Leishmania donovani modulates host miRNAs regulating cholesterol biosynthesis for its survival.

Cholesterol reduction by intracellular protozoan parasite Leishmania donovani (L. donovani), causative agent of leishmaniasis, impairs antigen presentation, pro-inflammatory cytokine secretion and host-protective membrane-receptor signaling in macrophages. Here, we studied the miRNA mediated regulation of cholesterol biosynthetic genes to understand the possible mechanism of L. donovani-induced cholesterol reduction and therapeutic importance of miRNAs in leishmaniasis. System-scale genome-wide microtranscriptome screening was performed to identify the miRNAs involved in the regulation of expression of key cholesterol biosynthesis regulatory genes through miRanda3.0. 11 miRNAs out of 2823, showing complementarity with cholesterol biosynthetic genes were finally selected for expression analysis. These selected miRNAs were differentially regulated in THP-1 derived macrophages and in primary human macrophages by L. donovani. Correlation of expression and target validation through luciferase assay suggested two key miRNAs, hsa-miR-1303 and hsa-miR-874-3p regulating the key genes hmgcr and hmgcs1 respectively. Inhibition of hsa-mir-1303 and hsa-miR-874-3p augmented the expression of targets and reduced the parasitemia in macrophages. This study will also provide the platform for the development of miRNA-based therapy against leishmaniasis.

Rehabilitating a severely worn dentition with removable prosthodontics.

In the next part of this series on tooth wear management, we discuss the indications and clinical stages for the provision of removable prostheses for the treatment of severely worn and depleted dentitions. The general design features of a complex prosthesis are described for reorganised occlusal schemes and maintenance guidelines are explained. In addition, the clinical stages for three different situations are described: removable-only approaches, and combined fixed and removable in the same arch and separate arches. The value of providing removable prostheses in worn dentitions allows the immediate rehabilitation of severely worn teeth taking a non-invasive and retrievable approach when the remaining dentition is of poor quality or structure and/or there are missing teeth.

Suspected Endodontic Failure in a Patient with Cleidocranial Dysplasia: A Case Report.

A patient's medical history and related dental manifestations can significantly contribute to confounding signs and symptoms leading to a diagnostic challenge. An 18-year-old female patient presented with persistent radiographic radiolucency associated with the apex of a previously treated tooth (tooth 9); asymptomatic apical periodontitis and endodontic failure were suspected. This report presents how a patient's condition of cleidocranial dysplasia had a profound effect on her dental history, which included the presence of multiple supernumerary teeth. Extensive surgical intervention during the patient's childhood was required to remove the supernumerary teeth, which resulted in an endodontic misdiagnosis in her adult life. After clinical and radiographic examination, the patient was diagnosed with a periapical scar. Periapical fibrous scars have a prevalence of between 2.5% and 12% and are a rare healing process with fibrous tissue after surgical and nonsurgical interventions. This report describes the diagnosis and pathophysiology of fibrous scars, including their risk factors and long-term monitoring approaches.

Recent advancements in fusion protein technologies in oncotherapy: A review.

Cancer is a complicated, adaptable, and heterogeneous disease caused by a wide variety of genetic changes that might impair ability of cells to function normally. The majority of the tumors can only be shrunk using conventional oncology therapies like chemotherapy, radiation, and surgical resection, and the tumor often recurs. The inability of conventional cancer therapies to completely destroy the Cancer Stem Cells (CSCs) that otherwise lead to therapy resistance is thus addressed by therapeutic approaches that concentrate on targeting CSCs and their micro-environmental niche. In this review, we summarize approaches that are used for the development of fusion proteins and their therapeutic applications for treating cancer. The main purpose of making advancements towards the fusion technology instead of using conventional treatment methods is to achieve a prolonged half-life of the therapeutic drugs. The fusion of drugs to the immune response enhancing cytokines or the fusion of antibody and cytokines not only increases half-life but also increase the stability of the anti-tumor drug. Several molecules including different fragments of antibodies, cytokines, Human Serum Albumin, transferrin, XTEN polymers, Elastin-like polypeptides (ELPs) can be employed as a fusion partner and the resulting fusion proteins are reported to show enhanced anti-tumor response.

The dental demolition derby: bruxism and its impact - part 3: repair and reconstruction.

Bruxism is a term that encompasses a range of presentations of rhythmic and repetitive muscular activity. For many, this is not a significant problem but for some, this behaviour leads to substantial impact and tissue damage that can be significant, compromising function and quality of life. This paper will review management methods for reconstructing the damaged dentition.

The dental demolition derby: bruxism and its impact - part 2: early management of bruxism.

Bruxism is a term that encompasses a range of presentations of rhythmic and repetitive muscular activity. For many, this is not a significant problem but for some, the behaviour leads to significant problems and extensive tissue damage. This is different to temporomandibular disorders. This paper will review methods of managing cases where bruxism is destructive, or potentially destructive, before needing to resort to full reconstruction.

The dental demolition derby: bruxism and its impact - part 1: background.

Bruxism may lead to changes or damage to the oral and perioral tissues. Bruxism may occur during sleep or when awake. Many patients will not require active management; however, for some, intervention is required. Control of bruxism may be difficult, if not impossible, but the need exists for preservation of the dentition and quality of life. A prediction of risk to the tissues for the planning of interventions is difficult and relies upon evidence of past damage and assessment of future risks. Treatment options may need to be imaginative and rescuable. This series of papers will review the aetiology of bruxism, its impacts and treatment strategies for persistent bruxers who are at risk of, or suffering, tissue damage.

Exploration of potential inhibitors for autophagy-related protein 8 as antileishmanial agents.

Leishmaniasis is considered a tropical neglected disease, which is caused by an intramacrophagic parasite, Leishmania. It is endemic in 89 different countries. Autophagy-related protein 8 (Ldatg8) is responsible for the transformation of parasites from promastigote to amastigote differentiation. Ldatg8 is one of the key drug targets of Leishmania donovani (L. donovani) responsible for the defense of parasites during stress conditions. Virtual screening of natural ligand library had been performed against Ldatg8 to identify novel and potent inhibitors. Molecular docking and molecular dynamics simulation studies showed that urolithin A stably blocked Ldatg8. Urolithins are combinations of coumarin and isocoumarin. Further, we evaluated the antileishmanial effects of urolithin A by antileishmanial assays. Urolithin A inhibited the growth and proliferation of L. donovani promastigotes with an IC50  value of 90.3 ± 6.014 µM. It also inhibited the intramacrophagic parasite significantly with an IC50  value of 78.67 ± 4.62 µM. It showed limited cytotoxicity to the human THP-1 differentiated macrophages with a CC50  value of 190.80 ± 16.89 µM. Further, we assayed reactive oxygen species (ROS) generation and annexin V/PI staining upon urolithin A treatment of parasites to have an insight into the mechanism of its action. It induced ROS significantly in a dose-dependent manner, which caused apoptosis partially in parasites. The potential inhibitors for Ldatg8, identified in this study, would provide the platform for the development of an effective and affordable antileishmanial drug.

Zygomatic implant-supported prosthodontic rehabilitation of edentulous patients with a history of cleft palate: A clinical report.

Zygomatic implants are an established treatment option in the management of the atrophic maxilla and in oncology rehabilitation, but evidence for their use in patients with a history of cleft palate is sparse. Zygomatic implants were used to retain a maxillary prosthesis in 7 edentulous patients with an unrepaired or repaired cleft lip and palate. Patient records were reviewed retrospectively to assess the survival rates. The mean follow-up time was 5 years with an implant survival of 100%. Most complications were associated with the prosthetic superstructures. This clinical report demonstrates that zygomatic implants can be successfully used to provide a maxillary prosthesis in patients with a history of cleft palate.

Sesamol Induces Apoptosis-Like Cell Death in Leishmania donovani.

Background: Visceral leishmaniasis (VL), caused by the protozoan parasite Leishmania donovani (L. donovani), is the most severe form of leishmaniasis. It is largely responsible for significant morbidity and mortality in tropical and subtropical countries. Currently, available therapeutics have lots of limitations including high-cost, adverse side-effects, painful route of administration, less efficacy, and resistance. Therefore, it is time to search for cheap and effective antileishmanial agents. In the present work, we evaluated the antileishmanial potential of sesamol against promastigotes as well as intracellular amastigotes. Further, we tried to work out its mechanism of antileishmanial action on parasites through different assays. Methodology: In vitro and ex vivo antileishmanial assays were performed to evaluate the antileishmanial potential of sesamol on L. donovani. Cytotoxicity was determined by MTT assay on human THP-1-derived macrophages. Sesamol-induced morphological and ultrastructural changes were determined by electron microscopy. H2DCFDA staining, JC-1dye staining, and MitoSOX red staining were performed for reactive oxygen assay (ROS), mitochondrial membrane potential, and mitochondrial superoxide, respectively. Annexin V/PI staining for apoptosis, TUNEL assay, and DNA laddering for studying sesamol-induced DNA fragmentation were performed. Conclusions: Sesamol inhibited the growth and proliferation of L. donovani promastigotes in a dose-dependent manner. It also reduced the intracellular parasite load without causing significant toxicity on host-macrophages. Overall, it showed antileishmanial effects through induction of ROS, mitochondrial dysfunction, DNA fragmentation, cell cycle arrest, and apoptosis-like cell death to parasites. Our results suggested the possible use of sesamol for the treatment of leishmaniasis after further in vivo validations.

Embilica officinalis L. inhibits the growth and proliferation of Leishmania donovani through the induction of ultrastructural changes, mitochondrial dysfunction, oxidative stress and apoptosis-like cell death.

Visceral leishmaniasis (VL) is caused by a protozoan parasite, Leishmania donovani (L. donovani). It affects around 1-2 million people around the world annually. There is an urgent need to investigate new medicament of it due to difficult method of drug administration, long period of treatment, high cost of the drug, adverse side-effects, low efficacy and development of parasite resistance to the available drugs. Medicinal plants have also been used for the treatment of different diseases in traditional system of medicines due to their holistic effects. The Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland has already started the program for identification of potential medicinal plant and plant products having antileishmanial potential. Keeping all these in consideration, we planned to study the antileishmanial activity of one of the medicinal plant, Embilica officinalis L. (EO) fruit extract. EO fruit extract inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes in dose-dependent manner. EO fruit extract induced morphological and ultrastructural changes in parasites as observed under Electron Microscope. It also induced the oxidative stress, mitochondrial dysfunction, DNA laddering and apotosis-like cell death in parasites. Here, we for the first time reported such a detailed mechanism of action of antileishmanial activity of EO fruit extract. Our results suggested that EO fruit extract could be used for the development of new phytomedicine against leishmaniasis.

Virtual screening of natural compounds for potential inhibitors of Sterol C-24 methyltransferase of Leishmania donovani to overcome leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by trypanosomatid parasite belonging to the genera Leishmania. Leishmaniasis is transmitted from one human to other through the bite of sandflies. It is endemic in around 98 countries including tropical and subtropical regions of Asia, Africa, Southern America, and the Mediterranean region. Sterol C-24 methyltransferase (LdSMT) of Leishmania donovani (L. donovani) mediates the transfer of CH3-group from S-adenosyl methionine to C-24 position of sterol side chain which makes the ergosterol different from cholesterol. Absence of ortholog in human made it potential druggable target. Here, we performed virtual screening of library of natural compounds against LdSMT to identify the potential inhibitor for it and to fight leishmaniasis. Gigantol, flavan-3-ol, and parthenolide showed the best binding affinity towards LdSMT. Further, based on absorption, distribution, metabolism, and excretion properties and biological activity prediction, gigantol showed the best lead-likeness and drug-likeness properties. Therefore, we further elucidated its antileishmanial properties. We found that gigantol inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes. Gigantol exerted its antileishmanial action through the induction of reactive oxygen species in dose-dependent manner. Our study, suggested the possible use of gigantol as antileishmanial drug after further validations to overcome leishmaniasis.

Hesperidin Targets Leishmania donovani Sterol C-24 Reductase to Fight against Leishmaniasis.

Hesperidin, a naturally occurring flavanoid, is present in citrus family of fruits. It was found effective against an array of pathogens including fungi, bacteria, viruses, and protozoa. Here, we evaluated its antileishmanial activity and possible mechanism of action through different in vitro and in silico experiments. It inhibited the growth and proliferation of the parasites significantly with a IC50 value of 1.019 ± 0.116 mM in vitro. It also reduced the growth of intra-macrophagic amastigotes with a IC50 value of 0.2858 ± 0.01398 mM. It induced the reactive oxygen species (ROS) in parasites in a dose-dependent manner. Through 2,7-dichloro dihydro fluorescein diacetate (H2DCFDA) staining, it was observed that around 96.9% parasites were ROS positive at 2.0 mM concentration of hesperidin. The ROS generated led to the apoptosis of parasites in a dose-dependent manner as observed by annexin/PI staining. Molecular docking with one of the very important and unique drug-targets of Leishmania donovani sterol C-24 reductase resulted in its significant inhibition. Here, we for the first time showed that hesperidin induced the antileishmanial response through the induction of apoptosis and inhibition of sterol C-24 reductase. Our study will be helpful in the development of a cost-effective antileishmanial lead with higher efficacy.

Antileishmanial Evaluation of Bark Methanolic Extract of Acacia nilotica: In Vitro and In Silico Studies.

Acacia nilotica (A. nilotica) is an important medicinal plant, found in Africa, the Middle East, and the Indian subcontinent. Every part of the plant possesses a wide array of biologically active and therapeutically important compounds. We reported the antileishmanial activity of A. nilotica bark methanolic extract through in vitro antileishmanial assays and dissected the mechanism of its action through in silico studies. Bark methanolic extract exhibited antipromastigote and antiamastigote potential in a time and dose-dependent manner with IC50 values of 19.6 ± 0.9037 and 77.52 ± 5.167 µg/mL, respectively. It showed cytotoxicity on THP-1-derived human macrophages at very high dose with a CC50 value of 432.7 ± 7.71 µg/mL. The major constituents identified by gas chromatography-mass spectrometry (GC-MS) analysis, 13-docosenoic acid, lupeol, 9,12-octadecadienoic acid, and 6-octadecanoic acid, showed effective binding with the potential drug targets of Leishmania donovani (L. donovani) including sterol 24-c-methyltransferase, trypanothione reductase, pteridine reductase, and adenine phosphoribosyltransferase, suggesting the possible mechanism of its antileishmanial action. Pharmacokinetic studies on major phytoconstituents analyzed by GC-MS supported their use as safe antileishmanial drug candidates. This study proved the antileishmanial potential of bark methanolic extract A. nilotica and its mechanism of action through the inhibition of potential drug targets of L. donovani.

Targeting sterol alpha-14 demethylase of Leishmania donovani to fight against leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by the protozoan parasite Leishmania. It is endemic in more than 89 different countries worldwide. Sterol alpha-14 demethylase (LdSDM), a sterol biosynthetic pathway enzyme in Leishmania donovani, plays an essential role in parasite survival and proliferation. Here, we used a drug repurposing approach to virtually screen the library of the Food and Drug Administration (FDA)-approved drugs against LdSDM to identify the potential lead-drug against leishmaniasis. Zafirlukast and avodart showed the best binding with LdSDM. Zafirlukast was tested for in vitro antileishmanial assay, but no significant effect on L. donovani promastigotes was observed even at higher concentrations. On the other hand, avodart profoundly inhibited parasite growth at relatively lower concentrations. Further, avodart showed a significant decrease in the number of intra-macrophagic amastigotes. Avodart-induced reactive oxygen species (ROS) in the parasites in a dose-dependent manner. ROS induced by avodart led to the induction of apoptosis-like cell death in the parasites as observed through annexin V/PI staining. Here, for the first time, we reported the antileishmanial activity and its possible mechanism of action of FDA-approved drug, avodart, establishing a nice example of the drug-repurposing approach. Our study suggested the possible use of avodart as an effective antileishmanial agent after further detailed validations.

Repurposing of FDA-approved drugs as inhibitors of sterol C-24 methyltransferase of Leishmania donovani to fight against leishmaniasis.

Leishmaniasis is a vector-borne disease caused by around 20 species of Leishmania. The main clinical forms of leishmaniasis are cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). VL is caused by Leishmania infantum in Central and South America, Mediterranean Basin, Middle East, and by L. donovani in Asia and Africa. Sterol C-24 methyltransferase (LdSMT) of L. donovani is a transferase enzyme of the sterol biosynthesis pathway. This pathway is one of the major targets for drug developments in Leishmania. Due to insufficient evidence about the exact function of SMT inside the cell and the uniqueness of the SMT enzyme in the Leishmania parasites made it a significant target for an effective drug development approach. We performed virtual screening of the Food and Drug Administration (FDA)-approved drug library against LdSMT and found simeprevir, an antiviral drug on top in the binding score. It showed a significant binding affinity with LdSMT. The binding was supported by hydrogen bonds and several other interactions. Simeprevir inhibited L. donovani growth of promastigotes with 50% inhibitory concentration (IC50 ) of 51.49 ± 5.87 µM. Further studies showed that simeprevir induced ROS generation in 44.7% of parasites at 125-µM concentration. Here, we for the first time reported simeprevir as an antileishmanial lead molecule using a drug repurposing approach.

Molecular docking analysis of rutin reveals possible inhibition of SARS-CoV-2 vital proteins.

BACKGROUND AND AIM: COVID-19 emerged by the end of 2019 in Wuhan, China. It spreaded and became a public health emergency all over the world by mid of April 2020. Flavonoids are specialized metabolites that have antimicrobial properties including anti-viral activity. Rutin, a medicinally important flavonoid belongs to one of the best natural antioxidant classes. It has antiprotozoal, antibacterial, and antiviral properties. Keeping the antimicrobial potential of rutin in mind, we studied its role in the inhibition of essential proteins of SARS-CoV-2 including main protease (Mpro), RNA-dependent RNA polymerase (RdRp), papain-like protease (PLpro), and spike (S)-protein through different in silico approaches. EXPERIMENTAL PROCEDURE: Molecular docking, inhibition constant, hydrogen bond calculations, and ADMET-properties prediction were performed using different softwares. RESULTS AND CONCLUSION: Molecular docking study showed significant binding of rutin with Mpro, RdRp, PLpro, and S-proteins of SARS-CoV-2. Out of these four proteins, Mpro exhibited the strongest binding affinity with the least binding energy (-8.9 kcal/mol) and stabilized through hydrogen bonds with bond lengths ranging from 1.18 Å to 3.17 Å as well as hydrophobic interactions. The predicted ADMET and bioactivity showed its optimal solubility, non-toxic, and non-carcinogenic properties. The values of the predicted inhibitory constant of the rutin with SARS-CoV-2 vital proteins ranged between 5.66 µM and 6.54 µM which suggested its promising drug candidature. This study suggested rutin alone or in combination as a dietary supplement may be used to fight against COVID-19 after detailed in vitro and in vivo studies.

Assessment of the Antileishmanial Potential of Cassia fistula Leaf Extract.

Cassia fistula has a wide array of biologically active and therapeutically important class of compounds. Leishmania donovani important drug targets, sterol 24-c methyltransferase (LdSMT), trypanothione reductase (LdTR), pteridine reductase (LdPTR1), and nucleoside hydrolase (LdNH), were modelled, and molecular docking was performed against the abundant phytochemicals of its leaf extract. Molecular docking results provided the significant prima facie evidence of the leaf extract to have antileishmanial potential. To confirm this, we performed in vitro antileishmanial and cytotoxicity assays. Methanolic extract of C. fistula leaves showed growth inhibition and proliferation of L. donovani promastigote with an IC50 value of 43.31 ± 4.202 µg/mL. It also inhibited the growth of intra-macrophagic amastigotes with an IC50 value of 80.76 ± 3.626 µg/mL. C. fistula extract was found cytotoxic at a very high concentration on human macrophages (CC50 = 626 ± 39 µg/mL). Annexin V/propidium iodide (PI) staining assay suggested partial apoptosis induction in parasites by C. fistula to exert its antileishmanial activity. Here, for the first time, we have shown the antileishmanial potential of C. fistula leaves. Overall, our results could open new insight for an affordable and natural antileishmanial with high efficacy and less toxicity.

Cynaroside inhibits Leishmania donovani UDP-galactopyranose mutase and induces reactive oxygen species to exert antileishmanial response.

Cynaroside, a flavonoid, has been shown to have antibacterial, antifungal and anticancer activities. Here, we evaluated its antileishmanial properties and its mechanism of action through different in silico and in vitro assays. Cynaroside exhibited antileishmanial activity in time- and dose-dependent manner with 50% of inhibitory concentration (IC50) value of 49.49 ± 3.515 µM in vitro. It inhibited the growth of parasite significantly at only 20 µM concentration when used in combination with miltefosine, a standard drug which has very high toxicity. It also inhibited the intra-macrophagic parasite significantly at low doses when used in combination with miltefosine. It showed less toxicity than the existing antileishmanial drug, miltefosine at similar doses. Propidium iodide staining showed that cynaroside inhibited the parasites in G0/G1 phase of cell cycle. 2,7-dichloro dihydro fluorescein diacetate (H2DCFDA) staining showed cynaroside induced antileishmanial activity through reactive oxygen species (ROS) generation in parasites. Molecular-docking studies with key drug targets of Leishmania donovani showed significant inhibition. Out of these targets, cynaroside showed strongest affinity with uridine diphosphate (UDP)-galactopyranose mutase with -10.4 kcal/mol which was further validated by molecular dynamics (MD) simulation. The bioactivity, ADMET (absorption, distribution, metabolism, excretion and toxicity) properties, Organisation for Economic Co-operation and Development (OECD) chemical classification and toxicity risk prediction showed cynaroside as an enzyme inhibitor having sufficient solubility and non-toxic properties. In conclusion, cynaroside may be used alone or in combination with existing drug, miltefosine to control leishmaniasis with less cytotoxicity.