Insights into the self-assembly of pi-conjugated systems

In Chapter 1 supramolecular polymerization of small molecules is introduced and exemplified. The self-assembly of these single component systems, for example by hydrogen bonding and p-p interactions, has reached a high level of understanding. However for the fabrication of complex architectures with specific properties, a perfect organization of multiple components in the aggregates needs to be achieved. In order to arrive at such complex architectures more insight into the specificities of the self- assembly of p-conjugated systems is a necessity, which is the subject of this thesis. A covalent strategy for the spatial organization of p-conjugated components by placement on a foldameric scaffold is discussed in Chapter 2. Although the synthesis of these foldamers was successful, the 30 synthetic steps make the synthesis of a specific foldamer a lengthy process while only achieving relatively small structures. It was found that placing the chromophores in a defined three dimensional environment hampered a uniform description of the charge transfer characteristics within the current theoretical models. Furthermore, the level of complexity in these synthetic systems pales in comparison with the examples found in Nature. A more promising approach could be the self-assembly of molecules. Supramolecular polymerization via the cooperative mechanism is generally observed for the examples discussed in this thesis. Cooperative mechanisms have a nucleation event preceding the formation of the selfassembled structures. The evidence presented in Chapter 3 showed that the nucleation of oligothiophenes can be either homogeneous or heterogeneous in nature, depending on sample purity. Especially when the supramolecular interactions are counterbalancing each other, heterogeneous nucleation is likely to be present. By changing the self-assembly protocol, e.g. altering the cooling rate and method, the outcome of the self-assembly was remarkably different. This showed that the oligothiophenes were able to self-assemble into multiple different structures thereby displaying the presence of a complex energy landscape in analogy to polymorphism in the crystallization of molecules and to protein folding. At higher concentration the internal structure of the oligothiophene assemblies was resolved by using a combination of small-angle X-ray scattering and linear birefringence on magnetically aligned assemblies as discussed in Chapter 4. Optical and chiroptical studies showed that the cooperative nature of the self-assembly at this higher concentration was maintained. Cylindrical assemblies were observed, where the thiophene molecules were radially organized and p-p stacking was in a tangential direction. Chapter 5 discusses the use of circular dichroism (CD) spectroscopy to monitor the coassembly of multiple components. For the achiral oligo(p-phenylene vinylene) ureidotriazine systems, artificial CD effects are observed for the self-assembled structures in dodecane that are a result of unwanted linear dichroic (LD) effects as a consequence of flow induced alignment in the cuvette. These LD artifacts interfere with the CD measurements. By using methylcyclohexane (MCH) the LD effects are absent and the observed CD effects can be considered as a result of exciton coupling between the chromophores. The influence of two preparation methods on the organization of mixed assemblies consisting of achiral and chiral OPV derivatives with different oligomeric length is studied with CD spectroscopy in MCH. The first preparation method allows the molecules to be fully mixed at high temperature, while subsequent cooling shows chiral amplification of the achiral longer oligomer by the shorter chiral oligomer. The study indicates the formation of enriched clusters of one of the two components either within one stack, or as separate stacks. The second method is based on the addition of the shorter oligomer at a temperature, where the assemblies of the achiral oligomer are already present and kinetically inert. In this case the chiral amplification is reduced, indicating the formation of more enriched clusters of compounds. However, to arrive at an unambiguous assignment of the internal structure of the assemblies, the CD data should be combined with complementary techniques. In Chapter 6 the influence of reduced dynamics on the self-assembly of p-conjugated star shaped molecules is discussed. Two different types of aggregates, A1 and A2, are distinguished for the selfassembly in MCH, where a transition from A1 to A2 occurs, thereby showing the increased thermodynamic stability of the A2 aggregate. The addition of a good solvent enhances the rate of the transition. SEC in these alkane solvents shows that the A1 state in the solvent mixture (4:1MCH/toluene) was far more dynamic than in pure MCH. Furthermore, it reveals an unusually high monomer content of 15 mol% in the solvent mixture, which is confirmed by 1H NMR. As expected, in pure MCH the monomer content is much lower and for that reason it could not be detected. In order to investigate the origin of the A1–A2 transition, the two enantiomers are mixed and chiral amplification in the form of the Majority Rules effect is shown to occur in MCH, while it is only weakly present in the solvent mixture. The presence of the other enantiomer significantly slows down the kinetics of the transition from A1 to A2, where the transition only occurred in the solvent mixture. Quite remarkably, chiral amplification is absent in the A2 state in the solvent mixture. The combined results suggest that the A1–A2 transition is related to a tightening of the internal structure, where the transition is facilitated by the presence of an enantiomerically pure cluster. In general the research described in this thesis reveals some important parameters that determine the self-assembly process and its outcome. Reflection on these parameters and their implication on the design and synthesis of complex multicomponent supramolecular architectures is given in an epilogue to this thesis

An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker’s MALDI Sepsityper kit, the commercially available Molzym’s MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification

Oropharyngeal Chlamydia trachomatis in women; Spontaneous clearance and cure after treatment (FemCure)

Objectives: Women attending STI clinics are not routinely tested for oropharyngeal Chlamydia trachomatis (CT) infections. We aimed to assess spontaneous clearance of oropharyngeal CT and cure after antibiotic treatment in women. Methods: Women with vaginal or rectal CT (n=560) were recruited at STI clinics in 2016-2017, as part of the FemCure study (prospective cohort study). We included participants' data from week -1, that is, the diagnosis at initial visit, when clinics applied selective oropharyngeal testing. At week -1, a total of 241 women were oropharyngeally tested (30 positive) and 319 were untested. All FemCure participants provided nurse-collected oropharyngeal samples at study enrolment, that is, week 0, just prior to treatment (n=560), and after treatment at weeks 4 (n=449), 8 (n=433) and 12 (n=427). Samples were tested by nucleic acid amplification test, and at week 0 also by viability testing by viability PCR. Proportions of oropharyngeal CT test results were presented to represent spontaneous clearance and cure. Results: Of 30 women diagnosed with oropharyngeal CT at week -1, fifteen (50%) were negative at week 0 after a median of 9 days, that is, € spontaneous clearance'. At week 0, a total of 560 participants were tested, and 46 (8.8%) were oropharyngeal CT positive; 12 of them (26.1%) had viable CT. Of the 46 positive, 36 women had an oropharyngeal test after treatment; 97.2% (35/36) were negative at week 4, that is, € cure'. Of all women with follow-up visits, the proportion of oropharyngeal CT positive was between 0.5% and 1.6% between weeks 4 and 12. Of those not tested at week -1 (n=319), 8.5% (n=27) were oropharyngeal positive at week 0. Conclusions: The clinical importance of oropharyngeal CT in women is debated. We demonstrated that spontaneous clearance of oropharyngeal CT among women is common; of those who did not clear for CT, three-quarters had non-viable CT. After regular treatment with azithromycin or doxycycline, cure rate (97%) of oropharyngeal CT is excellent. Trial registration number: NCT02694497

Spontaneous clearance of Chlamydia trachomatis accounting for bacterial viability in vaginally or rectally infected women (FemCure)

Objectives: Spontaneous clearance of Chlamydia trachomatis (CT) infections can occur between diagnosis and treatment. We followed CT patien

Advancing COVID-19 diagnostics:rapid detection of intact SARS-CoV-2 using viability RT-PCR assay

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness.</p

Design of the FemCure study: prospective multicentre study on the transmission of genital and extra-genital Chlamydia trachomatis infections in women receiving routine care

BACKGROUND: In women, anorectal infections with Chlamydia trachomatis (CT) are about as common as genital CT, yet the anorectal site remains largely untested in routine care. Anorectal CT frequently co-occurs with genital CT and may thus often be treated co-incidentally. Nevertheless, post-treatment detection of CT at both anatomic sites has been demonstrated. It is unknown whether anorectal CT may play a role in post-treatment transmission. This study, called FemCure, in women who receive routine treatment (either azithromycin or doxycycline) aims to understand the post-treatment transmission of anorectal CT infections, i.e., from their male sexual partner(s) and from and to the genital region of the same woman. The secondary objective is to evaluate other reasons for CT detection by nucleic acid amplification techniques (NAAT) such as treatment failure, in order to inform guidelines to optimize CT control. METHODS: A multicentre prospective cohort study (FemCure) is set up in which genital and/or anorectal CT positive women (n = 400) will be recruited at three large Dutch STI clinics located in South Limburg, Amsterdam and Rotterdam. The women self-collect anorectal and vaginal swabs before treatment, and at the end of weeks 1, 2, 4, 6, 8, 10, and 12. Samples are tested for presence of CT-DNA (by NAAT), load (by quantitative polymerase chain reaction -PCR), viability (by culture and viability PCR) and CT type (by multilocus sequence typing). Sexual exposure is assessed by online self-administered questionnaires and by testing samples for Y chromosomal DNA. Using logistic regression models, the impact of two key factors (i.e., sexual exposure and alternate anatomic site of infection) on detection of anorectal and genital CT will be assessed. DISCUSSION: The FemCure study will provide insight in the role of anorectal chlamydia infection in maintaining the CT burden in the context of treatment, and it will provide practical recommendations to reduce avoidable transmission. Implications will improve care strategies that take account of anorectal CT. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02694497

Participation, retention, and associated factors of women in a prospective multicenter study on Chlamydia trachomatis infections (FemCure)

Prospective studies are key study designs when attempting to unravel health mechanisms that are widely applicable. Understanding the internal validity of a prospective study is essential to judge a study's quality. Moreover, insights in possible sampling bias and the external validity of a prospective study are useful to judge the applicability of a study's findings. We evaluated participation, retention, and associated factors of women in a multicenter prospective cohort (FemCure) to understand the study's validity.Chlamydia trachomatis (CT) infected adult women, negative for HIV, syphilis, and Neisseria gonorrhoeae were eligible to be preselected and included at three sexually transmitted infection (STI) clinics in the Netherlands (2016-2017). The planned follow-up for participants was 3 months, with two weekly rectal and vaginal CT self-sampling and online questionnaires administered at home and at the clinic. We calculated the proportions of preselected, included, and retained (completed follow-up) women. Associations with non-preselection, noninclusion, and non-retention (called attrition) were assessed (logistic and Cox regression).Among the 4,916 women, 1,763 (35.9%) were preselected, of whom 560 (31.8%) were included. The study population had diverse baseline characteristics: study site, migration background, high education, and no STI history were associated with non-preselection and noninclusion. Retention was 76.3% (n = 427). Attrition was 10.71/100 person/month (95% confidence interval 9.97, 12.69) and was associated with young age and low education. In an outpatient clinical setting, it proved feasible to include and retain women in an intensive prospective cohort. External validity was limited as the study population was not representative (sampling bias), but this did not affect the internal validity. Selective attrition, however (potential selection bias), should be accounted for when interpreting the study results

Development of a Flow-Trough Microarray based Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Assay for the Detection of European Bunyaviruses

It is suspected that apart from tick-borne encephalitis virus several additional European Arboviruses such as the sandfly borne Toscana virus, sandfly fever Sicilian virus and sandfly fever Naples virus, mosquito-borne Tahyna virus, Inkoo virus, Batai virus and tick-borne Uukuniemi virus cause aseptic meningo-encephalitis or febrile disease in Europe. Currently, the microarray technology is developing rapidly and there are many efforts to apply it to infectious diseases diagnostics. In order to arrive at an assay system useful for high throughput analysis of samples from aseptic meningo-encephalitis cases the authors developed a combined multiplex ligation-dependent probe amplification and flow-through microarray assay for the detection of European Bunyaviruses. These results show that this combined assay indeed is highly sensitive, and specific for the accurate detection of multiple viruses