Sustainable photoelectrocatalytic oxidation of antibiotics using Ag-CoFe2O4@TiO2 heteronanostructures for eco-friendly wastewater remediation.

Developing high-performance and durable catalysts presents a significant challenge for oxidizing toxic inorganic and pharmaceutical compounds in wastewater. Recently, there has been a surge in the development of new heterogeneous catalysts for degrading pharmaceutical compounds, driven by advancements in electrocatalysts and photoelectrocatalysts. In this study, a plasmonic Ag nanoparticles decorated CoFe2O4@TiO2 heteronanostructures have been successfully designed to fabricate a high-performing photoelectrode for the oxidation of pharmaceutical compounds. The developed Ag-CoFe2O4@TiO2 possessed a higher electrochemical stability and effectively harvested the UV to visible and NIR radiation in sunlight which generates the enormous photochemical reactive species that involved in the oxidation of ibuprofen in wastewater. Under direct sunlight irradiation, Ag-CoFe2O4@TiO2 achieved complete oxidation of ibuprofen in wastewater at 0.8 V vs RHE. This indicates that metallic Ag nanoparticles are involved in the charge separation and transport of charge carriers from the photoactive sites of CoFe2O4@TiO2, promoting the generation of abundant hydroxy, oxy, and superoxide radicals that actively break the bonds of ibuprofen. Additionally, oxidation agents such as urea and H2O2 were utilized to enhance the formation of superoxide ions and hydroxyl radicals, which rapidly participate in the oxidation of ibuprofen. Significantly, testing for recyclability confirmed the stability of the Ag-CoFe2O4@TiO2 photoanode, ensuring its suitability for prolonged use in photoelectrochemical advanced oxidation processes. Integrating Ag-CoFe2O4@TiO2 photoanodes into water purification systems could enhance economic feasibility, reduce energy consumption, and improve efficiency.

Advancing nanoarchitectures of 2D WO3/MXene photoanode for enhanced photoelectrocatalytic oxidation of phenol and arsenic in synthetic wastewater.

The photoelectrocatalytic advanced oxidation process (PEAOP) necessitates high-performing and stable photoanodes for the effective oxidation of complex pollutants in industrial wastewater. This study presents the construction of 2D WO3/MXene heteronanostructures for the development of efficient and stable photoanode. The WO3/MXene heterostructure features well-ordered WO3 photoactive sites anchored on micron-sized MXene sheets, providing an increased visible light active catalytic surface area and enhanced electrocatalytic activities for pollutant oxidation. Phenol, a highly toxic compound, was completely oxidized at an applied potential of 0.8 V vs. RHE under visible light irradiation. Systematic optimization of operational conditions for the photoelectrocatalytic oxidation of phenol was conducted. The phenol oxidation mechanism was elucidated via high-performance liquid chromatography (HPLC) analysis and the identification of intermediate compounds. Additionally, a mixed model of phenol and arsenic (III) in polluted water demonstrated the capability of WO3/MXene photoanode for the simultaneous oxidation of both organic and inorganic pollutants, achieving complete conversion of phenol and As(III) to non-toxic As(V). The WO3/MXene photoanode facilitated water oxidation, generating a substantial amount of O2•- and •OH oxidative species, which are crucial for the concurrent oxidation of phenol and arsenic. Recyclability tests demonstrated a 99% retention of performance, confirming the WO3/MXene photoanode's suitability for long-term operation in PEAOPs. The findings suggest that integrating WO3/MXene photoanodes into water purification systems can enhance economic feasibility, reduce energy consumption, and improve efficiency. This PEAOP offers a viable solution to the critical issue of heavy metal and organic chemical pollution in various water bodies, given its scalability and ability to preserve ecosystems while conserving clean water resources.

Photoelectrochemical advanced oxidation processes for simultaneous removal of antibiotics and heavy metal ions in wastewater using 2D-on-2D WS2@CoFe2O4 heteronanostructures.

The presence of antibiotics in water poses significant threats to both human health and the environment. Addressing this issue requires the effective treatment of medical wastewater. Photoelectrochemical advanced oxidation processes (PEAOPs) are emerging as promising solutions for wastewater treatment. This process utilizes photocatalysts to convert charge carriers into reactive species such as hydroxyl radicals and superoxide ions, which are essential for degrading pollutants in wastewater. However, limitations in charge carrier separation and transport have hindered the efficiency of photoelectrochemical advanced oxidation processes. To overcome these limitations, we designed WS2@CoFe2O4 heterojunctions, optimizing their energy levels to enhance charge transport and separation. This improvement significantly increased the oxidation of antibiotics such as amoxicillin and azithromycin. Multiple reactions occurred at the WS2@CoFe2O4 heterojunctions during photoelectrochemical advanced oxidation processes, leading to the impressive degradation of up to 99% of antibiotics under visible light irradiation at 0.8 V. Urea and H2O2 acted as oxidation agents within photoelectrochemical advanced oxidation processes, amplifying the generation of hydroxyl radicals and superoxide ions, further enhancing antibiotic oxidation. Moreover, the WS2@CoFe2O4 photoanode efficiently oxidized toxic antibiotics while converting As(III) into the less harmful As(V). Crucially, recyclability tests confirmed the robustness of the WS2@CoFe2O4 photoanode, ensuring its suitability for prolonged use in photoelectrochemical advanced oxidation processes. Integrating WS2@CoFe2O4 photoanodes into water purification systems can enhance efficiency, reduce energy consumption, and improve economic viability. This technology's scalability and its ability to protect ecosystems while conserving water resources make it a promising solution for addressing the critical issue of antibiotic pollution in water environments.

Mobile phone ingestion requiring endoscopic retrieval.

A 24-year-old male was brought to our emergency with complaints of abdominal pain for two days. There was a history of foreign body ingestion five days earlier, details of which he refused to reveal. After investigation with abdominal X ray and ultrasound, the foreign body was detected to be a mobile phone containing a battery. Clinical evaluation revealed no signs of lithium toxicity due to battery leak. The patient underwent endoscopy for removal of the mobile phone. The case shows the importance of thorough investigation and prompt attempt at endoscopic removal in the event of ingestion of foreign bodies containing batteries.

Prosthetic valve thrombosis - association of genetic polymorphisms of VKORC1, CYP2C9 and CYP4F2 genes.

Prosthetic Valve Thrombosis (PVT), in spite of the advances in the valve design and the material used, remains a serious complication of mechanical cardiac valve replacement. The factors influencing the development of PVT are: thrombogenicity of the valve, hemodynamics of the transprosthetic blood flow and ineffective anticoagulation. Genetic polymorphism of the genes VKORC1 (-1639 G > A and 1173 C > T), CYP2C9 (*2 & *3 alleles) and CYP4F2 (1347 G > A) are known to influence the anticoagulant dose-effect response. Since there has not been any earlier study on the direct influence of gene polymorphism on the development of PVT, we investigated into this association.Genotyping for the genes VKORC1, CYP2C9 and CYP4F2 was carried out by conventional PCR-RFLP method for 91 consecutive PVT patients. Subjects of our earlier study served as controls (n = 136).Female patients and patients with smaller prosthetic valve size were more prone to developing PVT (68%, n = 62). Patients bearing A allele of CYP4F2 1347 G > A polymorphism exhibited a fivefold increased risk of PVT (OR = 5.022 (1.39-18.04), P = .013). G allele of VKORC1 when analyzed in combination of genotypes showed a fourteen fold increased risk for developing PVT (OR = 14.25 (5.52-36.77), P = 0.001). CYP2C9 (*2&*3) gene polymorphism did not show any significant association with PVT (OR = 1.54 (0.128 - 18.82), P = .731).Patients bearing A allele of CYP4F2 showed an increased risk of developing PVT in our case - control study.

Mango leaf extract incorporated chitosan antioxidant film for active food packaging.

Health hazards associated with usage of plastic films for food preservation demands for development of active films from non-toxic and antioxidant rich bio-sources. The reported work highlights the development, characterization and application studies of chitosan films enhanced for their antioxidant activity by mango leaf extract (MLE) incorporation. Effect of MLE variation (1-5%) on the morphology, optical nature, water exposure and mechanical characteristics of the chitosan-MLE composite films was studied. Increase in the MLE concentration resulted in films with increased thickness and decreased moisture content. Contact angle, water solubility and vapor permeability analysis demonstrated the reduced hydrophilicity and water vapor penetrability of the films due to MLE inclusion. MLE films possessed better tensile strength (maximum of 23.06 ±â€¯0.19 MPa) with reduced elongation ratio than the pure chitosan film (18.14 ±â€¯0.72 MPa). Antioxidants assessment in terms of total phenolic content, DPPH radical scavenging, ferric reducing power and ABTS radical scavenging showed improved antioxidant activity with the incremental amounts of MLE in the chitosan films. Microscopic studies revealed the smooth, compact and dense nature of the MLE-chitosan films favouring low oxygen transport rates. Application studies to cashew nuts preservation for 28 days storage indicated 56% higher oxidation resistance for the 5% MLE film than a commercial polyamide/polyethylene film. Results highlight the potential and promising nature of MLE impregnated chitosan films as suitable alternative for active packaging films for food preservation.

Efficacy of Low-level Laser Therapy, Hyaluronic Acid Gel, and Herbal Gel as Adjunctive Tools in Gingivectomy Wound Healing: A Randomized Comparative Clinical and Histological Study.

Introduction Gingival overgrowth is usually an inflammatory response to plaque present on tooth surfaces. The other causes could be drugs and other systemic conditions. When the local factors are responsible and subgingival scaling does not help, gingivectomy is performed. The gingivectomy wound is raw and heals slowly. Low-level laser therapy (LLLT), hyaluronic acid, and herbal gels aid in healing after a gingivectomy. This study compared the efficacy of LLLT, hyaluronic acid, and herbal gel when used topically after a gingivectomy. This was a single-arm, interventional trial wherein 30 patients aged between 18 and 40 years with moderate gingival overgrowth participated. The study was approved by the institutional ethical committee. (DN/0109-16). The participants signed the consent form and the study was also registered (NCT03569683). Materials and methods The samples were equally divided into three groups. Group A received LLLT immediately postop, and on the first, third, and seventh days after surgery. Group B received hyaluronic acid (Gengigel) while Group C received an herbal gel (Hiora SG) for the same time periods after surgery, respectively. Analysis of variance (ANOVA) followed by a post-hoc Bonferroni test was used to evaluate differences within groups. Intergroup comparison was performed using the independent t-test. A p-value of <0.05 was considered statistically significant. Results The plaque index (PI), gingival index (GI), and gingival enlargement index (GEI) showed good improvement postoperatively within the groups, which was statistically significant. However, on an intergroup comparison, the GEI pertaining only to the laser group showed significant changes. Also, the pain perception analyzed by the visual analog score (VAS) was the least, and histologically, the amount of mature collagen fibers laid down were more in the laser group. Conclusion Patients irradiated with laser after gingivectomy (Group A) showed superior results in the clinical, histological variables as compared to Groups B and C.

Design, synthesis and biological evaluation of novel pyrazolochalcones as potential modulators of PI3K/Akt/mTOR pathway and inducers of apoptosis in breast cancer cells.

Cancer has been established as the "Emperor of all maladies". In recent years, medicinal chemistry has focused on identifying novel anti-cancer compounds; though discovery of these compounds appears to be a herculean task. In present study, we synthesized forty pyrazolochalcone conjugates and explored their cytotoxic activity against a panel of sixty cancer cell lines. Fifteen conjugates of the series showed excellent growth inhibition (13b-e, 13h-j, 14c-d, 15 a, 15 c-d, 16b, 16d and 18f; GI50 for MCF-7: 0.4-20 µM). Conjugates 13b, 13c, 13d, 16b and 14d were also evaluated for their cytotoxic activity in human breast cancer cell line (MCF-7). The promising candidates induced cell cycle arrest, mitochondrial membrane depolarization and apoptosis in MCF-7 cells at a 2 µM concentration. Furthermore, inhibition of PI3K/Akt/mTOR pathway-regulators such as PI3K, p-PI3K, p-AKT, and mTOR were observed; as well as upregulation of p-GSK3ß and tumor-suppressor protein, PTEN. Our study indicates that pyrazolochalcone conjugates could serve as potential leads in the development of tailored cancer therapeutics.

2-Anilino-3-Aroylquinolines as Potent Tubulin Polymerization Inhibitors.

Several 2-anilino-3-aroylquinolines were designed, synthesized, and screened for their cytotoxic activity against five human cancer cell lines: HeLa, DU-145, A549, MDA-MB-231, and MCF-7. Their IC50 values ranged from 0.77 to 23.6 µm. Among the series, compounds 7 f [(4-fluorophenyl)(2-((4-fluorophenyl)amino)quinolin-3-yl)methanone] and 7 g [(4-chlorophenyl)(2-((4-fluorophenyl)amino)quinolin-3-yl)methanone] showed remarkable antiproliferative activity against human lung cancer and prostate cancer cell lines. The IC50 values for inhibiting tubulin polymerization were 2.24 and 2.10 µm for compounds 7 f and 7 g, respectively, and were much lower than that of the reference compound E7010 [N-(2-(4-hydroxyphenylamino)pyridin-3-yl)-4-methoxybenzenesulfonamide]. Furthermore, flow cytometric analysis revealed that these compounds arrest the cell cycle at the G2 /M phase, leading to apoptosis. Apoptosis was also confirmed by mitochondrial membrane potential, Annexin V-FITC assay, and intracellular ROS generation. Immunohistochemistry, western blot, and tubulin polymerization assays showed that these compounds disrupt tubulin polymerization. Molecular docking studies revealed that these compounds bind efficiently to ß-tubulin at the colchicine binding site.

Design, synthesis of phenstatin/isocombretastatin-oxindole conjugates as antimitotic agents.

A series of phenstatin/isocombretastatin-oxindole conjugates was synthesized and tested for their cytotoxic activity against five human cancer cells such as prostate (DU-145), lung (A549), colon (HT-29), breast (MCF-7), liver (HepG2) cancer cells with IC50 values ranging from 0.049 to 38.90 µM. Amongst them, two conjugates (5c and 5d) showed broad spectrum of antiproliferative efficacy on lung cancer cells with an IC50 value of 79 nM and 93 nM, respectively, whereas on colon cancer cells with an IC50 values 45 nM and 49 nM, respectively. In addition, cell cycle assay revealed that these conjugates (5c and 5d) arrest at the G2/M phase and leads to apoptotic cell death which was confirmed by Annexin V-FITC and mitochondrial membrane depolarization. Further, the tubulin polymerization assay analysis results suggest that these conjugates particularly 5c and 5d exhibit significant inhibitory effect on the tubulin assembly with an IC50 value of 1.23 µM and 1.01 µM, respectively. Molecular docking studies indicated that these compounds (5c and 5d) occupy the colchicine binding site of the tubulin.

Combretastatin linked 1,3,4-oxadiazole conjugates as a Potent Tubulin Polymerization inhibitors.

A new class of combretastatin linked 1,3,4-oxadiazoles were designed, synthesized and screened for their cytotoxic activity against five human cancer cell lines such as HeLa, DU-145, A549, MDA-MB-231 and B16. These compounds showed significant cytotoxicity with IC50 values in the range 0.118-54.32µM. Conjugate 5m displayed potent antiproliferative activity against DU-145 cell line. Flow cytometric analysis revealed that these compounds arrested the cell cycle in G2/M phase. Moreover, the tubulin polymerization assay and immunofluorescence analysis indicate that 5m exhibits potent inhibitory effect on the tubulin assembly. Further, DNA fragmentation and Hoecst staining assays confirm that 5m induces apoptosis. Molecular docking studies and competitive binding assay indicated that 5m effectively bind at the colchicine binding site of the tubulin.

Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4 (CDK4) inhibitors by catalyst-free, green approach.

Aiming to develop a new target for the anticancer treatment, a series of 5'H-spiro[indoline-3,4'-pyrrolo [1,2-a]quinoxalin]-2-ones has been synthesized by simple, highly efficient and environmentally friendly method in excellent yields under catalyst-free conditions using ethanol as a green solvent. A simple filtration of the reaction mixture and subsequent drying affords analytically pure products. The synthesized derivatives were evaluated for their antiproliferative activity against five different human cancer cell lines, among the congeners compound 3n showed significant cytotoxicity against the human prostate cancer (DU-145). Flow cytometric analysis revealed that this compound induces cell cycle arrest in the G0/G1 phase and Western blot analysis suggested that reduction in Cdk4 expression level leads to apoptotic cell death. This was further confirmed by mitochondrial membrane potential ((ΔΨm), Annexin V-FITC assay and docking experiments. Furthermore, it was observed that there is an increase in expression levels of cyclin dependent kinase inhibitors like Cip1/p21 and Kip1/p27.

Synthesis of phenstatin/isocombretastatin-chalcone conjugates as potent tubulin polymerization inhibitors and mitochondrial apoptotic inducers.

A series of phenstatin/isocombretastatin­chalcones were synthesized and screened for their cytotoxic activity against various human cancer cell lines. Some representative compounds exhibited significant antiproliferative activity against a panel of sixty human cancer cell lines of the NCI, with GI50 values in the range of 0.11 to 19.0 µM. Three compounds (3b, 3c and 3e) showed a broad spectrum of antiproliferative efficacy on most of the cell lines in the sub-micromolar range. In addition, all the synthesized compounds (3a­l and 4a­l) displayed moderate to excellent cytotoxicity against breast cancer cells such as MCF-7 and MDA-MB-231 with IC50 values in the range of 0.5 to 19.9 µM. Moreover, the tubulin polymerization assay and immunofluorescence analysis results suggest that some of these compounds like 3c and 3e exhibited significant inhibitory effect on the tubulin assembly with an IC50 value of 0.8 µM and 0.6 µM respectively. A competitive binding assay suggested that these compounds bind at the colchicine-binding site of tubulin. A cell cycle assay revealed that these compounds arrest at the G2/M phase and lead to apoptotic cell death. Furthermore, this was confirmed by Hoechst 33258 staining, activation of caspase 9, DNA fragmentation, Annexin V-FITC and mitochondrial membrane depolarization. Molecular docking studies indicated that compounds like 3e occupy the colchicine binding site of tubulin.

Synthesis of arylpyrazole linked benzimidazole conjugates as potential microtubule disruptors.

In an attempt to develop potent and selective anticancer agents, a series of twenty arylpyrazole linked benzimidazole conjugates (10a-t) were designed and synthesized as microtubule destabilizing agents. The joining of arylpyrazole to the benzimidazole moiety resulted in a four ring (A, B, C and D) molecular scaffold that comprises of polar heterocyclic rings in the middle associated with rotatable single bonds and substituted aryl rings placed in the opposite directions. These conjugates were evaluated for their ability to inhibit the growth of sixty cancer cell line panel of the NCI. Among these some conjugates like 10a, 10b, 10d, 10e, 10p and 10r exhibited significant growth inhibitory activity against most of the cell lines ranging from 0.3 to 13µM. Interestingly, the conjugate 10b with methoxy group on D-ring expressed appreciable cytotoxic potential. A549 cells treated with some of the potent conjugates like 10a, 10b and 10d arrested cells at G2/M phase apart from activating cyclin-B1 protein levels and disrupting microtubule network. Moreover, these conjugates effectively inhibited tubulin polymerization with IC50 values of 1.3-3.8µM. Whereas, the caspase assay revealed that they activate the casepase-3 leading to apoptosis. Particularly 10b having methoxy substituent induced activity almost 3 folds higher than CA-4. Furthermore, a competitive colchicine binding assay and molecular modeling analysis suggests that these conjugates bind to the tubulin successfully at the colchicine binding site. These investigations reveal that such conjugates having pyrazole and benzimidazole moieties have the potential in the development of newer chemotherapeutic agents.

Synthesis of (Z)-(arylamino)-pyrazolyl/isoxazolyl-2-propenones as tubulin targeting anticancer agents and apoptotic inducers.

A new class of pyrazole and isoxazole conjugates were synthesized and evaluated for their cytotoxic activity against various human cancer cell lines. These compounds have shown significant cytotoxicity with lower IC50 values. FACS results revealed that A549 cells treated with these compounds arrested cells at the G2/M phase of the cell cycle apart from activating cyclin B1 protein levels. Particularly, compounds 9a and 9b demonstrated a remarkable inhibitory effect on tubulin polymerization and showed a pronounced inhibitory effect on tubulin polymerization with IC50 values of 1.28 µM and 0.28 µM respectively, whereas nocodazole, a positive control, has shown lower antitubulin activity with an IC50 value of 2.64 µM. Furthermore, these compounds induced apoptosis by loss of mitochondrial membrane potential, propidium iodide (PI) staining and the activation of caspase-3. Results of a fluorescence based competitive colchicine binding assay suggest that these conjugates bind successfully at the colchicine binding site of tubulin. These investigations reveal that such conjugates containing pyrazole with a trimethoxy phenyl ring and indole moieties have potential for the development of newer chemotherapeutic agents.

Pyrazole-oxadiazole conjugates: synthesis, antiproliferative activity and inhibition of tubulin polymerization.

A number of pyrazole-oxadiazole conjugates were synthesized and evaluated for their ability to function as antiproliferative agents on various human cancer cell lines. These conjugates are comprised of pyrazole and oxadiazole scaffolds closely attached to each other without any spacer as two structural classes. The Type I class has a trimethoxy substituent and the type II class has a 3,4-(methylenedioxy) substituent on their A rings. Among these conjugates 11a, 11d and 11f manifest potent cytotoxicity with IC50 values ranging from 1.5 µM to 11.2 µM and inhibit tubulin polymerization with IC50 values of 1.3 µM, 3.9 µM and 2.4 µM respectively. The cell cycle assay showed that treatment with these conjugates results in accumulation of cells in the G2/M phase and disrupts the microtubule network. Elucidation of zebrafish embryos revealed that the conjugates cause developmental defects. Molecular docking simulations determined the binding modes of these potent conjugates at the colchicine site of tubulin.

Design and synthesis of aminostilbene-arylpropenones as tubulin polymerization inhibitors.

A series of aminostilbene-arylpropenones were designed and synthesized by Michael addition and were investigated for their cytotoxic activity against various human cancer cell lines. Some of the investigated compounds exhibited significant antiproliferative activity against a panel of 60 human cancer cell lines of the US National Cancer Institute, with 50 % growth inhibition (GI50) values in the range from < 0.01 to 19.9 µM. One of the compounds showed a broad spectrum of antiproliferative efficacy on most of the cell lines, with a GI50 value of < 0.01 µM. All of the synthesized compounds displayed cytotoxicity against A549 (non-small-cell lung cancer), HeLa (cervical carcinoma), MCF-7 (breast cancer), and HCT116 (colon carcinoma) with 50 % inhibitory concentration (IC50) values ranging from 0.011 to 8.56 µM. A cell cycle assay revealed that these compounds arrested the G2/M phase of the cell cycle. Two compounds exhibited strong inhibitory effects on tubulin assembly with IC50 values of 0.71 and 0.79 µM. Moreover, dot-blot analysis of cyclin B1 demonstrated that some of the congeners strongly induced cyclin B1 protein levels. Molecular docking studies indicated that these compounds occupy the colchicine binding site of tubulin.

Synthesis and biological evaluation of imidazopyridine-oxindole conjugates as microtubule-targeting agents.

A library of imidazopyridine-oxindole conjugates was synthesised and investigated for anticancer activity against various human cancer cell lines. Some of the tested compounds, such as 10 a, 10 e, 10 f, and 10 k, exhibited promising antiproliferative activity with GI50 values ranging from 0.17 to 9.31 µM. Flow cytometric analysis showed that MCF-7 cells treated by these compounds arrested in the G2 /M phase of the cell cycle in a concentration-dependent manner. More particularly, compound 10 f displayed a remarkable inhibitory effect on tubulin polymerisation. All the compounds depolarised mitochondrial membrane potential and caused apoptosis. These results are further supported by the decreased phosphorylation of Akt at Ser473. Studies on embryonic development revealed that the lead compounds 10 f and 10 k caused delay in the development of zebra fish embryos. Docking of compound 10 f with tubulin protein suggested that the imidazo[1,2-a]pyridine moiety occupies the colchicine binding site of tubulin.

Synthesis of terphenyl benzimidazoles as tubulin polymerization inhibitors.

A series of new terphenyl benzimidazoles (3a-z and 3aa-ad) were synthesized and evaluated for their anticancer activity. All the 30 compounds have shown moderate to good anticancer potency, however some of the compounds (3j, 3m-t and 3aa-ad) exhibited prominent anticancer potency with GI(50) values ranging from <0.1 to 9.72 µM. These compounds exhibit G2/M phase arrest and the analysis of tubulin by Western blot experiments in case of 3t and 3ad shows the disturbances that are caused in the ratio of soluble versus polymerized tubulin in cells. Compounds 3t and 3ad are the most promising candidates amongst the series and has the potential to be taken up for further detailed studies either alone or in combination with the existing therapies.