivatives as broad spectrum inhibitors of serine ß-lactamases and metallo-ß-lactamases: a computational, biophysical and biochemical study.

The ß-lactam antibiotics are the most effective medicines for treating bacterial infections. Resistance to them, particularly through the production of ß-lactamases, which can hydrolyse all kinds of ß-lactams, poses a threat to their continued use. The synthesised flavone and coumarin based isoxazole derivatives have the potential to be used as broad-spectrum inhibitors of the mechanistically different serine-(SBL) and metallo-ß-lactamases (MBL). The synthesised compounds were discovered as potent ß-lactamase inhibitors using molecular docking and in silico pharmacokinetic analysis. We studied the binding of chemically synthesised inhibitors to clinically significant ß-lactamases of class A, B, and C using biophysical and biochemical approaches, and computational analyses. These molecules follow Lipinski's rule of five and have acceptable solubility, permeability, and oral bioavailability. These molecules were found to be non-toxic and non-carcinogenic. MIC results suggest that these molecules restore the antibiotic efficacy against class A, B, and C ß-lactamases. Kinetics data showed that these molecules reduce the catalytic efficiency of clinically relevant class A, B, and C ß-lactamases. Fluorescence study showed significant interaction between these flavone-/coumarin-based isoxazole derivatives and class A/B/ C ß-lactamases. This study showed promising effect of these new generation compounds as broad spectrum ß-lactamase inhibitors of both SBLs and MBLs.Communicated by Ramaswamy H. Sarma.

Repurposing drug molecule against SARS-Cov-2 (COVID-19) through molecular docking and dynamics: a quick approach to pick FDA-approved drugs.

A novel coronavirus known as severe acute respiratory syndrome is rapidly spreading worldwide. The international health authorities are putting all their efforts on quick diagnosis and placing the patients in quarantine. Although different vaccines have come for quick use as prophylactics, drug repurposing seems to be of paramount importance because of inefficient therapeutic options and clinical trial limitations. Here, we used structure-based drug designing approach to find and check the efficacy of the possible drug that can inhibit coronavirus main protease which is involved in polypeptide processing to functional protein. We performed virtual screening, molecular docking and molecular dynamics simulations of the FDA-approved drugs against the main protease of SARS-CoV-2. Using well-defined computational methods, we identified amprenavir, cefoperazone, riboflavin, diosmin, nadide and troxerutin approved for human therapeutic uses, as COVID-19 main protease inhibitors. These drugs bind to the SARS-CoV-2 main protease conserved residues of substrate-binding pocket and formed a remarkable number of non-covalent interactions. We have found diosmin as an inhibitor which binds covalently to the COVID-19 main protease. This study provides enough evidences for therapeutic use of these drugs in controlling COVID-19 after experimental validation and clinical demonstration.

Designing multi-epitope vaccine candidates against functional amyloids in Pseudomonas aeruginosa through immunoinformatic and structural bioinformatics approach.

Pseudomonas aeruginosa (P. aeruginosa) displays high drug resistance and biofilm-mediated adaptability, which makes its infections difficult to treat. Alternative intervention methods and targets have made such infections treatment manageable. One of the biofilm components, functional amyloids of Pseudomonas (Fap) is correlated positively with virulence and mucoidy phenotype found in infection in cystic fibrosis (CF) patients. Extracellular accessibility, conservation across P. aeruginosa isolates and linkage with lung infections phenotype in CF patients, makes Fap a promising intervention target. Furthermore, the reported effect of bacterial amyloid on neuronal function and immune response makes it a targetable candidate. In the current study, Fap C protein and its immediate interactions were explored to extract antigenic T-cell and B-cell epitopes. A combination of epitopes and peptide adjuvants has been linked to derive vaccine candidate structures. The vaccine candidates were validated for antigenicity, allergenicity, physiochemical properties, stability and interactions with TLRs and MHC alleles. Immunosimulation studies have demonstrated that vaccines elicit Th1 dominated response, which can assist in good prognosis of infection in CF patients.

Patents in chemotherapy: nanoparticles as drug-delivery vehicles.

The high statistics of cancer cases and mortalities throughout the world signify the urgent need for an advanced technology to target tumor cells. Nanotechnology has emerged as one such revolutionary platform to specifically target cancerous tissues and to enhance the efficacy for various anticancer drugs. This report is a snapshot of the patents in chemotherapy from January 2010 to May 2020 involving nanoparticles, novel methods developed for their synthesis and their impact as efficient drug-delivery vehicles.

Generation of oxidative stress and induction of apoptotic like events in curcumin and thymoquinone treated adult Fasciola gigantica worms.

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.

Antibiotics versus biofilm: an emerging battleground in microbial communities.

Biofilm is a complex structure of microbiome having different bacterial colonies or single type of cells in a group; adhere to the surface. These cells are embedded in extracellular polymeric substances, a matrix which is generally composed of eDNA, proteins and polysaccharides, showed high resistance to antibiotics. It is one of the major causes of infection persistence especially in nosocomial settings through indwelling devices. Quorum sensing plays an important role in regulating the biofilm formation. There are many approaches being used to control infections by suppressing its formation but CRISPR-CAS (gene editing technique) and photo dynamic therapy (PDT) are proposed to be used as therapeutic approaches to subside bacterial biofim infections, especially caused by deadly drug resistant bad bugs.

Identification of factors involved in Enterococcus faecalis biofilm under quercetin stress.

Enterococcus faecalis is a gram positive enteric commensal bacteria or opportunistic pathogen and its infection involves biofilm formation. Quercetin, a plant origin polyphenol was found to inhibit E. faecalis biofilm. Crystal violet assay, SEM and CLSM microscopy confirmed biofilm inhibition by quercetin. Proteomics was used to elucidate the changes occurred in bacterial cell by quercetin treatment. 2D-Electrophorosis and MALDI-TOF analysis revealed that nineteen proteins were differentially expressed in quercetin treated sample. Glycolytic pathways, protein translation-elongation pathways and protein folding pathways were under differential expression after treatment. Real Time-PCR (RT-PCR) validated the proteomic data at genomic level except for the translation elongation factor G which showed opposite data to proteomics. Protein-protein interaction networks constructed using STRING 10.0 demonstrated strong connection of translation-elongation proteins with many important proteins. The results of the comparative analysis indicate that quercetin exerts its inhibitory effect by disturbing glycolytic, protein translation-elongation and protein folding pathways. This disturbs bacterial physiology and stops transition of planktonic cells to biofilm state.

Efficacy of photodynamic therapy against Streptococcus mutans biofilm: Role of singlet oxygen.

In photodynamic therapy (PDT), killing is entirely based on the ROS generation and among different types of ROS generated during PDT, singlet oxygen is considered as the most potential as illustrated in many studies and therefore it is predominantly responsible for photodamage and cytotoxic reactions. The aim of this study was to check whether singlet oxygen (Type II photochemistry) is more potential than free radicals (Type I photochemistry) against Streptococcus mutans biofilm. We have taken two phenothiazinium dyes i.e. toluidine blue O (TBO) and new methylene blue (NMB). TBO was found to have better antibacterial as well as antibiofilm effect than NMB. Antibacterial effect was evaluated by colony forming unit while antibiofilm action by crystal violet and congo red binding assays. We have also evaluated the disruption of preformed biofilm by biofilm reduction assay, confocal laser electron and scanning electron microscopy. More singlet oxygen production was detected in case of TBO than NMB while more Free radical (HO) was produced by NMB than TBO. TBO showed better antibacterial as well as antibiofilm effect than NMB so; we conclude that potency of a photosensitizer is correlated with the capability to produce singlet oxygen.

Retracted: Novel spiro-thiazolidin-4-one and thioether derivatives of benzylidene flavanones: New leads in cancer and microbial chemotherapy.

The above article from Archiv der Pharmazie, published online on 12 March 2018 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor-in-Chief, Prof. Holger Stark, and Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. The retraction has been agreed due to errors in the spectroscopic data of the investigated new compounds. REFERENCE TO RETRACTION: S. Mukhtar, M. A. Alsharif, M. I. Alahmdi, H. Parveen, A. U. Khan, Arch. Pharm. Chem. Life Sci. 2018;1-12. DOI: 10.1002/ardp.201700397.

Enhanced photodynamic therapy using light fractionation against Streptococcus mutans biofilm: type I and type II mechanism.

AIM: The objective of the study was to look the efficacy of fractionated light against Streptococcus mutans biofilm. MATERIALS & METHODS: Antibiofilm assays (crystal violet, congo red), electron microscopic, confocal and spectroscopic studies were performed to check the effect of fractionated light. RESULTS: 6-6.5 log10 reduction of planktonic and 3.6-4.2 log10 reduction in biofilm were observed after irradiation with fractionated as compared with continuous light. Increased permeability to propidium iodide and leakage of cellular constituent validate the greater antibiofilm effect of fractionated light. Spectroscopic studies confirmed the relative contribution of type I and type II photochemistry. CONCLUSION: Phenothiazinium dyes have a potential against bacterial biofilm in combination with light fractionation and it offers new opportunities to explore its clinical implication.

Nanomedicine for anticancer and antimicrobial treatment: an overview.

Nanoparticle-based treatment has become a potential therapeutic approach. The nanosize of these particles provides them with unique physicochemical properties and enhances their interaction with the biological system. Nanomaterials have the potential to overcome some of the major issues in the clinical world which may include cancer treatment and may be utilised to resolve the major problem of drug resistance in infection control. These particles are being used to improve present therapeutics by virtue of their shape, size and diverse intrinsic as well as chemical properties. The authors have discussed the use of nanoparticles in cancer treatment, infections caused by multidrug-resistant microbial strains and biofilm inhibition along with the detailed description of the current status of nanomaterials in the field of medicine.

Anthelmintic Potential of Thymoquinone and Curcumin on Fasciola gigantica.

Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 µM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence potential. Thus, both thymoquinone and curcumin appeared to be promising anthelmintic compounds for further investigations.

A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm.

BACKGROUND: Antimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm. MATERIAL AND METHODS: Enterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm. RESULTS: 8log10 reduction in bacterial count was observed in Enterococcus faecalis while 3log10 in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy. CONCLUSION: We conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells.

Understanding the interaction between human serum albumin and anti-bacterial/ anti-cancer compounds.

Human serum albumin (HSA) is the most important carrier of exogenous and endogenous molecules in human plasma. Understanding and characterizing the interaction of drugs with HSA has attracted enormous research interests from decades. The nature and magnitude of these bindings have direct consequence on drug delivery, pharmacokinetics, pharmacodynamics, therapeutic efficacy and drug designing. An overview of HSA and antibacterial/ anti-cancer ligands interaction is the need of the hour as these drugs together constitute more than half of the total drug consumption in the world. In this review, the information on the number of binding sites, binding strength, the nature of binding interactions and the location of binding sites of such drugs on the HSA are summarised. The effect of such drugs on the overall conformation, stability and function of HSA is also reviewed. This review will help to gain useful insights into the significance of the binding of anti-bacterial and anti-cancer drugs with plasma protein and the effect of binding on its overall distribution and pharmacological activities.

Studies on structure-based sequence alignment and phylogenies of beta-lactamases.

The ß-lactamases enzymes cleave the amide bond in ß-lactam ring, rendering ß-lactam antibiotics harmless to bacteria. In this communication we have studied structure-function relationship and phylogenies of class A, B and D beta-lactamases using structure-based sequence alignment and phylip programs respectively. The data of structure-based sequence alignment suggests that in different isolates of TEM-1, mutations did not occur at or near sequence motifs. Since deletions are reported to be lethal to structure and function of enzyme. Therefore, in these variants antibiotic hydrolysis profile and specificity will be affected. The alignment data of class A enzyme SHV-1, CTX-M-15, class D enzyme, OXA-10, and class B enzyme VIM-2 and SIM-1 show sequence motifs along with other part of polypeptide are essentially conserved. These results imply that conformations of betalactamases are close to native state and possess normal hydrolytic activities towards beta-lactam antibiotics. However, class B enzyme such as IMP-1 and NDM-1 are less conserved than other class A and D studied here because mutation and deletions occurred at critically important region such as active site. Therefore, the structure of these beta-lactamases will be altered and antibiotic hydrolysis profile will be affected. Phylogenetic studies suggest that class A and D beta-lactamases including TOHO-1 and OXA-10 respectively evolved by horizontal gene transfer (HGT) whereas other member of class A such as TEM-1 evolved by gene duplication mechanism. Taken together, these studies justify structure-function relationship of beta-lactamases and phylogenetic studies suggest these enzymes evolved by different mechanisms.

Designing and synthesis of novel antimicrobial heterocyclic analogs of fatty acids.

Novel series of long chain isoxazole derivatives were designed as inhibitors of Cytochrome P450-14DM14a-demethylase from Candida albicans and ribosomal subunit of S12 protein from Escherichia coli. The novel compounds (6-10) were synthesized through 1,3-dipolar cycloaddition of nitrile oxide to long chain alkynoic acid and alkenyl/hydroxyalkenyl esters and tested for their antimicrobial activity by disk diffusion assay and MIC by broth micro dilution method. After predicting the hidden potential and drug-likeness of compounds, ADMET-related descriptors were also calculated to predict pharmacokinetic properties. Molecular docking studies have been performed to evaluate possible mode of action of molecules in active site of receptor. Compounds (9 and 10) showed excellent antimicrobial activity nearly equivalent to the control compounds.