An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H₂O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 μg/mm². Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.     

Contact guidance as a consequence of coupled morphological evolution and motility of adherent cells

Biomechanics and Modeling in Mechanobiology - Adherent cells seeded on substrates spread and evolve their morphology while simultaneously displaying motility. Phenomena such as contact guidance,...     

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH₂), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.     

Albumin uptake and distribution in the zebrafish liver as observed via correlative imaging

Although liver transport routes have been extensively studied in rodents, live imaging under in situ and in vivo conditions of large volumes is still proven to be difficult. In this study, we took advantage of the optical transparency of zebrafish and their small size to explore their usefulness for correlative imaging studies and liver transport experimentations. First, we assessed the micro-architecture of the zebrafish liver and compared its fine structure to the rodent and humans’ literature. Next, we investigated the transport routes and cellular distribution of albumin using combined and correlative microscopy approaches. These methods permitted us to track the injected proteins at different time points through the process of liver uptake and clearance of albumin.We demonstrate strong structural and functional resemblance between the zebrafish liver and its rodents and humans’ counterparts. In as short as 5?min post-injection, albumin rapidly accumulated within the LSECs. Furthermore, albumin entered the space of Disse where it initially accumulated then subsequently was taken up by the hepatocytes. We propose the zebrafish as a viable alternative experimental model for hepatic transport studies, allowing swift multimodal imaging and direct quantification on the hepatic distribution of supramolecular complexes of interest.     

Plumage coloration and personality in early life: sexual differences in signalling

Several studies have shown that melanin‐based traits play a crucial role in social contexts as they are associated with dominance, personality and social behaviour. However, most of these studies have focused on adults, and the role of these traits in juveniles has rarely been explored. Here, we explore the association between two melanin‐based traits and nestling personality in Common Kestrels Falco tinnunculus. Our results show that female nestlings with blacker plumages displayed bolder personalities, providing evidence of sex‐dependent phenotypic integration of these two traits in males and females. We consider that this differential integration may arise from different selection pressures acting on males and females on plumage coloration during adulthood and that nestling coloration can act as a status signal within the juvenile age‐class.     

Zic1 mRNA is transiently upregulated in subcutaneous fat of acutely cold-exposed mice

In the mammalian adipose organ cold exposure not only activates typical brown adipose tissue, but also induces browning, that is the formation of thermogenic multilocular adipocytes in white, or predominantly white, adipose depots such as subcutaneous fat. Unlike typical brown adipocytes, newly formed thermogenic adipocytes have been reported not to express the gene zinc finger of the cerebellum 1 (Zic1). Here, a time course approach enabled us to document a significant increase in Zic1 messenger RNA in inguinal subcutaneous fat from acutely (24 hr) cold-exposed mice, which was paralleled by an increase in multilocular and paucilocular uncoupling protein 1-positive adipocytes and in parenchymal noradrenergic innervation. This transient, depot-specific molecular signature was associated not to Zic1 promoter demethylation, but to chromatin remodeling through an H3K9me3 histone modification. These findings challenge the notion that Zic1 is exclusively expressed by typical brown adipocytes and suggest its involvement in brown adipocyte precursor differentiation and/or white-to-brown adipocyte transdifferentiation.     

The small molecule SI113 hinders epithelial-to-mesenchymal transition and subverts cytoskeletal organization in human cancer cells

The small molecule SI113 is an inhibitor of the kinase activity of SGK1, a key biological regulator acting on the PI3K/mTOR signal transduction pathway. Several studies demonstrate that this compound is able to strongly restrain cancer growth in vitro and in vivo, alone or in associative antineoplastic treatments, being able to elicit an autophagic response, either cytotoxic or cytoprotective. To elucidate more exhaustively the molecular mechanisms targeted by SI113, we performed activity-based protein profiling (ABPP) proteomic analysis using a kinase enrichment procedure. This technique allowed the identification via mass spectrometry of novel targets of this compound, most of them involved in functions concerning cell motility and cytoskeletal architecture. Using a glioblastoma multiforme, hepatocarcinoma and colorectal carcinoma cell line, we recognized an inhibitory effect of SI113 on cell migration, invading, and epithelial-to-mesenchymal transition. In addition, these cancer cells, when exposed to this compound, showed a remarkable subversion of the cytoskeletal architecture characterized by F-actin destabilization, phospho-FAK delocalization, and tubulin depolimerization. These results were definitely concordant in attributing to SI113 a key role in hindering cancer cell malignancy and, due to its negligible in vivo toxicity, can sustain performing a Phase I clinical trial to employ this drug in associative cancer therapy.     

Long bone scaling in Captorhinidae: do limb bones scale according to elastic similarity in sprawling basal amniotes?

Captorhinids are a speciose clade of sauropsids that are crucial to understand several aspects of basal amniote general biology. Members of the Captorhinidae explored different diets and, amongst basal amniotes, were one of the first groups to demonstrate high‐fibre herbivory. Several papers have been published on the cranial anatomy of captorhinids, but there are relatively few studies which focus on the post‐cranium, especially on the appendicular skeleton and long bones. This contribution presents the first quantitative long bone scaling in Captorhinidae performed through morphometric analyses. From classical biomechanical research, it is well‐established that to accommodate an increase in size, gravity will result in elastic deformation of long bones. This outcome is especially significant in terrestrial tetrapods with a sprawling limb posture such as captorhinids, where great torsional stresses are applied to long bones, both during locomotion and in the resting phase. In this paper, we test whether the consistent evolutionary size increase in captorhinids led to major re‐patterning in long bone structure as theoretically expected, based on the theory of elastic similarity. Morphometric analysis shows that, apart from a small positive allometry in the humerus, captorhinid long bones scale geometrically as body size increases. Thus, the predicted elastic similarity to maintain similar levels in peak stress with an increase in dimensions does not seem not to apply to long bone evolution in captorhinids. We propose that, as already observed experimentally in larger‐bodied varanid lizards, large captorhinids could also mitigate size‐related increases in stress by reducing femur rotation and increasing the percentage of the stride cycle during which the right hindfoot was on the ground (i.e. the duty factor). In this way, large captorhinids could avoid reaching peak stress thresholds by sacrificing speed during locomotion and without a substantial long bone re‐patterning or postural change.     

Reproduction of the introduced oyster Crassostrea gigas (Bivalvia: Ostreidae) cultured on rafts in Spain

The oyster Crassostrea gigas was introduced in Spain for aquaculture purposes; however, until now, it is not known whether populations are established in the wild, being necessary to define whether this species is spawning and which environmental variables trigger this process. The influence of environmental parameters on the reproduction of C. gigas was evaluated from January 2008 to October 2009 with oysters grown on a raft in the Ría de Arousa (Galicia, NW Spain). Temperature and chlorophyll a are directly correlated to sexual maturation. Oysters can mature at temperatures below 14°C. The temperature necessary for spawning differs between the sexes, requiring a temperature above 15°C for males and 18°C for females. Females had a single massive spawn between June and September, while males had partial spawning from May to December with two peaks, one in May–September and another in October–December, with the second peak more pronounced. The first spawning peak is related to high temperatures and concentrations of chlorophyll a, and the second spawning peak is mainly related to the food availability in the water. The spawning asynchrony may be impeding establishment of wild C. gigas populations in Spain.     

Developmental staging table of the green iguana

Embryonic staging tables provide information to standardize embryological investigations and to subsidize discussions about evolution. We have established a developmental staging table for Iguana iguana iguana. The sample was composed of 142 embryos, incubated at a constant temperature and collected at regular intervals. Morphological features as pharyngeal arches, craniofacial structures, eyes, limbs, claws, pigmentation, scales and egg tooth were evaluated to determine development stages. The normal staging table includes 17 stages from oviposition to hatching, based on chronology and morphological external features. Stages from 1 to 27 occur before oviposition. Stage 28 was the first described, because all embryos presented limb bud anlage, key feature of the previous stage. We used pharyngeal arches and limb buds to describe the first stages; claws, genital papilla and scales to describe the middle stages; and pigmentation, size and egg tooth to describe the last stages. Incubation lasted approximately 2 months in a controlled environment. The results were similar to the data from other lizards, confirming the embryonic conservative pattern of the group.