| 1. |
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| 2. |
- Baker, Joseph, et al.
(författare)
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Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:21
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that Class 1a pili of uropathogenic E. coli (UPEC), as well as Class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC Class 5 pili. Examining structural and steered molecular dynamics simulation data, we find this difference in Class 1 pili subunit behavior originates from an alpha-helical motif that can unfold when exposed to force. A disulfide bond cross-linking beta-strands in Class 1 pili stabilizes subunits, allowing them to tolerate higher forces than Class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, Class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of Class 5 ETEC pili.
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| 3. |
- Baker, Joseph, et al.
(författare)
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Unveiling the Contributions of Secondary Structure and Disulfide Bonds for Bacterial Adhesion Pili Extension using a Multiscale Approach
- 2021
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial adhesion pili are essential virulence factors for many pathogenic Escherichia coli, including bacteria that cause urinary tract infections (UPEC) and diarrheal diseases (ETEC). To sustain adhesion under forces similar to those in the fluid environments of the urinary tract and gastrointestinal tract, these pili (also called fimbriae) can extend to over seven times their original length. Both UPEC and ETEC can uncoil their quaternary structure under pulling force and re-coil to their helical form when the force is reduced, as observed using optical tweezers. However, after extension to a linear polymer UPEC undergo an additional reversible conformational change, that is not seen in ETEC. The mechanism for this conformational change in UPEC is not known. Therefore, to obtain a comprehensive picture of pilus extension we have taken a synergistic approach that combines optical tweezer experiments, structural data from cryo-EM, and steered molecular dynamics simulations to investigate the response of pilin subunits to force.Our multi-faceted approach provides novel molecular-scale insights into the structural changes that occur in UPEC and ETEC pili under pulling forces. We find that the conformational change observed in UPEC pili in optical tweezer experiments is correlated with the presence of an alpha helix. In addition, structural analysis and steered molecular dynamics simulations show that there is a disulfide bond that provides additional stability of UPEC pilin subunits that is not observed in ETEC pilins, which lack cysteine residues. Together, these results suggest that the mechanism of extension of bacterial adhesion pili is related to their environmental niche, and the magnitude of fluid forces in the urinary tract versus the GI tract.
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| 4. |
- Dahlberg, Joen, et al.
(författare)
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Capacity-Driven Automatic Design of Dynamic Aircraft Arrival Routes
- 2018
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Ingår i: 2018 IEEE/AIAA 37TH DIGITAL AVIONICS SYSTEMS CONFERENCE (DASC). - : IEEE. - 9781538641125 ; , s. 1194-1202
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Konferensbidrag (refereegranskat)abstract
- We present a Mixed-Integer Programming framework for the design of aircraft arrival routes in a Terminal Maneuvering Area (TMA) that guarantee temporal separation of aircraft. The output routes constitute operationally feasible merge trees, and guarantee that the overall traffic pattern in the TMA can be monitored by air traffic controllers; in particular, we ensure that all aircraft on the arrival routes are separated in time and all merge points are spatially separated. We present a proof of concept of our approach, and demonstrate its feasibility by experiments for arrival routes during one hour at Stockholm TMA.
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| 5. |
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| 6. |
- Dahlberg, Rasmus, et al.
(författare)
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Aggregation-Based Certificate Transparency Gossip
- 2019
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Ingår i: Proceedings of the The Thirteenth International Conference on Emerging Security Information, Systems and Technologies - SECURWARE 2019, October 27, 2019 to October 31, 2019 - Nice, France. - : International Academy, Research and Industry Association (IARIA). - 9781713800521
-
Konferensbidrag (refereegranskat)abstract
- Certificate Transparency (CT) requires that every certificate which is issued by a certificate authority must be publicly logged. While a CT log can be untrusted in theory, it relies on the assumption that every client observes and cryptographically verifies the same log. As such, some form of gossip mechanism is needed in practice. Despite CT being adopted by several major browser vendors, no gossip mechanism is widely deployed. We suggest an aggregation-based gossip mechanism that passively observes cryptographic material that CT logs emit in plain text, aggregating at packet processors (such as routers and switches) to periodically verify log consistency off-path. In other words, gossip is provided as-a-service by the network. Our proposal can be implemented for a variety of programmable packet processors at line-speed without aggregation distinguishers (throughput), and, based on 20 days of RIPE Atlas measurements that represent clients from 3500 autonomous systems, we show that significant protection against split-viewing CT logs can be achieved with a realistic threat model and an incremental deployment scenario.
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| 7. |
- Dahlberg, Rasmus, et al.
(författare)
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Efficient Sparse Merkle Trees : Caching Strategies and Secure (Non-)Membership Proofs
- 2016
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Ingår i: Secure IT Systems. - Cham : Springer. - 9783319475592 ; , s. 199-215
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Konferensbidrag (refereegranskat)abstract
- A sparse Merkle tree is an authenticated data structure based on a perfect Merkle tree of intractable size. It contains a distinct leaf for every possible output from a cryptographic hash function, and can be simulated efficiently because the tree is sparse (i.e., most leaves are empty). We are the first to provide complete, succinct, and recursive definitions of a sparse Merkle tree and related operations. We show that our definitions enable efficient space-time trade-offs for different caching strategies, and that verifiable audit paths can be generated to prove (non-)membership in practically constant time (<4 ms) when using SHA-512/256. This is despite a limited amount of space for the cache—smaller than the size of the underlying data structure being authenticated—and full (concrete) security in the multi-instance setting.
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| 8. |
- Dahlberg, Rasmus
(författare)
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On Certificate Transparency Verification and Unlinkability of Websites Visited by Tor Users
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Certificate Transparency is an ecosystem of logs, monitors, and auditors that hold certificate authorities accountable while issuing certificates. We show how the amount of trust that TLS clients and domain owners need to place in Certificate Transparency can be reduced, both in the context of existing gradual deployments and the largely unexplored area of Tor. Our contributions include improved third-party monitoring, a gossip protocol plugging into Certificate Transparency over DNS, an incrementally deployable gossip-audit model tailored for Tor Browser, and using certificates with onion addresses. The methods used range from proof sketches to Internet measurements and prototype evaluations. An essential part of our evaluation in Tor is to assess how the protocols used during website visits—such as requesting an inclusion proof from a Certificate Transparency log—affect unlinkability between senders and receivers. We find that most false positives in website fingerprinting attacks can be eliminated for all but the most frequently visited sites. This is because the destination anonymity set can be reduced due to how Internet protocols work: communication is observable and often involves third-party interactions. Some of the used protocols can further be subject to side-channel analysis. For example, we show that remote (timeless) timing attacks against Tor’s DNS cache reliably reveal the timing of past exit traffic. The severity and practicality of our extension to website fingerprinting pose threats to the anonymity provided by Tor. We conclude that access to a so-called website oracle should be an assumed attacker capability when evaluating website fingerprinting defenses.
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| 9. |
- Dahlberg, Rasmus, et al.
(författare)
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Privacy-Preserving & Incrementally-Deployable Support for Certificate Transparency in Tor
- 2021
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Ingår i: Proceedings on Privacy Enhancing Technologies Symposium. - : Sciendo. - 2299-0984. ; , s. 194-213
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Konferensbidrag (refereegranskat)abstract
- The security of the web improved greatly throughout the last couple of years. A large majority of the web is now served encrypted as part of HTTPS, and web browsers accordingly moved from positive to negative security indicators that warn the user if a connection is insecure. A secure connection requires that the server presents a valid certificate that binds the domain name in question to a public key. A certificate used to be valid if signed by a trusted Certificate Authority (CA), but web browsers like Google Chrome and Apple's Safari have additionally started to mandate Certificate Transparency (CT) logging to overcome the weakest-link security of the CA ecosystem. Tor and the Firefox-based Tor Browser have yet to enforce CT.In this paper, we present privacy-preserving and incrementally-deployable designs that add support for CT in Tor. Our designs go beyond the currently deployed CT enforcements that are based on blind trust: if a user that uses Tor Browser is man-in-the-middled over HTTPS, we probabilistically detect and disclose cryptographic evidence of CA and/or CT log misbehavior. The first design increment allows Tor to play a vital role in the overall goal of CT: detect mis-issued certificates and hold CAs accountable. We achieve this by randomly cross-logging a subset of certificates into other CT logs. The final increments hold misbehaving CT logs accountable, initially assuming that some logs are benign and then without any such assumption. Given that the current CT deployment lacks strong mechanisms to verify if log operators play by the rules, exposing misbehavior is important for the web in general and not just Tor. The full design turns Tor into a system for maintaining a probabilistically-verified view of the CT log ecosystem available from Tor's consensus. Each increment leading up to it preserves privacy due to and how we use Tor.
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| 10. |
- Dahlberg, Rasmus, et al.
(författare)
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Standardized Syslog Processing : Revisiting Secure Reliable Data Transfer and Message Compression
- 2016
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Today's computer logs are like smoking guns and treasure maps in case of suspicious system activities: they document intrusions, and log crucial information such as failed system updates and crashed services. An adversary thus has a clear motive to observe, alter, and delete log entries, considering that she could (i) start by using the log's content to identify new security vulnerabilities, and (ii) exploit them without ever being detected. With this in mind we consider syslog standards and open source projects that safeguard events during the storage and transit phases, and examine how data compression effects security. We conclude that there are syslog standards in place that satisfy security on a hop-by-hop basis, that there are no such standards for secure storage, and that message compression is not recommended during transit.
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| 11. |
- Dahlberg, Rasmus, et al.
(författare)
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Timeless Timing Attacks and Preload Defenses in Tor's DNS Cache
- 2023
-
Ingår i: Proceedings of the 32nd USENIX Security Symposium. - : USENIX - The Advanced Computing Systems Association. - 9781939133373 ; , s. 2635-2652
-
Konferensbidrag (refereegranskat)abstract
- We show that Tor's DNS cache is vulnerable to a timeless timing attack, allowing anyone to determine if a domain is cached or not without any false positives. The attack requires sending a single TLS record. It can be repeated to determine when a domain is no longer cached to leak the insertion time. Our evaluation in the Tor network shows no instances of cached domains being reported as uncached and vice versa after 12M repetitions while only targeting our own domains. This shifts DNS in Tor from an unreliable side-channel---using traditional timing attacks with network jitter---to being perfectly reliable. We responsibly disclosed the attack and suggested two short-term mitigations.As a long-term defense for the DNS cache in Tor against all types of (timeless) timing attacks, we propose a redesign where only an allowlist of domains is preloaded to always be cached across circuits. We compare the performance of a preloaded DNS cache to Tor's current solution towards DNS by measuring aggregated statistics for four months from two exits (after engaging with the Tor Research Safety Board and our university ethical review process). The evaluated preload lists are variants of the following top-lists: Alexa, Cisco Umbrella, and Tranco. Our results show that four-months-old preload lists can be tuned to offer comparable performance under similar resource usage or to significantly improve shared cache-hit ratios (2--3x) with a modest increase in memory usage and resolver load compared to a 100 Mbit/s exit. We conclude that Tor's current DNS cache is mostly a privacy harm because the majority of cached domains are unlikely to lead to cache hits but remain there to be probed by attackers.
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| 12. |
- Dahlberg, Rasmus, et al.
(författare)
-
Verifiable Light-Weight Monitoring for Certificate Transparency Logs
- 2018
-
Ingår i: Secure IT Systems. NordSec 2018. - Cham : Springer. - 9783030036379 ; , s. 171-183
-
Konferensbidrag (refereegranskat)abstract
- Trust in publicly verifiable Certificate Transparency (CT) logs is reduced through cryptography, gossip, auditing, and monitoring. The role of a monitor is to observe each and every log entry, looking for suspicious certificates that interest the entity running the monitor. While anyone can run a monitor, it requires continuous operation and copies of the logs to be inspected. This has lead to the emergence of monitoring as-a-service: a trusted third-party runs the monitor and provides registered subjects with selective certificate notifications. We present a CT/bis extension for verifiable light-weight monitoring that enables subjects to verify the correctness of such certificate notifications, making it easier to distribute and reduce the trust which is otherwise placed in these monitors. Our extension supports verifiable monitoring of wild-card domains and piggybacks on CT’s existing gossip-audit security model.
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| 13. |
- Dahlberg, Tobias, et al.
(författare)
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3D printed water-soluble scaffolds for rapid production of PDMS micro-fluidic flow chambers
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- We report a novel method for fabrication of three-dimensional (3D) biocompatible micro-fluidic flow chambers in polydimethylsiloxane (PDMS) by 3D-printing water-soluble polyvinyl alcohol (PVA) filaments as master scaffolds. The scaffolds are first embedded in the PDMS and later residue-free dissolved in water leaving an inscription of the scaffolds in the hardened PDMS. We demonstrate the strength of our method using a regular, cheap 3D printer, and evaluate the inscription process and the channels micro-fluidic properties using image analysis and digital holographic microscopy. Furthermore, we provide a protocol that allows for direct printing on coverslips and we show that flow chambers with a channel cross section down to 40 x 300 μm can be realized within 60 min. These flow channels are perfectly transparent, biocompatible and can be used for microscopic applications without further treatment. Our proposed protocols facilitate an easy, fast and adaptable production of micro-fluidic channel designs that are cost-effective, do not require specialized training and can be used for a variety of cell and bacterial assays. To help readers reproduce our micro-fluidic devices, we provide: full preparation protocols, 3D-printing CAD files for channel scaffolds and our custom-made molding device, 3D printer build-plate leveling instructions, and G-code.
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| 14. |
- Dahlberg, Tobias, et al.
(författare)
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Biophysical Fingerprinting of Single Bacterial Spores using Laser Raman Optical Tweezers
- 2020
-
Ingår i: Chemical, biological, radiological, nuclear, and explosives (CBRNE) sensing XXI. - : SPIE-Intl Soc Optical Eng. - 9781510636101
-
Konferensbidrag (refereegranskat)abstract
- Spore-forming bacteria that cause diseases pose a danger in our society. When in spore form, bacteria can survive high temperatures and resist a plethora of disinfection chemicals. Effective disinfection approaches are thus critical. Since a population of bacterial spores is heterogeneous in many aspects, single spore analyzing methods are suitable when heterogeneous information cannot be neglected. We present in this work a high-resolution Laser Raman optical tweezers that can trap single spores and characterize their Raman spectra. We first evaluate our system by measuring Raman spectra of spores, and purified DNA and DPA. Thereafter, we expose Bacillus thuringiensis spores to peracetic acid, chlorine dioxide, and sodium hypochlorite, which are common disinfection chemicals. The data reveals how these agents change the constitutes of a spore over time, thus improving on the mode of action of these disinfection chemicals.
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| 15. |
- Dahlberg, Tobias, 1990-
(författare)
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KNOW YOUR ENEMY : Characterizing Pathogenic Biomaterials Using Laser Tweezers
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diseases caused by pathogenic agents such as bacteria and viruses result in devastating costs on personal and societal levels. However, it is not just the emergence of new diseases that is problematic. Antibiotic resistance among bacteria makes uncomplicated infections difficult and lethal. Resilient disease-causing spores spread in hospitals, the food industry, and water supplies requiring effective detection and disinfection methods. Further, we face complex neurological diseases where no effective treatment or diagnostic methods exist. Thus, we must increase our fundamental understanding of these diseases to develop effective diagnostic, detection, disinfection, and treatment methods.Classically, the methods used for detecting and studying the underlying mechanics of pathogenic agents work on a large scale, measuring the average macroscopic behavior and properties of these pathogens. However, just as with humans, the average behavior is not always representative of individual behavior. Therefore, it is also essential to investigate the characteristics of these pathogens on a single cell or particle level. This thesis develops and applies optical techniques to characterize pathogenic biomaterial on a single cell or particle level. At the heart of all these studies is our Optical Tweezers (OT) instrument. OT are a tool that allows us to reach into the microscopic world and interact with it. Finally, by combining OT with other experimental techniques, we can chemically characterize biomaterials and develop assays that mimic different biological settings. Using these tools, we investigate bacterial adhesion, disinfection, and detection of pathogenic spores and proteins.Hopefully, the insights of these studies can lessen the burden on society caused by diseases by helping others develop effective treatment, diagnostic, detection, and disinfection methods in the future.
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| 16. |
- Dahlberg, Tobias, et al.
(författare)
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Optical design for laser tweezers Raman spectroscopy setups for increased sensitivity and flexible spatial detection
- 2021
-
Ingår i: Applied Optics. - : Optical Society of America. - 1559-128X .- 2155-3165. ; 60:16, s. 4519-4523
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a method to double the collection efficiency in Laser Tweezers Raman Spectroscopy (LTRS) by collecting both the forward and back-scattered light in a single-shot multitrack measurement. Our method can collect signals at different sample volumes, granting both the pinpoint spatial selectivity of confocal Raman and the bulk sensitivity of non-confocal Raman simultaneously. Further, we display that our approach allows for reduced detector integration time and laser power. To show this, we measure the Raman spectra of both polystyrene beads and bacterial spores. For spores, we can trap them at 2.5 mW laser power and acquire a high signal-to-noise ratio Power spectrum of the CaDPA peaks using an integration time of 2 x 30 seconds. Thus, our method will enable the monitoring of biological samples sensitive to high intensities for longer times. Additionally, we demonstrate that by a simple modification, we can add polarization sensitivity and retrieve extra biochemical information.
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| 17. |
- Dahlberg, Tobias, 1990-, et al.
(författare)
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Unveiling molecular interactions that stabilize bacterial adhesion pili
- 2022
-
Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 121:11, s. 2096-2106
-
Tidskriftsartikel (refereegranskat)abstract
- Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers, we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal “staple” region of P pili, which provides interactions with pilins that are four and five subunits away, significantly stabilizes the helical filament structure. These data are consistent with previous structural data, and suggest that more layer-to-layer interactions could compensate for the lack of a staple in type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the function of pili is critically dependent on their structure and biophysical properties.
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| 18. |
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| 19. |
- Doran, Matthew, et al.
(författare)
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CS20 bridge the gap between class 1 and class 5 bacterial adhesion pili
- 2022
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Ingår i: Biophysical Journal. - : Cell press. - 0006-3495 .- 1542-0086. ; 121:3, suppl. 1, s. 168a-168a
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) are pathogenic bacteria that cause diarrheal disease that disrupts the nutrition and the growth of children under the age of 5 and causes illness in travelers to countries where these bacteria are endemic. ETEC express long thin helical filaments on their surface, ∼1 micron long and 8 nm in diameter, called pili or fimbriae. Often essential virulence factors, these filaments, including ETEC CS20 pili, are composed of approximately 1,000 copies of the major pilin protein and one copy of a tip protein that provides binding specificity. While the structures of ETEC pili from different strains are similar, there are critical differences that alter their biophysical properties.ETEC express Class 1 and/or Class 5 pilins. The Class 1 CS20 pilin, CsbA, is genetically similar to FimA from Type 1 pili that are expressed on many strains of Escherichia coli, including bacteria that infect the urinary tract or the gastrointestinal tract, and also to PapA pilins expressed on bacteria that infect the kidneys. Thus, despite CS20 being expressed on ETEC, its pilin is genetically distant from the Class 5 CFA/I pilin, CfaB, the most commonly expressed ETEC pilin.We show here the three-dimensional structure and surface coulombic charge of CS20 pili, determined at 3.4 Å resolution by electron cryomicroscopy (cryo-EM). Our force spectroscopy data show that CS20 pili have a helix unwinding force that is twice that of CFA/I pili, and half that of Type 1 pili. Molecular dynamics simulations are further used to unveil features along the unwinding pathway at an atomistic scale. We see that CS20 pili bridge the genetic and environmental gap between Class 1 and Class 5 adhesion pili that are expressed on pathogenic bacteria.
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| 20. |
- Doran, Matthew H., et al.
(författare)
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Three structural solutions for bacterial adhesion pilus stability and superelasticity
- 2023
-
Ingår i: Structure. - : Elsevier. - 0969-2126 .- 1878-4186.
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial adhesion pili are key virulence factors that mediate host-pathogen interactions in diverse epithelial environments. Deploying a multimodal approach, we probed the structural basis underpinning the biophysical properties of pili originating from enterotoxigenic (ETEC) and uropathogenic bacteria. Using cryo-electron microscopy we solved the structures of three vaccine target pili from ETEC bacteria, CFA/I, CS17, and CS20. Pairing these and previous pilus structures with force spectroscopy and steered molecular dynamics simulations, we find a strong correlation between subunit-subunit interaction energies and the force required for pilus unwinding, irrespective of genetic similarity. Pili integrate three structural solutions for stabilizing their assemblies: layer-to-layer interactions, N-terminal interactions to distant subunits, and extended loop interactions from adjacent subunits. Tuning of these structural solutions alters the biophysical properties of pili and promotes the superelastic behavior that is essential for sustained bacterial attachment.
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| 21. |
- Enevold, Jenny, et al.
(författare)
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Tunable two-dimensional patterning of a semiconducting Nanometer-Thin C60 fullerene film using a spatial light modulator
- 2020
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Ingår i: ACS Applied Nano Materials. - : Acoustical Society of America (ASA). - 2574-0970. ; 3:6, s. 2574-0970
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The photochemical coupling of fullerene molecules into covalently connected oligomeric or polymeric structures can result in drastically lowered solubility in common solvents with retained semiconductor properties. Here, we exploit this combination of properties for the utilization of fullerenes as a negative photoresist material with electronic functionality. Specifically, we develop an easily tunable exposure system, essentially comprising a laser and a computer-controlled spatial light modulator (SLM) featuring >8 million independently controlled pixels, for the spatially selective photochemical transformation of nanometer-thin C60 fullerene films. With a carefully designed laser-SLM-exposure/solvent-development cycle, we are able to realize well-resolved two-dimensional hexagonal or square patterns of circular C60 microdots with a center-to-center distance of 1–5 μm and a maximum thickness of 20–35 nm over several square-millimeter-sized areas on a substrate. The functionality of such a hexagonal C60 pattern was demonstrated by its inclusion in between the transparent electrode and the active material in a light-emitting electrochemical cell, which featured an enhanced light output by >50% in comparison to a reference device void of the patterned C60 layer.
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| 22. |
- Jonsmoen, Unni Lise, et al.
(författare)
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Endospore pili - flexible, stiff and sticky nanofibers
- 2023
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 122:13, s. 2696-2706
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Tidskriftsartikel (refereegranskat)abstract
- Species belonging to the Bacillus cereus group form endospores (spores) whose surface is decorated with micrometers-long and nanometers-wide endospore appendages (Enas). The Enas have recently been shown to represent a completely novel class of Gram-positive pili. They exhibit remarkable structural properties making them extremely resilient to proteolytic digestion and solubilization. However, little is known about their functional and biophysical properties. In this work, we apply optical tweezers to manipulate and assess how wild type and Ena-depleted mutant spores immobilize on a glass surface. Further, we utilize optical tweezers to extend S-Ena fibers to measure their flexibility and tensile stiffness. Finally, by oscillating single spores, we examine how the exosporium and Enas affect spores’ hydrodynamic properties. Our results show that S-Enas (μm long pili) are not as effective as L-Enas in immobilizing spores to glass surfaces but are involved in forming spore to spore connections, holding the spores together in a gel-like state. The measurements also show that S-Enas are flexible but tensile stiff fibers, which support structural data suggesting that the quaternary structure is composed of subunits arranged in a complex to produce a bendable fiber (helical turns can tilt against each other) with limited axial fiber extensibility. Lastly, the results show that the hydrodynamic drag is 1.5-times higher for wild type spores expressing S- and L-Enas compared to mutant spores expressing only L-Enas or ”bald spores” lacking Ena, and 2-times higher compared to spores of the exosporium deficient strain. This study unveils novel findings on the biophysics of S- and L-Enas, their role in spore aggregation, binding of spores to glass, and their mechanical behavior upon exposure to drag forces.
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| 23. |
- Malyshev, Dmitry, et al.
(författare)
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Laser induced degradation of bacterial spores during micro-Raman spectroscopy
- 2022
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Ingår i: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy. - : Elsevier. - 1386-1425 .- 1873-3557. ; 265
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Tidskriftsartikel (refereegranskat)abstract
- Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.
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| 24. |
- Malyshev, Dmitry, et al.
(författare)
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Mode of action of Disinfection chemicals on the bacterial spore structure and their Raman spectra
- 2021
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:6, s. 3146-3153
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Tidskriftsartikel (refereegranskat)abstract
- Contamination of toxic spore-forming bacteria is problematic since spores can survive a plethora of disinfection chemicals and it is hard to rapidly detect if the disinfection chemical has inactivated the spores. Thus, robust decontamination strategies and reliable detection methods to identify dead from viable spores are critical. In this work, we investigate the chemical changes of Bacillus thuringiensis spores treated with sporicidal agents such as chlorine dioxide, peracetic acid, and sodium hypochlorite using laser tweezers Raman spectroscopy. We also image treated spores using SEM and TEM to verify if we can correlate structural changes in the spores with changes to their Raman spectra. We found that over 30 min, chlorine dioxide did not change the Raman spectrum or the spore structure, peracetic acid showed a time-dependent decrease in the characteristic DNA/DPA peaks and ∼20% of the spores were degraded and collapsed, and spores treated with sodium hypochlorite showed an abrupt drop in DNA and DPA peaks within 20 min and some structural damage to the exosporium. Structural changes appeared in spores after 10 min, compared to the inactivation time of the spores, which is less than a minute. We conclude that vibrational spectroscopy provides powerful means to detect changes in spores but it might be problematic to identify if spores are live or dead after a decontamination procedure.
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| 25. |
- Malyshev, Dmitry, et al.
(författare)
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pH induced changes in Raman, UV-Vis absorbance, and fluorescence spectra of dipicolinic acid (DPA)
- 2022
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Ingår i: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy. - : Elsevier. - 1386-1425 .- 1873-3557. ; 271
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Tidskriftsartikel (refereegranskat)abstract
- Dipicolinic acid (DPA) is an essential component for the protection of DNA in bacterial endospores and is often used as a biomarker for spore detection. Depending upon the pH of the solution, DPA exists in different ionic forms. Therefore, it is important to understand how these ionic forms influence spectroscopic response. In this work, we characterize Raman and absorption spectra of DPA in a pH range of 2.0–10.5. We show that the ring breathing mode Raman peak of DPA shifts from 1003 cm−1 to 1017 cm−1 and then to 1000 cm−1 as pH increases from 2 to 5. The relative peak intensities related to the different ionic forms of DPA are used to experimentally derive the pKa values (2.3 and 4.8). We observe using UV–vis spectroscopy that the changes in the absorption spectrum of DPA as a function of pH correlate with the changes observed in Raman spectroscopy, and the same pKa values are verified. Lastly, using fluorescence spectroscopy and exciting a DPA solution at between 210–330 nm, we observe a shift in fluorescence emission from 375 nm to 425 nm between pH 2 and pH 6 when exciting at 320 nm. Our work shows that the different spectral responses from the three ionic forms of DPA may have to be taken into account in, e.g., spectral analysis and for detection applications.
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| 26. |
- Malyshev, Dmitry, et al.
(författare)
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Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores
- 2022
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Ingår i: Journal of Biophotonics. - : John Wiley & Sons. - 1864-063X .- 1864-0648. ; 15:8
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial spores are highly resistant to heat, radiation and various disinfection chemicals. The impact of these on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores' germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15% of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65%. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.
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| 27. |
- Malyshev, Dmitry, et al.
(författare)
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Reference Raman Spectrum and Mapping of Cryptosporidium parvum Oocysts
- 2022
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Ingår i: Journal of Raman Spectroscopy. - : John Wiley & Sons. - 0377-0486 .- 1097-4555. ; 53:7, s. 1293-1301
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Tidskriftsartikel (refereegranskat)abstract
- Cryptosporidium parvum is a protozoan parasite and among the most infectious diarrhea-causing pathogens, leading to severe health problems for malnourished children and immunocompromised individuals. Outbreaks are common even in developed countries, originating from water or food contamination and resulting in suffering and large costs for society. Therefore, robust, fast and highly specific detection strategies of Cryptosporidium are needed. Label-free detection techniques such as Raman spectroscopy have been suggested, however high-resolution reported spectra in the literature are limited. In this work, we report reference Raman spectra at 3 cm-1 resolution for viable and inactivated Cryptosporidium oocysts of the species C. parvum, gathered at a single oocyst level using a laser tweezers Raman spectroscopy system. We furthermore provide tentative Raman peak assignments for the Cryptosporidium oocysts, along with Raman mapping of the oocysts’ heterogeneous internal structure. Finally, we compare the C. parvum Raman spectrum with other common enterotoxigenic pathogens: Escherichia coli, Vibrio cholerae, Bacillus cereus and Clostridium difficile. Our results show a significant difference between C. parvum Raman spectra and the other pathogens.
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| 28. |
- Nilsson, Daniel P. G., et al.
(författare)
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Step-by-Step Guide to 3D Print Motorized Rotation Mounts for Optical Applications
- 2021
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Ingår i: Applied Optics. - : Optical Society of America. - 0003-6935 .- 1539-4522. ; 60:13, s. 3764-3771
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Tidskriftsartikel (refereegranskat)abstract
- Motorized rotation mounts and stages are versatile instruments that introduce computer control to optical systems, enabling automation and scanning actions. They can be used for intensity control and position adjustments, etc. However, these rotation mounts come with a hefty price tag, and this limits their use. This work shows how to build two different types of motorized rotation mounts for 1" optics, using a 3D printer and off-the-shelf components. The first is intended for reflective elements, like mirrors and gratings, and the second for transmissive elements, like polarizers and retarders. We evaluate and compare their performance to commercial systems based on velocity, resolution, precision, backlash, and axis wobble. Also, we investigate the angular stability using Allan variance analysis. The results show that our mounts perform similar to systems costing more than 2000 Euro, while also being quick to build and costing less than 200 Euro. As a proof of concept, we show how to control lasers used in an optical tweezers and Raman spectroscopy setup. When used for this, the 3D printed motorized rotational mounts provide intensity control with a resolution of 0.03 percentage points or better.
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| 29. |
- Nilsson, Daniel, et al.
(författare)
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Patient-specific brain arteries molded as a flexible phantom model using 3D printed water-soluble resin
- 2022
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Visualizing medical images from patients as physical 3D models (phantom models) have many roles in the medical field, from education to preclinical preparation and clinical research. However, current phantom models are generally generic, expensive, and time-consuming to fabricate. Thus, there is a need for a cost- and time-efficient pipeline from medical imaging to patient-specific phantom models. In this work, we present a method for creating complex 3D sacrificial molds using an off-the-shelf water-soluble resin and a low-cost desktop 3D printer. This enables us to recreate parts of the cerebral arterial tree as a full-scale phantom model (10×6×410×6×4 cm) in transparent silicone rubber (polydimethylsiloxane, PDMS) from computed tomography angiography images (CTA). We analyzed the model with magnetic resonance imaging (MRI) and compared it with the patient data. The results show good agreement and smooth surfaces for the arteries. We also evaluate our method by looking at its capability to reproduce 1 mm channels and sharp corners. We found that round shapes are well reproduced, whereas sharp features show some divergence. Our method can fabricate a patient-specific phantom model with less than 2 h of total labor time and at a low fabrication cost.
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| 30. |
- Näsström, Thomas, et al.
(författare)
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Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:17
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Tidskriftsartikel (refereegranskat)abstract
- Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.
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| 31. |
- Pakharukova, Natalia, et al.
(författare)
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Archaic chaperone-usher pili self-secrete into superelastic zigzag springs
- 2022
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 609:7926, s. 335-340
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Tidskriftsartikel (refereegranskat)abstract
- Adhesive pili assembled via the chaperone-usher pathway (CUP) are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria 1-3. Archaic CUP pili, the most diverse and widespread CUP adhesins, are promising vaccine and drug targets due to their prevalence in the most troublesome multidrug-resistant (MDR) pathogens 1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the 3.4 Å resolution cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii, a notorious MDR nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into a conceptually novel ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed now for the first time in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight MDR bacterial infections.
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| 32. |
- Patkowski, Jonasz B., et al.
(författare)
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The F-pilus biomechanical adaptability accelerates conjugative dissemination of antimicrobial resistance and biofilm formation
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Conjugation is used by bacteria to propagate antimicrobial resistance (AMR) in the environment. Central to this process are widespread conjugative F-pili that establish the connection between donor and recipient cells, thereby facilitating the spread of IncF plasmids among enteropathogenic bacteria. Here, we show that the F-pilus is highly flexible but robust at the same time, properties that increase its resistance to thermochemical and mechanical stresses. By a combination of biophysical and molecular dynamics methods, we establish that the presence of phosphatidylglycerol molecules in the F-pilus contributes to the structural stability of the polymer. Moreover, this structural stability is important for successful delivery of DNA during conjugation and facilitates rapid formation of biofilms in harsh environmental conditions. Thus, our work highlights the importance of F-pilus structural adaptations for the efficient spread of AMR genes in a bacterial population and for the formation of biofilms that protect against the action of antibiotics.
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| 33. |
- Pulls, Tobias, 1985-, et al.
(författare)
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Cryptology ePrint Archive: Report 2018/737
- 2018
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- We present Steady: an end-to-end secure logging system engineered to be simple in terms of design, implementation, and assumptions for real-world use. Steady gets its name from being based on a steady (heart)beat of events from a forward-secure device sent over an untrusted network through untrusted relays to a trusted collector. Properties include optional encryption and compression (with loss of confidentiality but significant gain in goodput), detection of tampering, relays that can function in unidirectional networks (e.g., as part of a data diode), cost-effective use of cloud services for relays, and publicly verifiable proofs of event authenticity. The design is formalized and security proven in the standard model. Our prototype implementation (about 2,200 loc) shows reliable goodput of over 1M events/s (about 160 MiB/s) for a realistic dataset with commodity hardware for a device on a GigE network using 16 MiB of memory connected to a relay running at Amazon EC2.
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| 34. |
- Pulls, Tobias, 1985-, et al.
(författare)
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Steady : A Simple End-to-End Secure Logging System
- 2018
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Ingår i: Secure IT Systems. NordSec 2018. - Cham : Springer. - 9783030036379 ; , s. 88-103
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Konferensbidrag (refereegranskat)abstract
- We present Steady: an end-to-end secure logging system engineered to be simple in terms of design, implementation, and assumptions for real-world use. Steady gets its name from being based on a steady (heart)beat of events from a forward-secure device sent over an untrusted network through untrusted relays to a trusted collector. Properties include optional encryption and compression (with loss of confidentiality but significant gain in goodput), detection of tampering, relays that can function in unidirectional networks (e.g., as part of a data diode), cost-effective use of cloud services for relays, and publicly verifiable proofs of event authenticity. The design is formalized and security proven in the standard model. Our prototype implementation (2,200 loc) shows reliable goodput of over 1M events/s (160 MiB/s) for a realistic dataset with commodity hardware for a device on a GigE network using 16 MiB of memory connected to a relay running at Amazon EC2.
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| 35. |
- Pulls, Tobias, 1985-, et al.
(författare)
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Website fingerprinting with website oracles
- 2020
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Ingår i: Proceedings on Privacy Enhancing Technologies. - : De Gruyter Open. - 2299-0984. ; 2020:1, s. 235-255
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Tidskriftsartikel (refereegranskat)abstract
- Website Fingerprinting (WF) attacks are a subset of traffic analysis attacks where a local passive attacker attempts to infer which websites a target victim is visiting over an encrypted tunnel, such as the anonymity network Tor. We introduce the security notion of a Website Oracle (WO) that gives a WF attacker the capability to determine whether a particular monitored website was among the websites visited by Tor clients at the time of a victim’s trace. Our simulations show that combining a WO with a WF attack—which we refer to as a WF+WO attack—significantly reduces false positives for about half of all website visits and for the vast majority of websites visited over Tor. The measured false positive rate is on the order one false positive per million classified website trace for websites around Alexa rank 10,000. Less popular monitored websites show orders of magnitude lower false positive rates.We argue that WOs are inherent to the setting of anonymity networks and should be an assumed capability of attackers when assessing WF attacks and defenses. Sources of WOs are abundant and available to a wide range of realistic attackers, e.g., due to the use of DNS, OCSP, and real-time bidding for online advertisement on the Internet, as well as the abundance of middleboxes and access logs. Access to a WO indicates that the evaluation of WF defenses in the open world should focus on the highest possible recall an attacker can achieve. Our simulations show that augmenting the Deep Fingerprinting WF attack by Sirinam et al. [60] with access to a WO significantly improves the attack against five state-of-the-art WF defenses, rendering some of them largely ineffective in this new WF+WO setting.
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| 36. |
- Schmidt, Tobias, et al.
(författare)
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Synovial monocytes contribute to chronic inflammation in childhood-onset arthritis via IL-6/STAT signalling and cell-cell interactions
- 2023
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Ingår i: Frontiers in Immunology. - 1664-3224. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Monocytes are key effector cells in inflammatory processes. We and others have previously shown that synovial monocytes in childhood-onset arthritis are activated. However, very little is known about how they contribute to disease and attain their pathological features. Therefore, we set out to investigate the functional alterations of synovial monocytes in childhood-onset arthritis, how they acquire this phenotype, and whether these mechanisms could be used to tailorize treatment.METHODS: The function of synovial monocytes was analysed by assays believed to reflect key pathological events, such as T-cell activation-, efferocytosis- and cytokine production assays using flow cytometry in untreated oligoarticular juvenile idiopathic arthritis (oJIA) patients (n=33). The effect of synovial fluid on healthy monocytes was investigated through mass spectrometry and functional assays. To characterize pathways induced by synovial fluid, we utilized broad-spectrum phosphorylation assays and flow cytometry, as well as inhibitors to block specific pathways. Additional effects on monocytes were studied through co-cultures with fibroblast-like synoviocytes or migration in transwell systems.RESULTS: Synovial monocytes display functional alterations with inflammatory and regulatory features, e.g., increased ability to induce T-cell activation, resistance to cytokine production following activation with LPS and increased efferocytosis. In vitro, synovial fluid from patients induced the regulatory features in healthy monocytes, such as resistance to cytokine production and increased efferocytosis. IL-6/JAK/STAT signalling was identified as the main pathway induced by synovial fluid, which also was responsible for a majority of the induced features. The magnitude of synovial IL-6 driven activation in monocytes was reflected in circulating cytokine levels, reflecting two groups of low vs. high local and systemic inflammation. Remaining features, such as an increased ability to induce T-cell activation and markers of antigen presentation, could be induced by cell-cell interactions, specifically via co-culture with fibroblast-like synoviocytes. CONCLUSIONS: Synovial monocytes in childhood-onset arthritis are functionally affected and contribute to chronic inflammation, e.g., via promoting adaptive immune responses. These data support a role of monocytes in the pathogenesis of oJIA and highlight a group of patients more likely to benefit from targeting the IL-6/JAK/STAT axis to restore synovial homeostasis.
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| 37. |
- Stangner, Tim, et al.
(författare)
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Cooke-Triplet-Tweezers : More compact, robust and efficient optical tweezers
- 2018
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Ingår i: Optics Letters. - 0146-9592 .- 1539-4794. ; 43:9, s. 1990-1993
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Tidskriftsartikel (refereegranskat)abstract
- We present a versatile three-lens optical design to improve the overall compactness, efficiency, and robustness for optical tweezers based applications. The design, inspired by the Cooke–Triplet configuration, allows for continuous beam magnifications of 2–10× , and axial as well as lateral focal shifts can be realized without switching lenses or introducing optical aberrations. We quantify the beam quality and trapping stiffness and compare the Cooke–Triplet design with the commonly used double Kepler design through simulations and direct experiments. Optical trapping of 1 and 2 μm beads shows that the Cooke–Triplet possesses an equally strong optical trap stiffness compared to the double Kepler lens design but reduces its lens system length by a factor of 2.6. Finally, we demonstrate how a Twyman–Green interferometer integrated in the Cooke–Triplet optical tweezers setup provides a fast and simple method to characterize the wavefront aberrations in the lens system and how it can help in aligning the optical components perfectly.
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| 38. |
- Stangner, Tim, et al.
(författare)
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Step-by-step guide to reduce spatial coherence of laser light using a rotating ground glass diffuser
- 2017
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Ingår i: Applied Optics. - : Optical Society of America. - 1559-128X .- 2155-3165. ; 56:19, s. 5427-5435
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Tidskriftsartikel (refereegranskat)abstract
- Wide field-of-view imaging of fast processes in a microscope requires high light intensities motivating the use of lasers as light sources. However, due to their long spatial coherence length, lasers are inappropriate for such applications, as they produce coherent noise and parasitic reflections, such as speckle, degrading image quality. Therefore, we provide a step-by-step guide for constructing a speckle-free and high-contrast laser illumination setup using a rotating ground glass diffuser driven by a stepper motor. The setup is easy to build, cheap, and allows a significant light throughput of 48%, which is 40% higher in comparison to a single lens collector commonly used in reported setups. This is achieved by using only one objective to collect the scattered light from the ground glass diffuser. We validate our setup in terms of image quality, speckle contrast, motor-induced vibrations, and light throughput. To highlight the latter, we record Brownian motion of micro-particles using a 100x oil immersion objective and a high-speed camera operating at 2000 Hz with a laser output power of only 22 mW. Moreover, by reducing the objective magnification to 50x, sampling rates up to 10,000 Hz are realized. To help readers with basic or advanced optics knowledge realize this setup, we provide a full component list, 3D-printing CAD files, setup protocol, and the code for running the stepper motor.
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| 39. |
- Wallentin, Jennie, et al.
(författare)
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Översvämning! : Samhällets krisberedskap och förebyggande arbete när det gäller översvämningar som drabbar jordbrukssektorn
- 2016
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Samhällets krisberedskap och förebyggande arbete när det gäller översvämningar som drabbar jordbrukssektornNaturolyckor såsom översvämning, torka och skogsbrand kan få allvarliga konsekvenser för jordbrukssektorn och trädgårdsnäringen. För fisket och vattenbruket kan till exempel stormar orsaka problem. I ett framtida förändrat klimat kommer de extrema väderhändelserna att bli mer omfattande och frekventa. Detta innebär att samhället behöver öka sin förmåga att bedöma konsekvenserna av översvämningar för jordbrukssektorn i ett brett perspektiv och hur konsekvenserna av extrema väderhändelser, som till exempel översvämningar, ska hanteras. Jordbruksverket har därför under åren 2013-2015 drivit ett projekt - ”Ökad förmåga att hantera konsekvenser av allvarliga väderhändelser - översvämningar som modell” - som finansierats av Jordbruksverket och Myndigheten för samhällsskydd och beredskap (MSB). Denna rapport sammanfattar resultat och slutsatser från projektet.
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| 40. |
- Öberg, Rasmus, et al.
(författare)
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Monitoring bacterial spore metabolic activity using heavy water-induced Raman peak evolution
- 2023
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Ingår i: The Analyst. - : Royal Society of Chemistry. - 0003-2654 .- 1364-5528. ; 148:9, s. 2141-2148
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Tidskriftsartikel (refereegranskat)abstract
- Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. However, current methods for tracking metabolic activity are time-consuming and resource intensive. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. Specifically, we monitor the Raman spectrum of enterotoxic \textit{B. cereus} spores undergoing germination and cell division in D2O-infused broth. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C-D bonds at 2190 cm-1. We find that a significant C-D peak appears after 2 h of incubation at 37◦C. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. Lastly, the germination and cell growth rate of spores were not affected by adding 30 % heavy water to the broth. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. In conclusion, our work proposes tracking the evolution of the C-D Raman peak in spores incubated with D2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided.
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