Towards cyclopalladated therapeutic oligonucleotides

Based on Watson—Crick base pairing principle, therapeutic oligonucleotides bear the potential to either inhibit or alter protein synthesis. Replacing or augmenting Watson—Crick base paring by metal coordination offers a promising way to increase the hybridization affinity of therapeutic oligonucleotides for target nucleic acids. Crosslinking of DNA by the anticancer agent cisplatin is a well-known example of utilization of metal—nucleic acid interactions for therapeutic purposes. Many other transition metal mediated interactions have also been widely studied but in most cases rapid ligand exchange makes applicability in the intracellular medium questionable. This thesis brings covalently palladated oligonucleotides in the light for the first time as a new addition to existing antisense techniques. This thesis describes the synthesis of a few artificial nucleosidic and nonnucleosidic structures that offer reactive sites for cyclopalladation. The respective palladacyclic derivatives were integrated into short oligonucleotides and their base pairing preferences and effect on duplex stability studied by various techniques, including UV melting experiments (with detailed thermodynamic analysis), CD spectroscopy and Förster resonance energy transfer (FRET) –based competition assay. Pd(II) mediated base pairing in duplexes was found to depend on the structure of organometallic residue, the base pairing partner and the location of the base pair within the oligonucleotide. The palladated oligonucleotides were also tested for their splice correction ability in three human cell lines. In HeLa cells, the palladacyclic oligonucleotide modestly outperformed the corresponding unmodified oligonucleotide.Watson—Crick –emäspariutumiseen perustuvat oligonukleotidilääkkeet kykenevät estämään tai muuttamaan proteiinisynteesiä. Watson—Crick –emäspariutumisen korvaaminen tai täydentäminen on lupaava keino lisätä oligonukleotidilääkkeiden affiniteettia kohdenukleiinihappoihin. Syöpälääke sisplatiinin aiheuttama DNA:n ristiinsilloittuminen on hyvin tunnettu esimerkki metallien ja nukleiinihappojen välisten vuorovaikutusten hyödyntämisestä lääketieteessä. Monia muitakin siirtymämetallien välittämiä vuorovaikutuksia on tutkittu mutta useimmissa tapauksissa soveltuvuus solunsisäiseen käyttöön on kyseenalaista nopean ligandinvaihdon vuoksi. Tämä väitöskirjatyö on ensimmäinen tutkimus mahdollisuudesta käyttää kovalenttisesti palladoituja oligonukleotideja antisense-sovelluksissa. Väitöskirjassa kuvataan joidenkin sellaisten nukleosidisten ja ei-nukleosidisten rakenteiden synteesi, joissa on syklopalladaatioon sopiva reaktiivinen paikka. Vastaavat palladasykliset johdokset liitettiin osaksi lyhyitä oligonukleotideja ja niiden emäspariutumistaipumuksia sekä vaikutusta kaksoiskierteen pysyvyyteen tutkittiin CD- ja UV-sulamismittauksin (sisältäen yksityiskohtaisen termodynaamisen analyysin) sekä käyttäen Försterin resonanssienergiansiirtoon (FRET) perustuvaa kilpailukoetta. Pd(II)-välitteisen emäspariutumisen kaksoiskierteessä havaittiin riippuvan palladasyklin rakenteesta, vastinemäksestä sekä emäsparin sijainnista. Myös palladoitujen oligonukleotidien kykyä korjata virheellinen silmukoituminen testattiin kolmessa ihmisen solulinjassa. HeLa-solulinjassa palladasyklinen oligonukleotidi toimi vähän paremmin kuin vastaava modifioimaton oligonukleotidi

Palladacyclic Conjugate Group Promotes Hybridization of Short Oligonucleotides

Short oligonucleotides with cyclopalladated benzylamine moieties at their 5-termini have been prepared to test the possibility of conferring palladacyclic anticancer agents sequence-selectivity by conjugation with a guiding oligonucleotide. Hybridization of these oligonucleotides with natural counterparts was studied by UV and CD (circular dichroism) melting experiments in the absence and presence of a competing ligand (2-mercaptoethanol). Cyclopalladated benzylamine proved to be strongly stabilizing relative to unmetalated benzylamine and modestly stabilizing relative to an extra A center dot T base pair. The stabilization was largely abolished in the presence of 2-mercaptoethanol, suggesting direct coordination of Pd(II) to a nucleobase of the complementary strand. In all cases, fidelity of Watson-Crick base pairing between the two strands was retained. Hybridization of the cyclopalladated oligonucleotides was characterized by relatively large negative enthalpy and entropy, consistent with stabilizing Pd(II) coordination partially offset by the entropic penalty of imposing conformational constraints on the flexible diethylene glycol linker between the oligonucleotide and the palladacyclic moiety

Sequence dependence of Pd(II)-mediated base pairing by palladacyclic nucleobase surrogates

A C-nucleoside derivative of phenylpyridine or the respective palladacycle was incorporated at either 3′- or 5′-terminus of a short oligodeoxynucleotide. Hybridization properties of these modified oligonucleotides were studied in a fluorescence-based competition assay in addition to conventional UV melting temperature analysis and compared with those of a previously prepared analogue featuring the modified nucleoside in the middle of the sequence. With the unpalladated phenylpyridine oligonucleotides, UV melting temperature qualitatively correlated with the ability to displace a strand from a double helix in the competition assay, decreasing in the order 5′ > 3′ > middle. Corresponding results on the palladacyclic oligonucleotides were more difficult to interpret but both UV melting and competition experiments revealed a decrease in the duplex stability upon palladation in most cases. On the other hand, dependence of the UV melting temperature on the identity of the canonical nucleobase opposite to the modified nucleobase analogue was much more pronounced with the palladacyclic duplexes than with their unpalladated counterparts. Furthermore, UV melting profiles of the palladacyclic duplexes featured an additional transition at a temperature exceeding the melting temperature of the unmodified part of the duplex. Taken together, these results lend support to the idea ofPd(II)-mediated base pairs that are highly stable but incompatible with the geometry of a double helix.Graphical abstractHybridization properties of oligonucleotides incorporating a phenylpyridine C-nucleoside or its palladacyclic derivative were studied by a fluorescence-based competition assay in addition to conventional melting temperature measurements. Position of the modification had a major impact on duplex stability even when the canonical base opposite to the modified base remained unchanged..</div

Palladacyclic Conjugate Group Promotes Hybridization of Short Oligonucleotides

Short oligonucleotides with cyclopalladated benzylamine moieties at their 5&prime;-termini have been prepared to test the possibility of conferring palladacyclic anticancer agents sequence-selectivity by conjugation with a guiding oligonucleotide. Hybridization of these oligonucleotides with natural counterparts was studied by UV and CD (circular dichroism) melting experiments in the absence and presence of a competing ligand (2-mercaptoethanol). Cyclopalladated benzylamine proved to be strongly stabilizing relative to unmetalated benzylamine and modestly stabilizing relative to an extra A&bull;T base pair. The stabilization was largely abolished in the presence of 2-mercaptoethanol, suggesting direct coordination of Pd(II) to a nucleobase of the complementary strand. In all cases, fidelity of Watson-Crick base pairing between the two strands was retained. Hybridization of the cyclopalladated oligonucleotides was characterized by relatively large negative enthalpy and entropy, consistent with stabilizing Pd(II) coordination partially offset by the entropic penalty of imposing conformational constraints on the flexible diethylene glycol linker between the oligonucleotide and the palladacyclic moiety

Palladacyclic Conjugate Group Promotes Hybridization of Short Oligonucleotides

Short oligonucleotides with cyclopalladated benzylamine moieties at their 5&prime;-termini have been prepared to test the possibility of conferring palladacyclic anticancer agents sequence-selectivity by conjugation with a guiding oligonucleotide. Hybridization of these oligonucleotides with natural counterparts was studied by UV and CD (circular dichroism) melting experiments in the absence and presence of a competing ligand (2-mercaptoethanol). Cyclopalladated benzylamine proved to be strongly stabilizing relative to unmetalated benzylamine and modestly stabilizing relative to an extra A&bull;T base pair. The stabilization was largely abolished in the presence of 2-mercaptoethanol, suggesting direct coordination of Pd(II) to a nucleobase of the complementary strand. In all cases, fidelity of Watson-Crick base pairing between the two strands was retained. Hybridization of the cyclopalladated oligonucleotides was characterized by relatively large negative enthalpy and entropy, consistent with stabilizing Pd(II) coordination partially offset by the entropic penalty of imposing conformational constraints on the flexible diethylene glycol linker between the oligonucleotide and the palladacyclic moiety

EVALUATION AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR THE USE OF BERBERINE FROM COPTIS TEETA BARK AS ANTI-CANCER PHYTOCHEMICAL

Objective: A new method of analysis using high-performance liquid reverse chromatography (HPLC) to calculate berberine in Coptis teeta bark was created in addition to the validation research. Methods: Berberine was analyzed with potassium hydrogen phosphate and acetonitrile using the SunFire C18 column at a flow rate of 1.0 ml/min; 5μ (4.6 × 250 mm) was detected at 347 nm. Results: For precision, selectivity, and linearity, the technique was tested. The quantification of berberine did not conflict with the existence of other chemical components. Between concentrations of 60 and 180 ppm, the calibration curve was linear. The precision ranged from 9.0% to 102.0% and 2% was the relative standard deviation. Conclusion: Finally, this method of HPLC enhancement is simple, effective and has been used to classify and quantify berberine successfully. This can be useful for the study of different berberine-containing plant extracts and can also be used to measure berberine from formulation to herbal formulation

Evaluation of relationship between periodontitis and various components of metabolic syndrome

Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors that include obesity, diabetes, hypertension, and dyslipidemia. Oxidative stress may act as a common link to explain the relationship between each component of MetS and periodontitis. MetS is characterized by oxidative stress, a condition in which the equilibrium between the production and the inactivation of reactive oxygen species (ROS) becomes disrupted. ROS contribute to cellular dysfunction and damage. Patients with more components of MetS had a significantly greater probing depth and clinical attachment loss. Evaluation of relationship between periodontitis and various components of Metabolic syndrome (MetS).This prospective study was carried out on study group consists of 30 subjects. All the patients will be evaluated for the risk factors of MetS which are: B.M.I., B.R, Triglycerides, R.B.S. increase in these risk factors is associated with increase in periodontal pocket depth, gingival recession and clinical loss of attachment. Metabolic alterations related to MetS causes an augmented response to bacterial plaque favouring periodontal insurgence. Oxidative stress acts as common link to explain the relationship between each component of MetS and periodontitis. Thus, periodontitis the harbinger of MetS which is more severe and disproportionate to number of local factors related to it

Granular Cell Ameloblastoma: A Rare Case Report and Literature Review

Ameloblastomas are rare, benign, aggressive neoplasms of odontogenic epithelial origin. Among the various types of ameloblastomas, granular cell ameloblastoma is rare and accounts for less than 5% of the total. Granular cells are epithelial in origin. The distinct histopathological characteristics of granular cells are associated with high lysosomal content in tumour cells cytoplasm. A 43-year-old female patient presented with a painful swelling on the left-side of the jaw. The diagnosis was based on clinical, radiological and histopathological examination. A complete surgical resection was done. Histopathology showed features of granular cell ameloblastoma. The operative procedure was uneventful. Granular cell ameloblastoma has a high tendency for recurrence. Around 33.3% recurrence is reported, according to various studies. This rate is much higher than other subtypes of ameloblastoma, which are more common. Hence, it is essential to provide appropriate surgical treatment on time to prevent tumour recurrence and metastasis.This case report aims to highlight the unique features of granular cell ameloblastoma, distinguishing it from other subtypes and discussing the pathogenesis and treatment modalities

Aneurysmal Bone Cyst with Ossifying Fibroma of the Mandible: A Case Report and Review of the Literature

Aneurysmal Bone Cysts (ABCs) are uncommon benign bone lesions primarily affecting children and adolescents. They are distinguished by severe bone breakdown and expansive tissue growth, resulting in clinical symptoms and potential complications. ABCs typically occur in long bones, but reports have also documented their presence in the jaws, particularly the mandible. These lesions are commonly considered non-cancerous and are characterized by cystic or blood-filled chambers. There are two clinicopathological variations of ABC: primary ABC and secondary ABC. Primary ABC originates independently, while secondary ABC develops as a result of a pre-existing lesion such as a cyst, tumor, or Fibro-Osseous Lesions (FOL) like solitary bone cyst, ossifying fibroma, or giant cell granuloma. When ABC coexists with another bone lesion believed to be its precursor, it is referred to as an “ABC plus lesion”. In this case report, a 75-year-old patient, presented with a painful growth in the lower anterior region of the jaw for the past 18 months. The diagnosis was established through clinical, radiological, and histopathological examinations. A complete surgical resection was performed, followed by uneventful reconstruction. Histopathological examination confirmed the presence of ABC with ossifying fibroma (ABC plus lesion). It is important to address ABC plus lesions as they can cause significant pain, deformity, and discomfort. Although non-cancerous, they can still disrupt normal bone structure and function. This case report emphasizes the clinical, radiographic, and histopathological features of ABC plus lesions, aiding in disease identification

CTLA4 Methylation and its Expression as a Prognostic Biomarker in Tobacco Users with and without Oral Squamous Cell Carcinoma- A Protocol for Cross-sectional Study

Introduction: Oral Squamous Cell Carcinoma (OSCC) is characterised by immunosuppression mediated by evasion of ‘immune checkpoints’ by tumour cells. Cytotoxic T Lymphocyte Associated Protein 4 (CTLA4) is one of the immune checkpoint molecule whose role in oral carcinogenesis remains to be elucidated. Deoxyribonucleic Acid (DNA) methylation of CTLA4 promoter region holds potential as a biomarker for diagnosis and assessment of individuals at risk of developing OSCC. Need for the study: Immunotherapy is an emerging treatment modality in some cancers. Thus, better understanding of role of CTLA4 methylation in OSCC paves a way for newer strategies in immunotherapeutic. Aim: To assess the DNA methylation pattern of promoter region of CTLA4 gene and evaluate its expression in tobacco users with and without OSCC so that it can contribute as biomarker for diagnosis and prognosis of OSCC. Materials and Methods: The proposed longitudinal cross-sectional study will be conducted at Sharad Pawar Dental College and Hospital, Maharashtra, India. It will evaluate promoter methylation and protein expression of CTLA4 gene in Smokeless Tobacco (SLT) users. There will be three groups: group A OSCC patients; group B normal individuals with SLT habit without any oral lesion; and group C normal individuals without SLT habit. Formalin Fixed Paraffin Embedded (FFPE) tissue blocks will be prepared from the biopsy obtained from total thirty nine participants. DNA methylation of CTLA4 promoter will be assessed using Methylation-Specific PCR (MS-PCR). In addition, the quantitative expression of CTLA4 will also be assessed using Immunohistochemistry (IHC)