Enabling MSI-Guided Laser Capture Microdissection

Introduction/Rationale: Coupling MALDI mass spectrometry imaging (MALDI-MSI) with Laser Capture Microdissection (LCM) allows for precise dissection of tissue regions based on molecular features [1]. Automated methods for alignment of the coordinate systems of the MSI and LCM platforms reduces errors associated with manual definition of ROI’s and increases throughput (a major bottleneck for LCM). Here we present the development of a method to transfer regions of interest from MALDI MSI images to an LCM platform, using consecutive tissue sections mounted on ITO conductive slides for MALDI MSI and on PEN-coated slides for LCM. Methods: The test system consists of a gelatin-embedded mouse liver. 12 µm slices were cut using a cryostat and two consecutive slices were mounted on ITO and PEN slides. The ITO slide was spray-coated with DHB (30mg/mL, MeOH 70%, water 30%, 0.2% TFA) and a MALDI image was acquired with an EP-MALDI source coupled to a Q-Exactive mass spectrometer. The MSI data was imported into MATLAB. The tissue mounted on the PEN slide was stained with hematoxylin and a high resolution optical image acquired using an Aperio Scanscope. The LCM instrument used was an Apotome 2 Axio Observer Z1 microscope equipped with a Palm Robomover LCM system (both Zeiss). Results: An image of an ion with a regular distribution on the tissue is used to align the MS image to the optical image of the hematoxylin-stained tissue section mounted on the PEN slide. The optical image of the PEN slide tissue section is imported in MATLAB and cropped to match the size of the MALDI image. An intensity-based co-registration algorithm is then used to align the MS image to the cropped optical image. The MS image is then rescaled to match to the original optical image. To obtain regions-of-interest to transfer to the LCM platform, the MSI data was TIC normalized and a k-means cluster analysis performed. The image of the cluster of interest was aligned to the PEN slide using the same transformations used for the whole MSI data, binarized and segmented to obtain the coordinates of the vertices of the cluster region. Vertex coordinates were expressed after setting the axes origin to a user-defined reference point on the slide. The coordinates of the origin in the Aperio reference system were then matched to the coordinates of the reference point in the Zeiss coordinate system and the same transformation applied. Coordinates were then formatted as an Element file readable by the LCM and exported as text files. Border coordinates were imported in the Zeiss PALMRobo software and regions of interest automatically dissected. Conclusions/Novelty: The presented method enables rapid transfer of coordinates from a MALDI image to an LCM instrument, increasing throughput and reducing errors due to freehand cutting. The method is applicable to consecutive tissue sections, and ROI’s can be defined either by MSI or via histopathological specification

Relative influence of the adeno-associated virus (AAV) type 2 p5 element for recombinant AAV vector site-specific integration.

The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo

Spectroscopy of 35^{35}P using the one-proton knockout reaction

The structure of $^{35}$P was studied with a one-proton knockout reaction at88~MeV/u from a $^{36}$S projectile beam at NSCL. The $\gamma$ rays from thedepopulation of excited states in $^{35}$P were detected with GRETINA, whilethe $^{35}$P nuclei were identified event-by-event in the focal plane of theS800 spectrograph. The level scheme of $^{35}$P was deduced up to 7.5 MeV using$\gamma-\gamma$ coincidences. The observed levels were attributed to protonremovals from the $sd$-shell and also from the deeply-bound $p\_{1/2}$ orbital.The orbital angular momentum of each state was derived from the comparisonbetween experimental and calculated shapes of individual ($\gamma$-gated)parallel momentum distributions. Despite the use of different reactions andtheir associate models, spectroscopic factors, $C^2S$, derived from the$^{36}$S $(-1p)$ knockout reaction agree with those obtained earlier from$^{36}$S($d$,\nuc{3}{He}) transfer, if a reduction factor $R\_s$, as deducedfrom inclusive one-nucleon removal cross sections, is applied to the knockout transitions.In addition to the expected proton-hole configurations, other states were observedwith individual cross sections of the order of 0.5~mb. Based on their shiftedparallel momentum distributions, their decay modes to negative parity states,their high excitation energy (around 4.7~MeV) and the fact that they were notobserved in the ($d$,\nuc{3}{He}) reaction, we propose that they may resultfrom a two-step mechanism or a nucleon-exchange reaction with subsequent neutronevaporation. Regardless of the mechanism, that could not yet be clarified, thesestates likely correspond to neutron core excitations in \nuc{35}{P}. Thisnewly-identified pathway, although weak, offers the possibility to selectivelypopulate certain intruder configurations that are otherwise hard to produceand identify.Comment: 5 figures, 1 table, accepted for publication in Physical Review

Unveiling the complexity of japanese metallic threads

In the framework of an extensive survey campaign on a collection of Japanese samurai armors, metallic threads from different parts of the traditional equipment were studied by several analytical techniques. The collection of armors belongs to Museo delle Culture (Lugano, Switzerland) and it is composed of ten elements, which date back from the 15th to 20th century. Metallic threads under study come from six of ten elements of the collection and represent a complex and unique multimaterial, which shows specific characteristics in Japanese tradition (kinran). The multianalytical approach based on ATR-FTIR spectroscopy and SEM-EDX analysis, together with a careful observation with optical and digital microscopy, permitted to obtain a complete characterization of materials, which have shown a great variability in metal foils and in organic adhesives (urushi, animal glue, starch). Gold and silver turned out to be not so largely used as scholars thought, while aluminum showed a great diffusion. Within the collection of analyzed armors, the obtained results allowed us for the first time to get a complete comprehension of materials and techniques used by Japanese craftsmen, and to observe differences in the quality of the materials and in manufacture technology over the centuries

Differentiating between Natural and Modified Cellulosic Fibres Using ATR-FTIR Spectroscopy

This paper presents the limitations and potential of ATR-FTIR spectroscopy applied to the study of cellulosic textile collections The technique helps to differentiate natural fibres according to the content of lignin, pectin, hemicellulose, and wax, although some problematic issues should be considered. The spectral differences derived from the environmental humidity uptake and the plant composition are reviewed and discussed in the light of new experimental data. Diagnostic bands are proposed that can discriminate between different fibres from different plants. The contribution of ageing is also considered, demonstrating that sometimes aged fibres cannot be reliably recognised. In contrast, the potential of ATR-FTIR spectroscopy to discriminate between natural and modified fibres is discussed and proven. The best results were obtained when microinvasive ATR-FTIR spectroscopy was coupled with SEM observations. The proposed protocol was tested on microsamples of various cellulosic materials from traditional Japanese samurai armours dating from the 16th to the 20th centuries (Morigi Collection, Museo delle Culture, Lugano, Switzerland). The results facilitated a complete characterisation of the materials and demonstrated that the protocol can be used to study a wide variety of cellulosic materials, including both natural and man-modified fibres, and paper

Clinical effects of Streptococcus salivarius K12 in hospitalized COVID-19 patients: results of a preliminary study

Anatomical and physiological considerations indicate that the oral cavity is a primary source of the lung microbiota community, and recent studies have shown that the microbiota in the lungs contributes to immunological homeostasis, potentially altering the organ’s susceptibility to viral infection, including SARS-CoV-2. It has been proposed that, in the case of viral infection, lung Gram-negative bacteria could promote the cytokine cascade with a better performance than a microbiota mainly constituted by Gram-positive bacteria. Recent observations also suggest that Prevotella-rich oral microbiotas would dominate the oral cavity of SARS-CoV-2-infected patients. In comparison, Streptococcus-rich microbiotas would dominate the oral cavity of healthy people. To verify if the modulation of the oral microbiota could have an impact on the current coronavirus disease, we administered for 14 days a well-recognized and oral-colonizing probiotic (S. salivarius K12) to hospitalized COVID-19 patients. The preliminary results of our randomized and controlled trial seem to prove the potential role of this oral strain in improving the course of the main markers of pathology, as well as its ability to apparently reduce the death rate from COVID-19. Although in a preliminary and only circumstantial way, our results seem to confirm the hypothesis of a direct involvement of the oral microbiota in the construction of a lung microbiota whose taxonomic structure could modulate the inflammatory processes generated at the pulmonary and systemic level by a viral infection