Development of fluorescent ligands for A1 adenosine receptor and cannabinoid receptors

Adenosine A1 receptor (A1AR), cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R) are class A G protein-coupled receptors (GPCRs) and play important roles in human pathophysiological conditions such as cardiovascular, neurological, metabolic and immunological disorders. Fluorescent ligands are powerful tools to investigate processes such as receptor expression, localisation, trafficking and receptor-protein interactions in the native cell environment. Fluorescent ligands can also be used as tracers in pharmacological assays, instead of the commonly used radioligands that carry inherent safety risks. The development of fluorescent ligands with high affinity, selectivity and suitable imaging properties for A1AR, CB1R and CB2R would greatly contribute to an increased understanding of receptor biology and thus facilitate the drug development process. Development of fluorescent ligands with sufficient polarity for cannabinoid receptors (CBRs), which have lipid-based endogenous ligands, is an especially challenging task. This thesis describes the development of small molecule-based fluorescent ligands for A1AR, CB1R and CB2R, via attachment of a linker and fluorophore to a ligand. (Benzimidazolyl)isoquinolinols, analogues of previously reported high affinity A1AR (benzimidazolyl)isoquinolines, were explored in chapter 2 for the development of A1AR fluorescent ligands. A procedure for 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) mediated aromatisation of tetrahydroisoquinolines and multistep synthesis for the preparation of (benzimidazolyl)isoquinolinols was developed. Based on the previously reported structure–activity relationship (SAR), linkers (and linker plus fluorophore conjugates) were introduced in the C-6 or C-7 position of (benzimidazolyl)isoquinolinols. Unfortunately, these fluorescent ligands did not exhibit any significant affinity for A1AR in a bioluminescence resonance energy transfer (BRET) assay using the NanoLuc luciferase and it was concluded that (benzimidazolyl)isoquinolinols might not be a suitable scaffold for the development of A1AR fluorescent ligands. NMR spectroscopy and reverse phase HPLC studies showed (benzimidazolyl)isoquinolinols exhibit tautomerism. Chromenopyrazoles, previously reported as high affinity CB1R ligands, were investigated for development into CB1R fluorescent ligands in chapter 3. Based on previous SAR, linkers then fluorophores were introduced at six different chromenopyrazole positions. Disappointingly, fluorescent chromenopyrazoles did not exhibit high affinity for CB1R in a radioligand binding assay. However, several chromenopyrazoles including a linker conjugate 3.22 and a peptide linker conjugate 3.39 exhibited high affinity for CB2R that were promising candidates for development of CB2R fluorescent ligands. All of the chromenopyrazoles that were evaluated in a cyclic adenosine monophosphate (cAMP) functional assay behaved as agonists at CB2R. Docking studies were carried out using a CB2R homology model and showed that high affinity CB2R chromenopyrazoles with linkers attached likely exit via a cavity located between transmembrane helix (TM) 1 and TM7. In chapter 4, efforts were made to develop high affinity CB2R fluorescent ligands and more polar CB2R linker conjugates that built on the results of chapter 3. The highest affinity CB2R linker conjugate 3.22 (obtained in chapter 3) was conjugated to four different fluorophores (BODIPY-FL, Cy5, TAMRA, BODIPY-630/650) and two high affinity CB2R fluorescent ligands (4.01, 4.02) were obtained. The highest affinity CB2R fluorescent ligand 4.02 (Ki = 41.8 ± 4.5 nM at hCB2R; 5856 ± 1264 nM at hCB1R) behaved as an inverse agonist (4.02, EC50 = 142.0 ± 13.1 nM at hCB2R, 196.7 ± 9.11 % of forskolin response at hCB2R) in the cAMP functional assay and showed CB2R-specific-binding in widefield imaging experiments using CB2R expressing HEK-293 cells. Fluorescent ligand 4.02 exhibited higher affinity for CB2R than any other reported CB2R fluorescent ligands and is the first high affinity CB2R fluorescent ligand for which functional data has been reported (as of July 2018). Fluorescent ligand 4.02 possesses suitable CB2R imaging properties and will be a useful tool for researchers studying CB2R biology in techniques such as fluorescence-based assays, widefield microscopy and flow-cytometry and can be used in resonance energy transfer experiments with other fluorescent partners. In addition, three high-moderate affinity peptide linker conjugates (4.06, 4.07, 4.08) with considerably higher polarity compared to commonly used cannabinoid receptor ligand CP55,940 were obtained, all of which behaved as CB2R agonists. Development of high affinity CB1R ligands based on a reported pyridyl scaffold was explored in chapter 5. Fluorescent ligands were designed using previously reported SAR coupled with results obtained from CB1R docking using the reported CB1R crystal structure. O-Linker pyridyl-2-carboxamides and corresponding fluorescent conjugates were prepared via a multistep synthesis. Unfortunately, none of the fluorescent ligands exhibited any significant affinity for CB1R, however, three moderate affinity CB1R linker conjugates (5.33, 5.34, 5.35) were obtained. It was therefore concluded that optimised derivatives of pyridyl-2-carboxamide with different O-linkers or with linkers conjugated at different positions of the pyridine could be another strategy for the development of CB1R fluorescent ligands

Inflammation-Induced Oxidative Stress Mediates Gene Fusion Formation in Prostate Cancer.

Approximately 50% of prostate cancers are associated with gene fusions of the androgen-regulated gene TMPRSS2 to the oncogenic erythroblast transformation-specific (ETS) transcription factor ERG. The three-dimensional proximity of TMPRSS2 and ERG genes, in combination with DNA breaks, facilitates the formation of TMPRSS2-ERG gene fusions. However, the origins of DNA breaks that underlie gene fusion formation in prostate cancers are far from clear. We demonstrate a role for inflammation-induced oxidative stress in the formation of DNA breaks leading to recurrent TMPRSS2-ERG gene fusions. The transcriptional status and epigenetic features of the target genes influence this effect. Importantly, inflammation-induced de novo genomic rearrangements are blocked by homologous recombination (HR) and promoted by non-homologous end-joining (NHEJ) pathways. In conjunction with the association of proliferative inflammatory atrophy (PIA) with human prostate cancer, our results support a working model in which recurrent genomic rearrangements induced by inflammatory stimuli lead to the development of prostate cancer

Features of holiday practice from geography of soil in the conditions of girsk and step Сrimea

UK: Стан грунтів пов’язан з різноманіттям навколишнього середовища та впливом анторопогенного фактору, чим обумовлюється виділення категорій придатності грунтів для використання у землеробстві. EN: Field soil surveys occupy an important place in the system of training geography teachers. The skills and experience gained in this practice are used by senior students, as well as for further work in general education schools. In conducting field practice, the focus is on the methodology of field research: the proper choice of the location of the soil and geographic profile and soil cuts, the description of the soil morphological properties, as well as methods for studying the soil cover, including ground mapping issues

A modified tubularised incised plate urethroplasty technique and a revised hypospadias algorithm

To simplify and standardize surgical management of hypospadias, a modified tubularised incised plate (TIP) urethroplasty (Snodgrass) technique has been described and a revised hypospadias management algorithm has been formulated. The study aims to evaluate the viability of the described procedure in different types of hypospadias and tests the validity of the algorithm. The modification described is recruitment of penile and glandular skin lateral to the urethral plate to facilitate tubularisation. The algorithm starts with penile degloving with preservation of urethral plate. Snodgrass repair was done in cases with no chordee and where skin chordee resolved by skin take down. Modified Snodgrass repair was done in cases where urethral plate was narrow. Another modification proposed by us is single layer penile skin closure instead of an added dartos flap, which was done in both classical and modified Snodgrass repair. Cases of severe chordee not resolved by skin take down were repaired by transverse preputial island flap (TPIF) and Bracka′s technique. Dorsal plication was not used as an orthoplasty modality. It was possible to repair 68.89% of the cases by Snodgrass repair. These patients either had no chordee or had superficial skin tethering (skin chordee) which resolved on degolving. All these cases were coronal, distal and mid penile hypospadias. Remaining cases were mid, proximal and penoscrotal with true fibrous chordee and were repaired by TPIF or Bracka′s technique. The Snodgrass technique had a fistula rate of 9.67%. Acceptably, low fistula rate and simple execution make the proposed modification of classical Snodgrass repair a viable option. The proposed algorithm proves to be a useful tool for standardised and logical preoperative decision making. It also defines indications of the three techniques vis-a-vis the type of hypospadias

A modified tubularised incised plate urethroplasty technique and a revised hypospadias algorithm

To simplify and standardize surgical management of hypospadias, a modified tubularised incised plate (TIP) urethroplasty (Snodgrass) technique has been described and a revised hypospadias management algorithm has been formulated. The study aims to evaluate the viability of the described procedure in different types of hypospadias and tests the validity of the algorithm. The modification described is recruitment of penile and glandular skin lateral to the urethral plate to facilitate tubularisation. The algorithm starts with penile degloving with preservation of urethral plate. Snodgrass repair was done in cases with no chordee and where skin chordee resolved by skin take down. Modified Snodgrass repair was done in cases where urethral plate was narrow. Another modification proposed by us is single layer penile skin closure instead of an added dartos flap, which was done in both classical and modified Snodgrass repair. Cases of severe chordee not resolved by skin take down were repaired by transverse preputial island flap (TPIF) and Bracka′s technique. Dorsal plication was not used as an orthoplasty modality. It was possible to repair 68.89% of the cases by Snodgrass repair. These patients either had no chordee or had superficial skin tethering (skin chordee) which resolved on degolving. All these cases were coronal, distal and mid penile hypospadias. Remaining cases were mid, proximal and penoscrotal with true fibrous chordee and were repaired by TPIF or Bracka′s technique. The Snodgrass technique had a fistula rate of 9.67%. Acceptably, low fistula rate and simple execution make the proposed modification of classical Snodgrass repair a viable option. The proposed algorithm proves to be a useful tool for standardised and logical preoperative decision making. It also defines indications of the three techniques vis-a-vis the type of hypospadias

Avoiding unfavorable results in postburn contracture hand

Deformities of the hands are a fairly common sequel of burn especially in the developing world. This is because of high incidence of burns, limited access to standard treatment and rehabilitation. The best outcome of a burnt hand is when deformities are prevented from developing. A good functional result is possible when due consideration is paid to hands during resuscitation, excisional surgery, reconstructive surgery and physiotherapy. The post-burns deformities of hand develop due direct thermal damage or secondary to intrinsic minus position due to oedema or vascular insufficiency. During the acute phase the concerns are, maintenance circulation minimize oedema prevent unphysiological positioning and wound closure with autogenous tissue as soon as possible. The rehabilitation program during the acute phase starts from day one and goes on till the hand has healed and has regained full range of motion. Full blown hand contractures are challenging to correct and become more difficult as time passes. Long-standing cases often land up with attenuation of extensor apparatus leading to swan neck and boutonniere deformity, muscle shortening and bony ankylosis. The major and most common pitfall after contracture release is relapse. The treatment protocol of contracture is solely directed towards countering this tendency. This article aims to guide a surgeon in obtaining optimal hand function and avoid pit falls at different stages of management of hand burns. The reasons of an unfavourable outcome of a burnt hand are possible lack of optimal care in the acute phase, while planning and performing reconstructive procedure and during aftercare and rehabilitation