<p dir="ltr">Nucleic acid testing has become a prominent method for rapid microbial detection. Unlike polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) is a simple method of nucleic acid amplification where the reaction can be performed at a constant temperature and the output provided in a colorimetric format. A transparent water bath heater is a desirable instrument to perform the heating and observe the visual results of nucleic acid amplification. However, existing methods of heating the water are not convenient for loading and unloading the nucleic acid samples. Here, we developed a field-deployable water bath heating device—an isothermal heater called IsoHeat for short–which is solely dedicated to performing LAMP reactions and can heat the water up to 85 °C (if needed). Using 3D-printing and LASER-cutting technology, we fabricated different parts of the device and mechanically assembled the parts to develop the entire device. Users can commence the heating by pressing the start button on the screen after entering the target temperature. Subsequently, the device heats up the water bath and maintains the target temperature through a PID algorithm-based control system. We demonstrate that IsoHeat can operate in environmental temperatures ranging from 5-33 °C and it can conduct LAMP reactions in a liquid format as well as in paper-based devices. IsoHeat is more efficient and user-friendly compared to a commercially available immersion-heating device, which is often used to perform LAMP reactions. This newly developed device would be helpful to detect pathogens conveniently in the field (e.g., at the point-of-care for human applications, on farms for plant and animal applications, and in production facilities for food safety applications).</p>

Feline coronavirus (FCoV) is endemic in cat populations worldwide. Persistently, subclinically infected cats play a significant role in spreading the infection. Testing fecal samples of cats may facilitate efforts to decrease the viral burden within a population. Real-time RT-PCR is highly sensitive and specific for the detection of FCoV but must be performed in a fully equipped laboratory. A simple and accurate assay is needed to identify FCoV at the point-of-need. The aim of this study was to develop a rapid FCoV detection assay based on isothermal amplification technology, i.e., reverse transcription-recombinase polymerase amplification (RT-RPA). Primers were designed to target the highly conserved 3′ untranslated region of the 7b gene. Running on a constant temperature of 42 °C, reverse transcription as well as DNA amplification and detection was achieved in a maximum of 15 min. A probit analysis revealed a detection limit of 58.5 RNA copies/reaction. For cross-detection, nucleic acids from 19 viruses were tested. Both RT-RPA and real-time RT-PCR showed cross-detection with canine coronavirus and transmissible gastroenteritis virus, but not with other pathogens. To evaluate clinical performance, RNA was extracted from 39 fecal samples from cats. All samples were tested simultaneously with real-time RT-PCR resulting in a RT-RPA sensitivity and specificity of 90.9% and 100%, respectively. RT-RPA can be considered a promising simple method for rapid detection of FCoV.

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

After oral exposure of cattle with classical bovine spongiform encephalopathy (C-BSE), the infectious agent ascends from the gut to the central nervous system (CNS) primarily via the autonomic nervous system. However, the timeline of this progression has thus far remained widely undetermined. Previous studies were focused on later time points after oral exposure of animals that were already 4 to 6 months old when challenged. In contrast, in this present study, we have orally inoculated 4 to 6 weeks old unweaned calves with high doses of BSE to identify any possible BSE infectivity and/or PrPBSE in peripheral nervous tissues during the first eight months postinoculation (mpi). For the detection of BSE infectivity, we used a bovine PrP transgenic mouse bioassay, while PrPBSE depositions were analyzed by immunohistochemistry (IHC) and by protein misfolding cyclic amplification (PMCA). We were able to show that as early as 8 mpi the thoracic spinal cord as well as the parasympathetic nodal ganglion of these animals contained PrPBSE and BSE infectivity. This shows that the centripetal prion spread starts early after challenge at least in this age group, which represents an essential piece of information for the risk assessments for food, feed, and pharmaceutical products produced from young calves.

An unprecedented use of high-throughput sequencing for routine monitoring of SARS-CoV-2 viruses in patient samples has created a dataset of over 6 million SARS-CoV-2 genomes. To monitor genomes, deposited in the GISAID database, and to track the continuous sequence evolution of molecular assay oligonucleotide target sequences. A simple pipeline tool for non-experts was developed to mine this database for nucleotide changes in oligonucleotides and tested with the long oligonucleotides of a Recombinase polymerase amplification (RPA) assay targeting the RNA-dependent RNA polymerase (RdRP) gene of the SARS-CoV-2. Results indicate the emergence of a single nucleotide change in the reverse oligonucleotide from 0.03 to 26.23% (January to May 2021) in Alpha variant genomes, which however reduced to 17.64% by September after which the Alpha variant was completely displaced by the Delta variant. For all other variants, no relevant nucleotide changes were observed. The oligonucleotide screening pipeline allows efficient screening of nucleotide changes in oligonucleotides of all sizes in minutes.

I used a theoretical model to determine the conditions under which Cactoblastis cactorum populations would be expected to experience positive population growth. Results from simulations suggest that host species richness, host quality, and the C. cactorum death rate interact to determine the probability of C. cactorum positive population growth. I also studied the influence of host diversity empirically. Cactoblastis cactorum prevalence was significantly higher when O. stricta was present in the community. Also, higher species richness within host assemblages led to a higher prevalence of infestation than in single-species host assemblages. Finally, I explored cooccurrence patterns of native cactuseeding insects in an effort to document the impact of C. cactorum to native insect assemblages. The presence of C. cactorum in a community did not appear to affect the structure of native cactuseeding insect assemblages.

No description available.

No description available.

Interfaces play a crucial role in the exchange of energy and matter in various physical, chemical and biological systems. A particular interest has been to study interfaces between aqueous phases and various minerals because of their importance in understanding geochemical phenomena as well as for applications such as enhanced oil recovery. The nonlinear optical technique of vibrational sum-frequency generation (SFG) spectroscopy, introduced over 20 years ago, has become a powerful tool to investigate various surfaces, in particular, mineral-water interfaces. One of the challenges of the SFG spectroscopy of aqueous surfaces is the need to tune the central frequency of relatively narrowband IR lasers through the broad range of the OH-stretch frequencies of water molecules (3000 - 4000 cm-1). We have developed a novel ultrabroadband IR laser source that generates infrared pulses in the ~2800-6000 cm-1 range (lambda~3300-1800 nm) with bandwidths Delta(nu)&gt;1000 cm-1, and bandwidths &gt;2000 cm-1 in the near-IR range (lambda~1000-2000 nm). Pulse front tilt of signal pulse has been corrected allowing for compression of signal pulses down to 25 fsec. Such ultrabroadband IR pulses allow us to perform SFG spectroscopy of aqueous surfaces over the entire frequency range of water molecule spectrum (extending from ~2900 cm -1 to ~3800 cm -1) simultaneously, without tuning the laser ("in one shot"). We have used this novel ultrabroadband IR source to investigate the vibrational SFG spectra of silica/water interfaces. The high signal-to-noise ratio of our spectroscopic setup has allowed us to study low-intensity features that were not studied in detail, or recognized previously in the SFG-spectroscopy investigations, including: 1) non-hydrogen bonded OH vibrations at hydrophilic silica/water interfaces; 2) combination [stretch+bend] bands of water at the silica surface appearing at ~5000-5200 cm -1. 3) Overtones of water stretching modes at silica/water interfaces. The most important conclusions from these studies are outlined below. 1. Non-hydrogen bonded hydroxyls at silica/water interface. Typically SFG-studies of mineral/water interfaces (in particular, silica/water) have focused on the most pronounced features - peaks of H-bonded hydroxyls at ~3150 and ~3450 cm -1. We have been able to systematically observe and study a weaker peak at ~3670 - 3700 cm -1. This peak becomes more pronounced as the pH of aqueous phase decreases, as well as the ionic strength increases, indicating that the hydroxyls corresponding to this spectral feature are situated in a very close proximity to the surface. Isotopic dilution experiments indicate that the 3700 cm -1 feature is not due to asymmetric OH stretches as was suggested before. Based on our results, we suggest that this spectral feature corresponds to hydroxyls of water molecules at the silica surface that cannot hydrogen bond with silanol groups because of the lower density of silanols compared to H2O. We believe this to be the first surface-specific study of non-hydrogen bonded hydroxyls at silica, a surface widely accepted as hydrophilic. 2. SFG spectroscopy of [ν(OH)+δ(HOH)] combination bands of water at silica surface. We have extended SFG spectroscopy of the interfacial hydroxyls at mineral/water surfaces into the near-IR frequency range. The studies of overtones of interfacial OH(OD) groups will provide information on the anharmonicity of such species, and thus on the energy of dissociation. In addition, the positions of the overtone frequencies of the hydroxyls are more sensitive to interactions with the environment than the fundamental stretch frequencies. Our particular focus has been to study the stretch+bend combination band nu comb nu;(OH)+delta;(HOH) of liquid water which occurs in the near-IR spectral range at ~5000-5200 cm -1. It is typically much weaker in the FTIR absorption spectra than the fundamental transitions of the OH stretches or HOH bending, similar to overtones of these modes. We have performed, what we believe to be, the first surface-specific vibrational SFG spectroscopic measurements of combination bands of water molecules at silica surfaces. SFG spectroscopy of water combination band allows access to the water bending mode (delta~1600 cm -1), which still has not been observed in sum-frequency. / Chemistry

Centrosomes are microtubule-based organelles composed of two centrioles and peri-centriolar material, involved in the formation and organization of the mitotic spindle, serving as microtubule-organizing center and involved in ciliogenesis. Supernumerary centrosomes are detrimental for cell physiology and activate the PIDDosome, a multi-protein complex that serves as a platform for the activation of Caspase-2, composed of: PIDD1, RAIDD and Caspase-2 itself. Caspase-2’s preferred cleavage site based on peptide screening is VDVAD, however Caspase-2, when activated via the PIDDosome, cleaves its bona fide substrate MDM2 (negative p53 regulator) in the FDVPD sequence. Here, I present evidence for VDVADase activity in apoptotic cells lacking Caspase-2, which suggests that this cleavage site is not Caspase-2 specific when the Caspase-2 activation occurs via the PIDDosome. In order to investigate if the mode of activation of Caspase-2 determines its substrate specificities I performed a Caspase-2 rescue experiment and introduced several mutations affecting the Caspase-2 autoproteolytic-processing. Furthermore, I present evidence that exogenous Caspase-2 is able to form the PIDDosome and cleaves MDM2 but when key autoproteolytic sites are mutated no MDM2 cleavage is detectable. Supernumerary centrosomes also accumulate upon overexpression of PLK4 (a kinase regulator of the centriole duplication). Immunofluorescence images of cells overexpressing PLK4 were taken following the centrioles quantification over time. Consequently, a large amount of image data was accumulated, which necessitated the development of a semi-automated pipeline for centrioles counting. This pipeline was generated using the image processing and analysis tool ImageJ and the deep learning segmentation tool MitoS together with the pretrained MitoSegNet model, which was finetuned to count centrioles stained against different centrosomal epitopes, namely Centrin 1, γ-Tubulin and ANKRD26. This semi-automated method of centrioles quantification is easy to use, reproducible and faster than manual quantification. Using this pipeline to quantify centrioles in p53, SCLT1 or ANKRD26 lacking cells we demonstrate accumulation of supernumerary centrosomes in these cells similar to parental cells. / I centrosomi sono organelli cellulari a base di microtubuli, composti da due centrioli e dal materiale pericentriolare che li circonda. I centrosomi sono coinvolti nell'organizzazione dei microtubuli, nella formazione del fuso mitotico e nella ciliogenesi. I centrosomi soprannumerari sono dannosi per la fisiologia cellulare e attivano il PIDDosoma, un complesso multiproteico, composto da PIDD1, RAIDD e Caspasi-2, che funge da piattaforma per l'attivazione della caspasi stessa. Il sito preferenziale di proteolisi di Caspasi-2 è stato individuato tramite screening peptidico nella sequenza VDVAD. Nonostante ciò, quando attivata tramite il PIDDosoma, Caspasi-2 scinde il suo substrato di elezione MDM2 (regolatore negativo di p53) a livello della sequenza FDVPD. In questa tesi presento evidenze di attività VDVAD-asica in cellule apoptotiche prive di Caspasi-2, suggerendo che questo sito di taglio non sia specifico di Caspasi-2 quando la sua attivazione avviene tramite il PIDDosoma. Al fine di indagare se la modalità di attivazione della proteasi determina le sue specificità di substrato, ho eseguito esperimenti di complementazione di Caspasi-2 facendo uso di diversi mutanti che influenzano il suo processamento autoproteolitico. Inoltre, presento prove che Caspasi-2 esogena è in grado di assemblare il PIDDosoma e proteolizzare MDM2 ma quando i suoi siti chiave di autoproteolisi sono mutati non è rilevabile il taglio di MDM2. I centrosomi soprannumerari si accumulano anche in caso di sovraespressione di PLK4 (chinasi regolatrice della duplicazione dei centrioli). Immagini di immunofluorescenza di cellule che sovraesprimono PLK4 sono state acquisite seguendo la cinetica di accumulo dei centrioli nel tempo. Di conseguenza, l’ingente mole di dati generati ha reso necessario lo sviluppo di una procedura semiautomatica per la conta dei centrioli. Questa pipeline è stata generata utilizzando il programma di elaborazione e analisi di immagini ImageJ e il programma di segmentazione basato su deep learning MitoS, insieme al modello MitoSegNet, che è stato affinato per la conta dei centrioli evidenziati tramite immunofluorescenza diretta contro diversi epitopi centrosomiali, ossia: Centrin 1, γ-Tubulina e ANKRD26. Questo metodo semiautomatico di quantificazione dei centrioli è facile da usare, riproducibile e più veloce della quantificazione manuale. Utilizzando questa procedura per quantificare i centrioli nelle cellule prive di p53, SCLT1 o ANKRD26, dimostriamo che l'accumulo di centrosomi soprannumerari in queste cellule è simile a quello riscontrato nelle cellule parentali.