Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting

Amphiphilic molecules emerged as versatile building blocks for the generation of nano-sized architectures in water, as they can be programmed to self-assemble into a wide range of different topologies. In this thesis the generation of auto-fluorescent heterovalent nano-sized structures was explored using two types of amphiphilic scaffolds: disc-shaped and linear amphiphiles self-assembling in water into columnar polymers or into amorphous spherical nanoparticles, respectively. Numerous applications for self-assembling nanostructures were reported in literature based on amphiphilic molecules, ranging from imaging to diagnostics, and from drug delivery to tissue engineering. Many of these applications require the capability of the supramolecular system to actively target specific cell surface receptors. This is typically achieved through decoration with bioactive epitopes such as small molecules, peptides, and proteins. As discussed in chapter 1, the bioactive epitopes can either already be part of the monomeric supramolecular building blocks (pre-functionalization) or introduced after self-assembly via covalent attachment to appending reactive groups (post-functionalization). Selective and multivalent binding of disc-shaped amphiphiles to bacterial receptors was previously shown through the introduction of three functional groups at the periphery of the ethylene oxide tails and subsequent functionalization with bioactive ligands. Here, to expand the library of scaffolds, amphiphiles containing either nine amine functionalities or a single amine, azide and propargyl group were synthesized. Their decoration with bioactive ligands such as peptides, carbohydrates, small molecules and fluorescent dyes using both amide coupling and copper-catalyzed azide-alkyne cycloaddition is described in chapter 2. The orthogonality of the copper-catalyzed azide-alkyne cycloaddition allowed the functionalization with unprotected ligands. Whereas the functionalization of discotics with a carbohydrate was quantitative, the coupling of peptides proceeded with at best 40% conversion. This was probably due to steric crowding of peripheral functionalities in the self-assembly inducing solvent, which is required for solubility of unprotected ligands. In contrast, discotics bearing a single amine emerged as a versatile non-sterically hindered scaffold for ligand attachment as they were rapidly and quantitatively functionalized with a range of peptidic- and non-peptidic ligands using both NHS ester and HBTU activation techniques under non-assembling solvent conditions. The ability to fine-tune the density and display of bioactive epitopes and thereby creating more complex dynamic and heterovalent structures without interfering with the self-assembling process is a key prerequisite for the development of a platform technology for targeting. A versatile and non-sterically hindered scaffold for ligand attachment, such as the presented discotic bearing a single amine, might constitute the basis for such a technology. The functionalization of this discotic leads to monovalent ligand functionalized discotics. The display of multiple ligands, which is important for enhanced binding affinities, will be accomplished upon self-assembly into columnar stacks. This so-called multivalency upon self-assembly has been probed with a number of monovalent ligand-functionalized discotics in chapter 3. Enzyme-linked lectin assay revealed a three-fold increase in binding activity compared with the non self-assembling counterpart. The self-assembly into a columnar stack and the accompanied display of multiple ligands was as well confirmed studying the binding of monovalent streptavidin to discotics functionalized with a single biotin using Förster resonance energy transfer and SDS-PAGE. The formation of heterovalent supramolecular polymers through dynamic intermixing of different functionalized building blocks was shown using mixtures of biotin and fluorescein functionalized discotics incubated with streptavidin coated magnetic beads. Thus the self-assembly into supramolecular polymers not only generates a multivalent, but as well a heterovalent system. The possibility to generate heterovalent supramolecular polymers via simple intermixing of discotics has a great potential in view of advanced biological applications, for example in the field of targeted imaging. To gain further inside into the dynamics of this intermixing process, discotics bearing a single O6 benzylguanine moiety were covalently post-functionalized with two FRET-pairing fluorescent proteins. Firstly, the covalent post-functionalization with proteins, ligands which are incompatible with the pre-functionalization strategy, was confirmed with several analytical techniques such as SDS-PAGE and LC-MS in chapter 4. The covalent protein conjugation at the same time leads to Förster resonance energy transfer from the auto-fluorescent discotic scaffold to the yellow fluorescent protein and allows on-line monitoring of the conjugation. At the same time the protein conjugation does not interfere with the self-assembling process, leading to a multivalent protein display on a supramolecular wire, as visualized via energy transfer from the cyan to the yellow fluorescent protein. Secondly, the system maintains its intermixing dynamics, which allows the formation of hetero-functionalized supramolecular protein-conjugated polymers through exchange of the protein-functionalized discotics over time. The supramolecular wires act as dynamic framework on which the two proteins can assemble and exchange in a dynamic manner, leading to effective protein interactions, as observed by energy transfer. The cellular uptake of amine-decorated discotics and the dependence of cellular uptake on the peripheral amine density were explored in chapter 5. Using the auto-fluorescence of the discotic scaffolds, their internalization was studied using live cell multiphoton fluorescence microscopy Discotics bearing three or nine amine groups at their periphery efficiently translocated through the plasma membrane via endocytosis. Additionally, the knowledge about the formation of intermixed supramolecular polymers obtained in chapter 3 and 4 was applied to generate multi-functional supramolecular polymers consisting of up to three different cell-permeable and non cell-permeable discotic monomers. Through intermixing with cell-permeable discotic monomers in the supramolecular polymer, the cellular uptake of non-cell permeable discotics was induced and each of the components could be individually visualized, demonstrating the potential of dynamic multi-component supramolecular polymers. The functionalization of self-assembling p-conjugated nanoparticles with bioactive epitopes, a prerequisite for applications in targeted multimodal imaging, was investigated in the last chapter. Upon microinjection into water, these linear and auto-fluorescent amphiphiles self-assemble into highly-fluorescent amorphous nanoparticles of 80-100 nm. Azide and mannose groups were introduced at the periphery of the ethylene glycol chains of the amphiphile and did not interfere with the self-assembly process. The binding of mannose functionalized nanoparticles to proteins and bacteria confirmed the accessibility of the introduced ligand. Co-assembly of different amphiphiles enabled the fine-tuning of ligand density, which was confirmed with Förster resonance energy transfer. Additionally, using copper catalyzed azide-alkyne cycloaddition reaction, azide bearing nanoparticles were post-functionalized with different ligands. Successful combination of both functionalization strategies via intermixing of mannose and azide bearing amphiphiles and subsequent copper catalyzed azide-alkyne cycloaddition led to heterovalent nanoparticles. Nano-sized columnar and spherical supramolecular assemblies were functionalized with a wide range of ligands such as carbohydrates, peptides, and proteins using both pre- and post-functionalization strategies. This allowed for expanding the ligand diversity at two independent stages in the fabrication process of these bioactive nano-structures. Supramolecular synthesis enabled the facile generation of complex heterovalent bioactive assemblies; in the case of nanoparticles via co-assembly of different amphiphiles and in the case of discotics via dynamic intermixing of building blocks between the supramolecular stacks. With this knowledge in hand advanced applications of complex multitargeting and multimodal supramolecular nano-sized structures in imaging can be envisioned; carrying for example several targeting ligands as well as an alternative imaging probe. The ability to tune the optical properties in the case of the nanoparticles should additionally enable multi-color imaging. At the same time, the self-assembling nature of these nanoparticles allows the incorporation of hydrophobic (drug) molecules and functionalized lipids, expanding the scope of functionalization strategies and with it of possible applications. The absence of unspecific adsorption of the bare scaffolds of both the disc-shaped and linear amphiphiles proves their broad potential as selective biological targeting tools

Melanoma and pregnancy

The purpose of the study is to analyze data on the incidence of melanoma during pregnancy in the Sverdlovsk region.Цель исследования – провести анализ данных по заболеваемости меланомой во время беременности в Свердловской области

Evolutionary Dynamics of Vibrio cholerae O1 following a Single-Source Introduction to Haiti

ABSTRACT Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics

Malignant neoplasms associated with pregnancy

The purpose of the study is to estimate the incidence of malignant neoplasms associated with pregnancy at the population level.Цель исследования — оценка частоты встречаемости злокачественных новообразований, ассоциированных с беременностью на популяционном уровне

White matter predicts functional connectivity in premanifest Huntington's disease

Objectives The distribution of pathology in neurodegenerative disease can be predicted by the organizational characteristics of white matter in healthy brains. However, we have very little evidence for the impact these pathological changes have on brain function. Understanding any such link between structure and function is critical for understanding how underlying brain pathology influences the progressive behavioral changes associated with neurodegeneration. Here, we demonstrate such a link between structure and function in individuals with premanifest Huntington's. Methods Using diffusion tractography and resting state functional magnetic resonance imaging to characterize white matter organization and functional connectivity, we investigate whether characteristic patterns of white matter organization in the healthy human brain shape the changes in functional coupling between brain regions in premanifest Huntington's disease. Results We find changes in functional connectivity in premanifest Huntington's disease that link directly to underlying patterns of white matter organization in healthy brains. Specifically, brain areas with strong structural connectivity show decreases in functional connectivity in premanifest Huntington's disease relative to controls, while regions with weak structural connectivity show increases in functional connectivity. Furthermore, we identify a pattern of dissociation in the strongest functional connections between anterior and posterior brain regions such that anterior functional connectivity increases in strength in premanifest Huntington's disease, while posterior functional connectivity decreases. Interpretation Our findings demonstrate that organizational principles of white matter underlie changes in functional connectivity in premanifest Huntington's disease. Furthermore, we demonstrate functional antero–posterior dissociation that is in keeping with the caudo–rostral gradient of striatal pathology in HD. The distribution of pathology in neurodegenerative disease can be predicted by the organizational characteristics of white matter in healthy brains. However, we have very little evidence for the impact these pathological changes have on brain function. Understanding any such link between structure and function is critical for understanding how underlying brain pathology influences the progressive behavioral changes associated with neurodegeneration. Here, we demonstrate such a link between structure and function in individuals with premanifest Huntington's. Methods Using diffusion tractography and resting state functional magnetic resonance imaging to characterize white matter organization and functional connectivity, we investigate whether characteristic patterns of white matter organization in the healthy human brain shape the changes in functional coupling between brain regions in premanifest Huntington's disease. Results We find changes in functional connectivity in premanifest Huntington's disease that link directly to underlying patterns of white matter organization in healthy brains. Specifically, brain areas with strong structural connectivity show decreases in functional connectivity in premanifest Huntington's disease relative to controls, while regions with weak structural connectivity show increases in functional connectivity. Furthermore, we identify a pattern of dissociation in the strongest functional connections between anterior and posterior brain regions such that anterior functional connectivity increases in strength in premanifest Huntington's disease, while posterior functional connectivity decreases. Interpretation Our findings demonstrate that organizational principles of white matter underlie changes in functional connectivity in premanifest Huntington's disease. Furthermore, we demonstrate functional antero–posterior dissociation that is in keeping with the caudo–rostral gradient of striatal pathology in HD

Влияние мультидисциплинарного подхода и маршрутизации пациентов на результаты лечения больных гепатоцеллюлярным раком

Introduction: Hepatocellular cancer (HCC) is the sixth most common form of cancer worldwide and the third most common cause of cancer death. The long-term results of treatment are influenced by organizational decisions aimed at increasing the availability of specialized care. The purpose of our study is to evaluate the impact of rerouting and the introduction of a multidisciplinary approach on overall survival (OS) of patients with HCC.Materials and methods. A retrospective study was conducted to study the treatment tactics and results of treatment in all patients with HCC registered in the Sverdlovsk Regional Oncological Cancer Registry with a diagnosis of HCC from 2015 to 2021. A comparative analysis was carried out in 3 groups of patients: the control group, before changes, group of patients after changes in routing, and a group of patients with multidisciplinary approach involving a gastroenterologist / hepatologist and an interventional oncologist.Results: There is an increase in the number of patients who received advice from an oncologist and referred for specialized treatment: 12.0 %, 19.5 % and 34.9 %, respectively (p < 0.001). Median OS increased significantly only in the multidisciplinary approach group of 3.1 versus 2.2 months (p = 0.002). Low overall survival rates are associated with a large proportion of late stages. A significant increase in OS was registered in the group of patients with BCLC C stage: from 3.4 months to 12.5 months (p = 0.046).Conclusions: Simplifying the patient’s route from a gastroenterologist to an oncologist shortens the time to treatment start, increases the number of patients who receive advisory assistance in an oncological dispensary, but does not affect OS. A multidisciplinary approach allows more patients to receive specialized care. At the same time, the maximum effect on OS is noted at the BCLC C stage.Введение: Гепатоцеллюлярный рак (ГЦР) является шестой наиболее распространенной формой рака во всем мире и третьей наиболее распространенной причиной смертности от рака. На отдаленные результаты лечения влияют организационные решения, направленные на повышение доступности специализированной помощи. Цель нашего исследования — оценка влияния изменения маршрутизации и внедрения мультидисциплинарного подхода на общую выживаемость (ОВ) пациентов с ГЦР.Материалы и методы: Проведено ретроспективное исследование по изучению лечебной тактики и результатов лечения у всех пациентов с ГЦР, поставленных на учет в Свердловский областной онкологический канцер-регистр с диагнозом ГЦР с 2015 г. по 2021 г. Проведен сравнительный анализ в 3 группах пациентов: группа контроля (до изменений в маршрутизации), группа пациентов после изменений в маршрутизации и группа больных с мультидисциплинарным подходом с подключением гастроэнтеролога / гепатолога и интервенционного онколога.Результаты: Отмечается увеличение числа пациентов, получивших консультацию онколога и направленных на специализированное лечение: 12,0 %, 19,5 % и 34,9 % соответственно (р < 0,001). Медиана ОВ статистически значимо выросла только в группе с мультидисциплинарным подходом — 3,1 мес. против 2,2 мес. (р = 0,002). Низкие показатели общей выживаемости связаны с большим удельным весом поздних стадий. Значимое увеличение ОВ было в группе пациентов со стадией BCLC C: с 3,4 мес. до 12,5 мес. (р = 0,046).Выводы. Упрощение маршрута пациента от гастроэнтеролога до онколога сокращает сроки до начала лечения, увеличивает число больных, которые получают консультативную помощь в онкологическом диспансере, но не влияет на ОВ. Мультидисциплинарный подход позволяет большему числу больных получить специализированную помощь. При этом максимальное влияние на ОВ отмечается при стадии BCLC C.

Lenvatinib Therapy in Patients with Unresectable Hepatocellular Carcinoma in Real Clinical Practice

Aim. To determine lenvatinib treatment outcomes in patients with advanced unresectable hepatocellular carcinoma (uHCC) in real clinical practice.Patients and methods. A multicenter retrospective observational study included 58 patients with a confirmed uHCC diagnosis receiving lenvatinib. At baseline, ECOG, Child-Pugh and BCLC scores were assessed. The objective response rate (ORR), disease control rate (DCR), median overall survival (OS) and median progression-free survival (PFS) rates were assessed. In addition, adverse effects (AE) during treatment were monitored.Results. The median OS and PFS comprised 14.6 (95 % CI 10.6–18.6) and 11.1 months (95 % CI 8.31–13.8), respectively. The ORR amounted to 32.8 %, while DCR reached the level of 79.3 %. The levels of ORR and DCR were not statistically significantly different between the patients with stages B and C according to the BCLC staging system, with grades 0 and 1 according to ECOG, with classes A and B according to the Child-Pugh score, with viral and non-viral HCC etiology, with and without extrahepatic spread, and with and without portal vein invasion. Patients with alpha-fetoprotein (AFP) blood levels <200 ng/mL showed significantly higher ORR and DCR compared to those with AFP levels >200 ng/mL (44.4 % vs. 13.6 %, p = 0.015; and 88.9 % vs. 63.6 %, p = 0.021, respectively). The uHCC stage according to BCLC, ECOG functional status, Child-Pugh class, presence or absence of extrahepatic spread and viral etiology had no effect on the OS and PFS median levels. Patients with macroscopic portal vein invasion had a significantly lower PFS compared with those lacking this complication: 3.97 (0.00-8.07) vs. 11.1 (8.46-13.7), p = 0.053. AFP levels ≥200 ng/mL adversely affected survival rates: median OS comprised 12.0 (5.95-18.9) months in the group of patients with AFP ≥200 ng/mL vs. 16.1 (8.73-23.5) months in the group of patients having AFP <200 ng/mL, p = 0.020. AEs were registered in 81.0% (n = 47) of patients. Among the most common AEs were arterial hypertension (32.8 %), weakness (24.1 %), weight loss (12.1 %) and appetite loss (10.3 %). Due to AEs, Lenvatinib was withdrawn in 5 (8.6 %) patients.Conclusion. Lenvatinib confirmed its efficacy and safety in patients with uHCC in real clinical practice. The treatment outcome might be affected by AFP levels and the presence of macroscopic portal vein invasion. Further comparative studies into treatment regimens applied in real clinical practice are required

Злокачественные новообразования, ассоциированные с беременностью: популяционное исследование

Introduction: Pregnancy-associated cancers are malignant neoplasms (MNs) diagnosed during pregnancy or within 12 months after delivery. The study aimed to evaluate the prevalence of pregnancy-associated cancers at the population level.Materials and methods: The population database was created from databases of two regional systems: the automated system Pregnancy Monitoring Program and the ONCOR system.Results: Overall, 357 female patients with pregnancy-associated cancers were identified. The incidence rate was 1.659 per 1,000 pregnancies. The most common MN localizations were cervix, breast, thyroid, and ovary. Most female patients were between 45 and 49 years of age. Stage IV breast cancer was identified in a large proportion of women undergoing delivery. Conversely, cervical and ovarian cancers were commonly diagnosed at early stages. The 1-year survival rate of patients with pregnancy-associated cancers was higher than in the entire cancer population.Conclusions: Pregnancy-associated cancers are still a complex and poorly understood issue that requires additional studies, disease-specific monitoring and treatment protocols.Введение: К злокачественным новообразованиям (ЗНО), ассоциированным с беременностью, относят ЗНО, выявленные во время беременности или в течение 12 месяцев после родов. Целью работы была оценка частоты встречаемости ЗНО, ассоциированных с беременностью, на популяционном уровне.Материалы и методы: Для формирования популяционной базы данных использовались информационные массивы из двух региональных систем: автоматизированной системы «Программа мониторинга беременных» и системы «ОНКОР».Результаты: ЗНО, ассоциированные с беременностью, были выявлены у 357 пациенток. Заболеваемость составила 1,659 на 1000 беременностей. Наиболее распространенными локализациями ЗНО были шейка матки, молочная железа, щитовидная железа, яичник. Наибольшая заболеваемость пришлась на возрастную группу 45–49 лет. В случае рака молочной железы у женщин на фоне родов заболевание часто выявлялось на IV стадии. При ЗНО шейки матки и яичников, наоборот, отмечен высокий процент начальных стадий. Сравнение 1‑годичной выживаемости у больных с ЗНО, ассоциированными с беременностью, и в общей популяции показало, что выживаемость при беременности была выше.Выводы: Ассоциирование ЗНО с беременностью остается сложным и малоизученным вопросом, требующим проведения дополнительных исследований и формирования протоколов наблюдения и лечения по каждой нозологии