Biosynthesis of capsaicinoids by recombinant Saccharomyces cerevisiae

Throughout history, people have used products from different microorganisms and plants for many medicinal and nutritional applications. Chilli peppers have been used for spicing up food, but also for their pharmacological properties. They contain alkaloid molecules called capsaicinoids, which have been shown to activate and desensitise the heat receptor - Transient Receptor Potential Vanilloid type 1 (TRPV1) cation channel. The TRPV1 receptor is a target for pain relief treatments for a range of health conditions, therefore capsaicinoids are important drug candidates.Capsaicinoids can be manufactured by extraction directly from the chili pepper,which typically results in relatively small amounts and therefore requires large areas of land. Furthermore, capsaicinoids yield can be affected by environmental and genotypical factors. Another way of production can be through the use of synthetic biology and metabolic engineering strategies to introduce enzymatic reactions in a model microorganism. This way, plant-derived or novel capsaicinoids with potentially improved therapeutic properties can be produced from given precursors, simple sugars or renewable raw materials.The aim of this work was to engineer baker’s yeast Saccharomyces cerevisiae for a whole-cell biocatalytic production of capsaicinoids. The structure of thesecompounds is comprised of a vanilloid moiety and a fatty acyl chain joined by anamide bond. The last step for synthesis of these compounds in the plant is through condensation of the precursors vanillylamine (derived from vanillin) and a fatty acyl-CoA forming the amide bond. Amide forming reactions are very important in chemical synthesis and research has been directed towards finding efficient enzymes to perform them. Furthermore, yeast as a model microorganism has been modified to produce vanillin from glucose; however, reductive amination of vanillin to vanillylamine, or production of capsaicinoids in yeast has not been achieved previously.This thesis addresses how capsaicinoids can be produced, the challenges for in vivo reductive amination of vanillin to vanillylamine and the identification andcharacterisation of several heterologous N-acyltransferase (NAT) and CoA-ligase(CL) amide-forming enzymes. Vanillylamine was produced from vanillin, byoverexpressing a vanillin aminotransferase. The transamination reaction wasimproved by the co-expression of an alanine dehydrogenase, which also removed the need for amine donor supplementation. Cultivation conditions were shown to have an effect on the transamination, with anaerobic conditions and ethanol as a cosubstrate leading to decrease in by-product formation and improved reductive amination of vanillin to vanillylamine. Moreover, combinations of NAT and CL enzymes were evaluated for production of nonivamide (a model capsaicinoid) from vanillylamine and nonanoic acid. Finally, nonivamide, was successfully produced in vivo from precursors vanillin and nonanoic acid, after implementing strain engineering strategies and evaluating reaction conditions. Even though the NAT and CL enzyme cascade effectively produced nonivamide, the titres and yields of the process can be further improved, indicating that the amidation step is the current bottleneck in the production and requires further optimisation.Lastly, the potential of S. cerevisiae as a biosensor was evaluated by overexpressing the TRPV1 receptor and modulating its activation. This was done as a first step towards the goal of using yeast as a screening platform for strains producing capsaicinoids with activity on the TRPV1 receptor.Transferring the capsaicinoid enzyme cascade in a cell host is not straightforward and requires optimal expression of several heterologous enzymes, adjustments of reaction conditions and strategies in reducing inhibitory effects and by-products. However, biosynthesis in whole-cells may offer advantages due to simple cell derived generation of enzymes, intermediates and co-factors, positively affecting the cost and performing the reaction in aqueous environment as a more environmentally sustainable way of production

Интердисциплинарен пристап и план на терапија кај пациенти со олигодонција

Oligodontia is a rare developmental dental anomaly in humans characterized by the absence of six or more teeth. In European populations the estimated prevalence of both syndromic and non- syndromic oligodontia is 0.14%. Clinical  features of oligodontia include six or more missing teeth, lack of development of maxillary and mandibular alveolar bone height and reduced lower facial height. Variation in tooth morphology, anomalies of the enamel, reduced size and aberrant form, delayed eruption have also been observed. Oligodontia is also associated with reduced salivary secretion rates.  These bring a functional  and esthetic  limitations and impact on emotional well-being. The aim of this study was to present a case of a 12-year-old girl with oligodontia and therapeutic procedures for orthodontic-prosthetic rehabilitation and normal orofacial function. A multidisciplinary approach that includes orthodontic and prosthetic therapy is often necessary for dental management in young patients with oligodontia. Oral rehabilitation and maintenance care in individuals with many missing permanent teeth is a long-standing commitment that requires involvement of different specialists. Methods used are age-dependent, and early diagnosis is crucial. Orthodontic treatment,  autotransplantation,  dental implants, avoiding tooth preparations, and partial prosthetic dentures are treatment choices.Олигодонцијата е ретка дентална аномалија кај хуманата популација, која се карактеризира со недостаток на шест или повеќе заби. Во Европа, преваленцијата на синдромската и несиндромската олигодонција е 0,14%. Клиничките манифестации вклучуваат недостаток на шест или повеќе заби, недоволен развој на максиларниот и мандибуларниот алвеоларен гребен и редуцирана долна третина од лицето. Варијациите во денталната морфологија, редуцираната големина и аберантна форма, аномалиите во емајлот, како и  доцната ерупција, додатно ја влошуваат клиничката слика. Олигодонцијата е асоцирана и со редуцирана саливарна секреција. Сето ова генерира функционални и естетски проблеми, со импакт на емоционалното здравје. Целта на студијата е да се прикаже случај на 12-годишно девојче со олигодонција, и преземените тераписки процедури за ортодонтско-протетска рехабилитација и воспоставување на нормална орофацијална функција. Може да се заклучи дека оралната рехабилитација кај индивидуите со олигодонција  е долготраен процес во кој се вклучени различни специјалисти. Третманот зависи од возраста, а раната дијагноза е круцијална. Превенцијата на кариес, ортодонтскиот третман, автотрансплантацијата, денталните импланти, избегнувањето на препарации на забите и парцијалните протетски надоместоци се препорачани методи

Primary Seminoma Localized in Mediastinum: Case Report

BACKGROUND: Primary mediastinal seminomas most commonly occur in young men, and they are localised in the anterior mediastinum. CASE PRESENTATION: The presented study is a case report of a 34-year-old man suffering from a mediastinal tumour in size of 19 cm, with pleural and pericardial effusion. The patient complains of cough, difficulty breathing, weight loss, and pronounced tiredness. CT of lungs and biopsy of the mediastinal change was performed. The histopathological analysis was in favour of a primary mediastinal seminoma. The patient initially had pronouncedly increased levels of LDH and β-hCG tumour marker. Pericardiocentesis was realised due to threatening tamponade of the heart, followed by 4 cycles of chemotherapy by BEP protocol. Following 2 cycles of chemotherapy, normalisation of LDH and β-HCG levels and significantly improved the clinical condition in the patient was found. Upon completion of 4 chemotherapy cycles by BEP protocol, the patient performed an FDG-PET scan with partial response and reduced dimension of a primary tumour in the mediastinum. Radiotherapy of residual tumour mass up to a total dose of 40Gy in 20 fractions was realised. Control FDG-PET scan had a finding of complete response to a tumour and absence of FDG uptake. The last follow-up examination was performed in October 2018, and the patient was disease-free for 54 months. CONCLUSION: Multimodality treatment approach of chemotherapy followed by radiation consolidation ensured long-term survival in primary advanced mediastinal seminoma

Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin‐derived substrates

Whole‐cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco‐efficient route to produce phenolic amines. Here, a novel cell growth‐based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp‐SpuC‐II and ATA from Chromobacterium violaceum (Cv‐ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp‐SpuC‐II and Cv‐ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94‐ and 92‐fold increased specific transaminase activity, respectively. Whole‐cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp‐SpuC‐II and Cv‐ATA, respectively. Still, amine production was limited by a substantial re‐assimilation of the product and formation of the by‐products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv‐ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re‐assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement

Testiranje spojnog smicanja dubokih okluzalnih kompozitnih restauracija postavljenih preko podloge od Dycal, Dycal LC, konvencionalnog ili smolom modifikovanog glas-jonomer cementa

Background/Aim. The longevity of a dental restoration may be predicted to some degree by its adhesive ability, and this, in turn, can be measured by bond strength testing between restorative materials and tooth structure. The aim of this study was to test an innovative joggle lap shearing jig that integrates the tooth and the entire biomechanical unit into testing, to compare the shear bond strengths of Class I occlusal composite restorations in deep cavity preparations lined with Dycal, Dycal LC, conventional glass ionomer or resin-modified glass ionomer. The mode of failure (adhesive, cohesive, mixed) after debonding was determined by stereomicroscopy. Methods. A total of 150 standardized occlusal cavities were prepared and divided into five groups. The group I cavities (n = 30) were coated with adhesive (ExciTE®F) and filled directly with composite (TetricEvoCeram). The group II and III cavities were lined with Dycal (n = 30) or Dycal LC (n = 30) before placing composite. The groups IV and V specimens were based with Fuji IX (n = 30) or Fuji II LC (n = 30). Shear bond strengths were determined with a universal testing machine and fractured bonding sites were analyzed under stereomicroscope. The mean bond strengths were analyzed using one-way ANOVA test (p lt 0.05) and the means between the groups were analyzed with Student's t-test. Results. The shear bond strength (MPa) of composite restorations in cavities without base (23.91 ± 4.54) was higher than cavities lined with Fuji II LC (17.45 ± 2.74), Fuji IX (8.76 ± 2.57), Dycal LC (13.07 ± 1.84) or Dycal (6.12 ± 1.28). The results using the jogged lap shearing jig were consistent with the literature. Conclusion. The shear bond strength of occlusal composite restorations in deep cavities without liners was greater than cavities lined with Fuji II LC > Fuji IX > Dycal LC > Dycal.Uvod/Cilj. Trajnost zubnih nadoknada može se donekle predvideti vstom adhezivne sposobnosti materijala i može se meriti testiranjem snage adhezije restorativnih materijala i zubnih struktura. Cilj ove studije bio je da se da se testira preklapanje spoja koji povezuje zub i biomehaničku jedinicu i da se uporedi jačina veze okluzalnih kompozitnih ispuna postavljenih u duboke kavitete preko podloge od Dycal, Dycal LC, konvencionalnog ili smolom-modifikovanog glas-jonomer cementa. Metode. Ukupno 150 standardizovanih okluzalnih kaviteta bilo je podeljeno u pet grupa (n = 30): I - kaviteti premazani adhezivom (ExciTE®F) i direktno ispunjeni kompozitom (TetricEvoCeram); II i III - kaviteti sa podlogom od Dycal ili Dycal LC pre postavljanja kompozitnog materijala; IV i V - uzorci sa bazom od Fuji IX ili Fuji II LC (n = 30). Jačina vezivne snage određena je pomoću univerzalne mašine, a način neuspeha (adhezivna, kohezivna, mešovita fraktura) određen je stereomikroskopom. Srednje vrednosti su analizirane pomoću ANOVA testa (p lt 0,05), a značajnost razlika između grupa analizirana je Student-ovim t-testom. Rezultati. Jačina vezivne snage (MPa) kompozitnih ispuna u kavitetima bez podloge (23,91 ± 4,54) bila je veća u poređenju sa ispunima postavljenim preko Fuji II LC (17,45 ± 2,74), Fuji IX (8,76 ± 2,57), Dycal LC (13,07 ± 1,84) ili Dycal (6.12 ± 1.28). Zaključak. Smicanje ili pomaknuće okluzalnih kompozitnih ispuna u dubokim kavitetima bez lajnera je veće nego u kavitetima postavljenim preko Fuji II LC > Fuji IX > Dycal LC > Dycal

Platelet-Rich Fibrin in Oral Surgery and Endodontic Procedures as a Regenerative Biomaterial: A Review Article

Background: One of the major challenges in clinical research is to incorporate materials and procedures into dentistry that can improve clinical outcomes, increasing percentages of success. Platelet-rich fibrin (PRF) is a surgical, biological additive prepared by manipulation of autologous blood, which stimulates and regulates inflammation during and after oral surgery. This study aims to assess the potential benefits of using PRF in modern oral and endodontic surgery to accelerate radiographic healing and reduce postoperative discomfort. Methods and Results: Electronic literature research was conducted using the biomedical search engine “National Library of Medicine,” PubMed/MEDLINE database from March 2000 to June 2023. To obtain results that involved the whole scope of dentistry and PRF, the keywords used for the search were “PRF,” “PRF in dentistry,” and “PRF on apical surgery.” The articles were selected by reviewing the titles and abstracts of the articles with the word “platelet-rich fibrin (PRF).” Articles related only to clinical applications in general dentistry and its different application fields were hand-searched in applicable, significant journals, and reference lists of included studies were broadcast with no language limitation. The inclusion criteria set for this review were as follows: all case reports, case series, original research papers, review papers, in vitro/in vivo studies, animal studies, and controlled clinical trials on PRF used in dentistry-related studies. Current studies, in vitro and in vivo, have confirmed safe and encouraging results, without opposing outcomes, related to the use of PRF alone or in a mixture with other biomaterials. Currently, the use of PRF treatment has been shown to enhance and promote natural tissue healing support

Case report: Primary hyperparathyroidism due to mediastinal parathyroid adenoma, our pint of view

Primary hyperparathyroidism is defined by elevated parathyroid hormone and calcium levels, most usually caused by a parathyroid adenoma. Parathyroid adenomas are most commonly detected in the neck or an ectopic site, seldom in the mediastinum. The parathyroid adenoma can occur in ectopic locations such as the mediastinum, thymus, or retro oesophagal area in 6-16% of cases. We presented the example of a 73-year-old woman who was found to have hypercalcemia during a regular test. The patient’s serum calcium (3.11 mmol/L), alkaline phosphatase (162 U/L), parathyroid hormone (PTH: 379 pg/mL) and creatinine (111.6 umol/L) levels were higher than the reference values. A chest computerized tomography scan revealed an anterior mediastinal mass, and nuclear scintigraphy revealed functioning parathyroid tissue in the mediastinum. The mediastinal parathyroid adenoma was effectively removed surgically, and the PTH level began to fall. Any hypercalcemia and high PTH levels in the absence of a parathyroid adenoma in the neck should prompt clinicians to look for ectopic sites using a mix of imaging modalities

Три Ñлучаи на Syndroma Willi Prader Labhardt во доенечка возраÑÑ‚

Authors presented three cases of Willi Prader Labhardt Syndroma whose cardinal symptoms are: psychomotor retardation, proportional obesity, hypogenitalism, a small bifrontal circumference, and carp-like mouth.During the pregnancy, children's mothers felt weak intrauterine movements and poor crying after delivery as well as no interests for surrounding environment.There is a poor prognosis, due to cardiopulmonary failure.ПретÑтавени Ñе ттри Ñлучаи на  Prader Labhardt Ñиндромот, чии Ñимптоми Ñе: пÑихомоторна ретардација, пропорционален обезитаÑ, хипогени тализам, мала бифронтала циркумференција, уÑта како шаран.За време на бременоÑта, мајките чувÑтвувале Ñлаби интраутерини движења, и Ñлабо плачење на децата по породувањето, како и отÑуÑтво на Ð¸Ñ‚ÐµÑ€ÐµÑ Ð·Ð° околината. Има лоша прогноза и води кон кардио-пулмонална ÑлабоÑÑ‚

Clinical Therapeutic Effects of the Application of Doxycycline in the Treatment of Periodontal Disease

OBJECTIVE: To compare the therapeutic effects of the application of doxycycline-full dose (100 mg) and sub-dose (20 mg) in the treatment of periodontal disease.MATERIAL AND METHODS: A total of 60 patients with periodontal disease were examined. Patients are divided into two groups: A) treated with antimicrobial dose of 100 mg doxycycline once daily for 30 days, and B) treated with 2 x 20 mg/day. doxycycline, during 75 days. Among all patients a conservative treatment was carried out and ordinated the proper dose doxycycline in total dose during treatment from 3 gr. Index of dental plaque by Löe-Sillness, index of gingival inflammation and gingival bleeding by Cowell were followed.RESULTS: Values of dental plaque in relation first examination, 10th, 20th day, 1 month and 2.5 months, showed that after 2.5 months, average value (x = 0.83) of dental plaque in second group is slightly less than the value (x = 0.93) of dental plaque in the first group. The average value (x = 0.17) of gingival inflammation in second group is significantly less than the value (x = 0.50) of gingival inflammation in the first group. The average value (x = 0.97) of gingival bleeding in patients from the first group was significantly higher than value(x = 0.37) of gingival bleeding in the second group.CONCLUSION: Patients whose therapy was helped by a sub-dose doxycycline demonstrated positive therapeutic effects on gingival inflammation and bleeding

Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism

<p>Abstract</p> <p>Background</p> <p>Many SNP discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. However, SNPs are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. In this study, a simple, sequence-adaptable SNP discrimination mechanism between a 'wild-type' and 'mutant' template is demonstrated. This model differs from other artificial restriction endonuclease models as <it>cis- </it>rather than <it>trans-</it>orientated regions of single stranded DNA were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted.</p> <p>Results</p> <p>A series of mismatch 'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to S1 nuclease. In this model, generation of oligonucleotide-mediated ssDNA mismatch 'bubbles' in the presence of S1 nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. Increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. This level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in Tm (~5.0°C per base-pair mismatch) and to optimise annealing conditions (~18.38°C below Tm) of the mismatched oligonucleotide sets.</p> <p>Conclusions</p> <p>The <it>in vitro </it>'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more SNPs. Furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine Tm and annealing rates as an alternative to conventional hybridisation detection strategies.</p