Distinct evolutionary trajectories of SARS-CoV-2-interacting proteins in bats and primates identify important host determinants of COVID-19.

The coronavirus disease 19 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a coronavirus that spilled over from the bat reservoir. Despite numerous clinical trials and vaccines, the burden remains immense, and the host determinants of SARS-CoV-2 susceptibility and COVID-19 severity remain largely unknown. Signatures of positive selection detected by comparative functional genetic analyses in primate and bat genomes can uncover important and specific adaptations that occurred at virus-host interfaces. We performed high-throughput evolutionary analyses of 334 SARS-CoV-2-interacting proteins to identify SARS-CoV adaptive loci and uncover functional differences between modern humans, primates, and bats. Using DGINN (Detection of Genetic INNovation), we identified 38 bat and 81 primate proteins with marks of positive selection. Seventeen genes, including the ACE2 receptor, present adaptive marks in both mammalian orders, suggesting common virus-host interfaces and past epidemics of coronaviruses shaping their genomes. Yet, 84 genes presented distinct adaptations in bats and primates. Notably, residues involved in ubiquitination and phosphorylation of the inflammatory RIPK1 have rapidly evolved in bats but not primates, suggesting different inflammation regulation versus humans. Furthermore, we discovered residues with typical virus-host arms race marks in primates, such as in the entry factor TMPRSS2 or the autophagy adaptor FYCO1, pointing to host-specific in vivo interfaces that may be drug targets. Finally, we found that FYCO1 sites under adaptation in primates are those associated with severe COVID-19, supporting their importance in pathogenesis and replication. Overall, we identified adaptations involved in SARS-CoV-2 infection in bats and primates, enlightening modern genetic determinants of virus susceptibility and severity.

Loss of intermediate filament IFB-1 reduces mobility, density, and physiological function of mitochondria in Caenorhabditis elegans sensory neurons.

Mitochondria and intermediate filament (IF) accumulations often occur during imbalanced axonal transport leading to various types of neurological diseases. It is still poorly understood whether a link between neuronal IFs and mitochondrial mobility exist. In Caenorhabditis elegans, among the 11 cytoplasmic IF family proteins, IFB-1 is of particular interest as it is expressed in a subset of sensory neurons. Depletion of IFB-1 leads to mild dye-filling and significant chemotaxis defects as well as reduced life span. Sensory neuron development is affected and mitochondrial transport is slowed down leading to reduced densities of these organelles. Mitochondria tend to cluster in neurons of IFB-1 mutants likely independent of the fission and fusion machinery. Oxygen consumption and mitochondrial membrane potential is measurably reduced in worms carrying mutations in the ifb-1 gene. Membrane potential also seems to play a role in transport such as carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone treatment led to increased directional switching of mitochondria. Mitochondria co-localize with IFB-1 in worm neurons and appear in a complex with IFB-1 in pull-down assays. In summary, we propose a model in which neuronal IFs may serve as critical (transient) anchor points for mitochondria during their long-range transport in neurons for steady and balanced transport.

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors.

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

On the Mechanism of Self-Assembly of Fibrinogen in Thrombin-free Aqueous Solution.

Fibrinogen dissolved in 0.12 M aqueous NaCl solution at a pH of 6.6 exhibits self-assembly in response to a lowering of the NaCl concentration to values equal to or lower than 60 mM. As has been established in a preceding work (Langmuir 2019, 35, and 12113), a characteristic signature of the self-assembly triggered by a drop in ionic strength is the formation of large globular particles. Growth of these particles most likely obeys a coalescence-like process also termed a step growth process. In order to extend this knowledge, the present work first optimized the protocol, leading to highly reproducible self-assembly experiments. Based on this optimization, the work succeeded in identifying an initial stage, not yet accessible, during which rigid short fibrils grow in close analogy to the thrombin-catalyzed polymerization of fibrin. In addition, first suggestions could be made on the transformation of these fibrils into larger aggregates, which upon drying turn into thick fiber-like ropes.

Non-pharmaceutical interventions to reduce COVID-19 transmission in the UK: a rapid mapping review and interactive evidence gap map.

BACKGROUND: Non-pharmaceutical interventions (NPIs) were crucial in the response to the COVID-19 pandemic, although uncertainties about their effectiveness remain. This work aimed to better understand the evidence generated during the pandemic on the effectiveness of NPIs implemented in the UK. METHODS: We conducted a rapid mapping review (search date: 1 March 2023) to identify primary studies reporting on the effectiveness of NPIs to reduce COVID-19 transmission. Included studies were displayed in an interactive evidence gap map. RESULTS: After removal of duplicates, 11 752 records were screened. Of these, 151 were included, including 100 modelling studies but only 2 randomized controlled trials and 10 longitudinal observational studies.Most studies reported on NPIs to identify and isolate those who are or may become infectious, and on NPIs to reduce the number of contacts. There was an evidence gap for hand and respiratory hygiene, ventilation and cleaning. CONCLUSIONS: Our findings show that despite the large number of studies published, there is still a lack of robust evaluations of the NPIs implemented in the UK. There is a need to build evaluation into the design and implementation of public health interventions and policies from the start of any future pandemic or other public health emergency.

Help-seeking following a flooding event: a cross-sectional analysis of adults affected by flooding in England in winter 2013/14.

BACKGROUND: Flooding can cause long-term, significant impacts on mental health in affected populations. We explored help-seeking behaviour of households affected by flooding. METHODS: A cross-sectional analysis was conducted on National Study of Flooding and Health data on households flooded in England in winter 2013/14. Participants (Year 1: n = 2006; Year 2: n = 988; Year 3: n = 819) were asked if they sought help from health services and other sources. Logistic regression was conducted to calculate odds ratios (ORs) of help-seeking in flooded and disrupted participants compared to unaffected, adjusted for a priori confounders. RESULTS: The odds of seeking help from any source 1 year after flooding were greater for flooded participants [adjusted OR (aOR): 1.71, 95% confidence interval (CI): 1.19-1.45] and those disrupted by flooding (aOR: 1.92, 95% CI: 1.37-2.68) compared to unaffected participants. This continued in the second year (flooded: aOR 6.24, 95% CI: 3.18-13.34; disrupted: aOR: 2.22, 95% CI: 1.14-4.68), and help-seeking remained greater in flooded than unaffected participants in the third year. Flooded and disrupted participants were particularly likely to seek help from informal sources. Help-seeking was more prevalent amongst participants with mental health outcomes, but a notable proportion of individuals with any mental health outcome did not seek help (Year 1: 15.0%; Year 2: 33.3%; Year 3: 40.3%). CONCLUSIONS: Flooding is associated with increased demand for formal and informal support, persisting for at least 3 years, and an unmet need for help amongst affected individuals. Our findings should be considered in flood response planning to reduce the long-term adverse health impacts of flooding.

Characterization of TAG-63 and its role on axonal transport in C. elegans.

Model organisms are increasingly used to study and understand how neurofilament (NF)-based neurological diseases develop. However, whether a NF homolog exists in C. elegans remains unclear. We characterize TAG-63 as a NF-like protein with sequence homologies to human NEFH carrying various coiled coils as well as clustered phosphorylation sites. TAG-63 also exhibits features of NFL such as a molecular weight of around 70 kD, the lack of KSP repeats and the ability to form 10 nm filamentous structures in transmission electron micrographs. An anti-NEFH antibody detects a band at the predicted molecular weight of TAG-63 in Western blots of whole worm lysates and this band cannot be detected in tag-63 knockout worms. A transcriptional tag-63 reporter expresses in a broad range of neurons, and various anti-NFH antibodies stain worm neurons with an overlapping expression of axonal vesicle transporter UNC-104(KIF1A). Cultured neurons grow shorter axons when incubating with drugs known to disintegrate the NF network and rhodamine-labeled in vitro reconstituted TAG-63 filaments disintegrate upon drug exposure. Speeds of UNC-104 motors are diminished in tag-63 mutant worms with visibly increased accumulations of motors along axons. UNC-104/TAG-63 and SNB-1/TAG-63 not only colocalize in neurons but also revealed positive BiFC (bimolecular fluorescence assay) signals. In summary, we identified and characterized TAG-63 in C. elegans, and demonstrate that lack of this protein limits axonal transport efficiencies. Additionally, this study would aid in developing NF-related disease models in the future.

Targeted Synthesis of the Type-A Particle Substructure from Enzymatically Produced Eumelanin.

Eumelanin exhibits a defined supramolecular buildup that is deprived of at least three distinct particle species. To enable the full potential of its promising material properties, access to all particle types is crucial. In this work, the first protocol for the synthesis of the intermediate type-A particles in pure and stable dispersion form is described. It is found that aggregation of type-A particles into the larger type-B variant can be inhibited by a strict pH control during the synthesis. The exact influence of pH on the supramolecular buildup is investigated via a combination of time-resolved light scattering, electron microscopy, and UV-vis spectroscopy. It is observed that a rapid buildup of type-B particles occurs without pH control and is generally dominant at lower pH values. At pH values above 6.2 however, type-A particles are gained, and no further aggregation occurs. Even more, lowering the pH of such a stable type-A dispersion at a later stage lifts the inhibition and again leads to the formation of larger particle species. The results confirm that it is easily possible to halt the aggregation of eumelanin substructures and to access them in the form of a stable dispersion. Moreover, a profound additional understanding of the supramolecular buildup is gained by the in-depth investigation of the pH influence.

Learning about COVID-19 across borders: public health information and adherence among international travellers to the UK.

OBJECTIVE: Public health control measures at borders have long been central to national strategies for the prevention and containment of infectious diseases. Travel was inevitably associated with the rapid global transmission of COVID-19. In the UK, public health authorities tried to reduce the risks of travel-associated spread by providing public health information at ports of entry. This study investigates risk assessment processes, decision-making and adherence to official advice among international travellers, to provide evidence for future policy on the provision of public health information to facilitate safer international travel. STUDY DESIGN: This study is a qualitative study evaluation. METHOD: International air passengers arriving at the London Heathrow Airport on scheduled flights from China and Singapore were approached for interview after consenting to contact in completed surveys. Semi-structured interviews were conducted by telephone, using two topic guides to explore views of official public health information and self-isolation. Interview transcripts were coded and analysed thematically. RESULTS: Participants regarded official advice from Public Health England as adequate at the time, despite observing differences with intervention measures implemented in their countries of departure. Most participants also described adopting precautionary measures, including self-isolation and the use of face coverings that went beyond official advice, but reported adherence to guidance on contacting health authorities was more variable. Adherence to the official guidance was informed by the perceived salience of specific transmission possibilities and containment measures assessed in relation to participants' local social and institutional environments. CONCLUSION: Analysis of study findings demonstrates that international air travellers' responses to public health advice constitute a proactive process of risk assessment and rationalised decision-making to guide preventive action. This process incorporates consideration of the current living situation, trust in information sources, correspondence with cultural logics and willingness to accept potential risk to self and significant others. Our findings concerning international passengers' understanding of, and compliance with, official advice and mitigation measures provide valuable evidence to inform future policy and generate recommendations on the presentation of public health information to facilitate safer international travel. Access to a central source of regularly updated official information would help minimise confusion between different national guidelines. Greater attention to the differentiated information needs of diverse groups in creating future public-facing guidance would help to minimise the uncertainties generated by the receipt of generic information.

Self-Assembled Fibrinogen Hydro- and Aerogels with Fibrin-like 3D Structures.

The natural blood protein fibrinogen is a highly potent precursor for the production of various biomaterials due to its supreme biocompatibility and cell interaction. To gain actual materials from fibrinogen, the protein needs to undergo fibrillogenesis, which is mostly triggered via enzymatic processing to fibrin, electrospinning, or drying processes. All of those techniques, however, strongly limit the available structures or the applicability of the material. To overcome the current issues of fibrin(ogen) as material, we herein present a highly feasible, quick, and inexpensive technique for self-assembly of fibrinogen in solution into defined, nanofibrous three-dimensional (3D) patterns. Upon interaction with specific anions in controlled environments, stable and flexible hydrogel-like structures are formed without any further processing. Moreover, the material can be converted into highly porous and elastic aerogels by lyophilization. Both of these material classes have never been described before from native fibrinogen. The observed phenomenon also represents the first enzyme-free process of fibrillogenesis from fibrinogen with significant yield in solution. The produced hydrogels and aerogels were investigated via electron microscopy, IR spectroscopy, and fluorescence spectroscopy, which also confirms the native state of the protein. Additionally, their mechanical properties were compared with actual fibrin and unstructured fibrinogen. The structural features show a striking analogy to actual fibrin, both as hydro- and aerogel. This renders the new material a highly promising alternative for fibrin in biomaterial applications. A much faster initiation of fiber formation, exclusion of possible thrombin residuals, and low-cost reagents are great advantages.

Splicing-factor oncoprotein SRSF1 stabilizes p53 via RPL5 and induces cellular senescence.

Splicing and translation are highly regulated steps of gene expression. Altered expression of proteins involved in these processes can be deleterious. Therefore, the cell has many safeguards against such misregulation. We report that the oncogenic splicing factor SRSF1, which is overexpressed in many cancers, stabilizes the tumor suppressor protein p53 by abrogating its MDM2-dependent proteasomal degradation. We show that SRSF1 is a necessary component of an MDM2/ribosomal protein complex, separate from the ribosome, that functions in a p53-dependent ribosomal-stress checkpoint pathway. Consistent with the stabilization of p53, increased SRSF1 expression in primary human fibroblasts decreases cellular proliferation and ultimately triggers oncogene-induced senescence (OIS). These findings underscore the deleterious outcome of SRSF1 overexpression and identify a cellular defense mechanism against its aberrant function. Furthermore, they implicate the RPL5-MDM2 complex in OIS and demonstrate a link between spliceosomal and ribosomal components, functioning independently of their canonical roles, to monitor cellular physiology and cell-cycle progression.

Determining the acceptability of testing contacts of confirmed COVID-19 cases to improve secondary case ascertainment.

BACKGROUND: UK asymptomatic contacts of confirmed COVID-19 cases are not routinely tested for SARS-CoV-2. Testing contacts may improve case ascertainment and reduce onward transmission. This study investigated the acceptability of SARS-CoV-2 testing among contacts of confirmed cases as an integral part of the contact-tracing process. METHODS: A cross-sectional descriptive survey of case contacts was conducted in the UK. All contacts who completed a telephone call with the NHS Test and Trace Agile Lighthouse team were eligible for inclusion and were offered a molecular test. Consenting participants were sent a self-swab kit. RESULTS: Of the 1523 individuals contacted, 602 (39.5%) accepted the test offer. Of the 240 (39.9%) samples returned for testing, 16.3% tested polymerase chain reaction-positive for SARS-CoV-2.Most individuals who declined with a reason (638/905; 70.5%) reported they had already taken or booked a SARS-CoV-2 test, or were part of a testing programme. Matched laboratory records confirmed 73.1% of those who declined were tested by another route. CONCLUSIONS: Most case contacts were tested, either through arranging a test by themselves or by accepting the study offer. Results demonstrate high acceptability, with substantial test positivity, indicating that there is public health benefit in offering tests to contacts as a routine part of the contact-tracing process.

Public health information on COVID-19 for international travellers: lessons learned from a mixed-method evaluation.

OBJECTIVES: In the containment phase of the response to the COVID-19 outbreak, Public Health England (PHE) delivered advice to travellers arriving at major UK ports. We aimed to rapidly evaluate the impact and effectiveness of these communication materials for passengers in the early stages of the pandemic. STUDY DESIGN: The study design used is the mixed-methods evaluation. METHODS: A questionnaire survey and follow-up interviews with passengers arriving at London Heathrow Airport on scheduled flights from China and Singapore. The survey assessed passengers' knowledge of symptoms, actions to take, and attitudes towards PHE COVID-19 public health information; interviews explored their views of official public health information and self-isolation. RESULTS: One hundred and twenty-one passengers participated in the survey and 15 in follow-up interviews. Eighty three percentage of surveyed passengers correctly identified all three COVID-19 associated symptoms listed in PHE information at that time. Most could identify the recommended actions and found the advice understandable and trustworthy. Interviews revealed that passengers shared concerns about the lack of wider official action, and that passengers' knowledge had been acquired elsewhere as much from PHE. Respondents also noted their own agency in choosing to self-isolate, partially as a self-protective measure. CONCLUSION: PHE COVID-19 public health information was perceived as clear and acceptable, but we found that passengers acquired knowledge from various sources and they saw the provision of information alone on arrival as an insufficient official response. Our study provides fresh insights into the importance of taking greater account of diverse information sources and of the need for public assurance in creating public health information materials to address global health threats.

Impact of the novel powered circular stapler on risk of anastomotic leakage in colorectal anastomosis: a propensity score-matched study.

BACKGROUND: Several risk factors for anastomotic leakage (AL) following colorectal surgery have been described. Improvement in devices for performing anastomosis is a modifiable factor that could reduce AL rates. The aim of this study was to assess the impact of technical improvements in the Echelon Circular™ powered stapler (ECPS) on the left-sided colorectal AL rate compared to current manual circular staplers (MCS). METHODS: A cohort study was carried out on consecutive patients between January 2017 and February 2020 in whom left-sided stapled colorectal anastomosis above 5 cm from anal verge was performed. The primary end point was the risk of AL depending on the type of circular stapler used. The ECPS cases were matched to MCS cases by propensity score matching to obtain comparable groups of patients. RESULTS: Two hundred seventy-nine patients met the inclusion criteria. A MCS anastomosis was performed in 218 patients and ECPS anastomosis in 61 (21.9%). Overall, AL was observed in 25 (9%) cases. Factors significantly associated with AL were American Society of Anesthesiologists score (p = 0.025) and type of circular stapler used (p = 0.021). After adjusting the cases with propensity score matching (119 cases MCS versus 60 ECPS), AL was observed in 14 (11.8%) patients in MCS group and in 1 (1.7%) patient in the ECPS group (p = 0.022). AL in the MCS group required reoperation in seven cases (5.8%), the remaining seven patients were treated conservatively. The patient in the ECSP group required an urgent Hartmann's procedure CONCLUSIONS: The ECPS device could have a positive impact by reducing AL rates in left-sided colorectal anastomosis. Multicenter controlled trials are needed for stronger evidence to change practice.

Dissolved hyperpolarized xenon-129 MRI in human kidneys.

PURPOSE: To assess the feasibility of using dissolved hyperpolarized xenon-129 (129 Xe) MRI to study renal physiology in humans at 3 T. METHODS: Using a flexible transceiver RF coil, dynamic and spatially resolved 129 Xe spectroscopy was performed in the abdomen after inhalation of hyperpolarized 129 Xe gas with 3 healthy male volunteers. A transmit-only receive-only RF coil array was purpose-built to focus RF excitation and enhance sensitivity for dynamic imaging of 129 Xe uptake in the kidneys using spoiled gradient echo and balanced steady-state sequences. RESULTS: Using spatially resolved spectroscopy, different magnitudes of signal from 129 Xe dissolved in red blood cells and tissue/plasma could be identified in the kidneys and the aorta. The spectra from both kidneys showed peaks with similar amplitudes and chemical shift values. Imaging with the purpose-built coil array was shown to provide more than a 3-fold higher SNR in the kidneys when compared with surrounding tissues, while further physiological information from the dissolved 129 Xe in the lungs and in transit to the kidneys was provided with the transceiver coil. The signal of dissolved hyperpolarized 129 Xe could be imaged with both tested sequences for about 40 seconds after inhalation. CONCLUSION: The uptake of 129 Xe dissolved in the human kidneys was measured with spectroscopic and imaging experiments, demonstrating the potential of hyperpolarized 129 Xe MR as a novel, noninvasive technique to image human kidney tissue perfusion.

Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells.

Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

Factors associated with adherence to self-isolation and lockdown measures in the UK: a cross-sectional survey.

OBJECTIVES: To investigate factors associated with adherence to self-isolation and lockdown measures due to COVID-19 in the UK. STUDY DESIGN: Online cross-sectional survey. METHODS: Data were collected between 6th and 7th May 2020. A total of 2240 participants living in the UK aged 18 years or older were recruited from YouGov's online research panel. RESULTS: A total of 217 people (9.7%) reported that they or someone in their household had symptoms of COVID-19 (cough or high temperature/fever) in the last 7 days. Of these people, 75.1% had left the home in the last 24 h (defined as non-adherent). Men were more likely to be non-adherent, as were people who were less worried about COVID-19, and who perceived a smaller risk of catching COVID-19. Adherence was associated with having received help from someone outside your household. Results should be taken with caution as there was no evidence for associations when controlling for multiple analyses. Of people reporting no symptoms in the household, 24.5% had gone out shopping for non-essentials in the last week (defined as non-adherent). Factors associated with non-adherence and with a higher total number of outings in the last week included decreased perceived effectiveness of government 'lockdown' measures, decreased perceived severity of COVID-19 and decreased estimates of how many other people were following lockdown rules. Having received help was associated with better adherence. CONCLUSIONS: Adherence to self-isolation is poor. As we move into a new phase of contact tracing and self-isolation, it is essential that adherence is improved. Communications should aim to increase knowledge about actions to take when symptomatic or if you have been in contact with a possible COVID-19 case. They should also emphasise the risk of catching and spreading COVID-19 when out and about and the effectiveness of preventative measures. Using volunteer networks effectively to support people in isolation may promote adherence.

Insights on UNC-104-dynein/dynactin interactions and their implications on axonal transport in Caenorhabditis elegans.

Bidirectional cargo transport in neurons can be explained by two models: the "tug-of-war model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons.

Easily Accessible Protein Nanostructures via Enzyme Mediated Addressing.

Site-specific formation of nanoscaled protein structures is a challenging task. Most known structuring methods are either complex and hardly upscalable or do not apply to biological matter at all. The presented combination of enzyme mediated autodeposition and nanosphere lithography provides an easy-to-apply approach for the buildup of protein nanostructures over a large scale. The key factor is the tethering of enzyme to the support in designated areas. Those areas are provided via prepatterning of enzymatically active antidots with variable diameters. Enzymatically triggered protein addressing occurs exclusively at the intended areas and continues until the entire active area is coated. After this, the reaction self-terminates. The major advantage of the presented method lies in its easy applicability and upscalability. Large-area structuring of entire support surfaces with features on the nanometer scale is performed efficiently and without the necessity of harsh conditions. These are valuable premises for large-scale applications with potentials in biosensor technology, nanoelectronics, and life sciences.

Insight into the Final Step of the Supramolecular Buildup of Eumelanin.

The final step in the supramolecular buildup of eumelanin particles is investigated regarding the involved species and mechanism. Time-resolved in situ light scattering and scanning electron microscopy reveal an aggregation of particles with a narrow size distribution around 40 nm, previously only observed as substructures. These form larger particles with again very uniform size and diameters around 200 nm. Aggregation of each single particle takes only a few minutes to complete, whereas the entire process goes on for at least 3 h, partly due to the kinetics of the precursors. The individual particles also undergo an additional consolidation step toward their final form, which takes up to 24 h. Atomic force microscopy shows that the size before consolidation is around twice the size of the final particles, due to free space between the substructures. Light scattering also reveals that the aggregation is random with respect to where the particles attach, as the shape of aggregates changes from sphere to coil, before it returns to a spherical shape at the end. Application of enzyme mediated autodeposition finally shows the potential to stop the supramolecular buildup at each level, and therefore enables isolation of the respective eumelanin particles at will. This may enable the full potential for melanin materials in nanotechnology deriving from its unique (for biological polymers) properties like paramagnetism, electrical conductivity, and many more.