Characterization and Future Distribution Prospects of "Carciofo di Malegno" Landrace for Its In Situ Conservation.

"Carciofo di Malegno" is a little-known landrace of Cynara cardunculus subsp. scolymus cultivated in Camonica Valley (northern Italy). The morphological and phytochemical characteristics of this landrace were investigated; furthermore, a species distribution model (MaxEnt algorithm) was used to explore its ecological niche and the geographical area where it could be grown in the future. Due to its spiky shape, "Carciofo di Malegno" was distinct from any other artichoke sample considered, and it appears to be similar to those belonging to the "Spinosi" group. The concentration of chlorogenic acid (497.2 ± 116.0 mg/100 g DW) and cynarine (7.4 ± 1.2 mg/100 g DW) in "Carciofo di Malegno" was comparable to that of the commercial cultivars. In "Carciofo di Malegno," luteolin was detected in a significant amount (9.4 ± 1.5 mg/100 g DW) only in the stems and in the edible parts of the capitula. A MaxEnt distribution model showed that in the coming decades (2040-2060s), the cultivation of this landrace could expand to the pre-Alps and Alps of Lombardy. Climate change may promote the diffusion of "Carciofo di Malegno", contributing to preservation and the enhancement of this landrace and generating sustainable income opportunities in mountain areas through exploring new food or medicinal applications.

Machine learning interpretable models of cell mechanics from protein images.

Cellular form and function emerge from complex mechanochemical systems within the cytoplasm. Currently, no systematic strategy exists to infer large-scale physical properties of a cell from its molecular components. This is an obstacle to understanding processes such as cell adhesion and migration. Here, we develop a data-driven modeling pipeline to learn the mechanical behavior of adherent cells. We first train neural networks to predict cellular forces from images of cytoskeletal proteins. Strikingly, experimental images of a single focal adhesion (FA) protein, such as zyxin, are sufficient to predict forces and can generalize to unseen biological regimes. Using this observation, we develop two approaches-one constrained by physics and the other agnostic-to construct data-driven continuum models of cellular forces. Both reveal how cellular forces are encoded by two distinct length scales. Beyond adherent cell mechanics, our work serves as a case study for integrating neural networks into predictive models for cell biology.

Formal analyses are fundamental for the definition of honey, a product representing specific territories and their changes: the case of North Tyrrhenian dunes (Italy).

Honey is a variegate matrix depending significantly on the floral origin, and it could become an important agri-food product to valorise specific territories. Being so diverse, different analytical techniques are necessary for its description. Herein we characterized the honey produced in one of the Italian sand dunes systems hosting beekeeping activities. In terms of floristic origin, phytochemical characterization, and sensory and colour analysis, honey collected in 2021 and 2022 was comparable. Honey was polyfloral, with several pollens from dune habitat plants classified as minor. The presence of the allochthonous Amorpha fruticosa L. and the ruderal Rubus fruticosus L. pollens in the category of the secondary pollens testifies the alteration of the park vegetation. The phytochemical profile was rich in polyphenols. Other interesting compounds were coumarine derivatives, likely attributable to resin-laden plants as rockroses, long chain hydroxyacids typical of royal jelly and nicotinic acid and its analogues (2-hydroxynicotinic acid and 2-hydroxyquinoline). The above-mentioned honey showed interesting features and was a good representation of the vegetation of this area. Our study pointed out the importance of relying on multiple analytical techniques for the characterization of honey and the advisability of a technical support toward beekeepers to correctly describe and valorise their product.

Repair of noise-induced damage to stereocilia F-actin cores is facilitated by XIRP2 and its novel mechanosensor domain.

Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as 'gaps' in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.

LIM domain proteins.

Sala and Oakes introduce LIM domain proteins and discuss their roles in transcription, cytokinesis, adhesion, motility and mechanosignaling.

Zyxin is all you need: machine learning adherent cell mechanics.

Cellular form and function emerge from complex mechanochemical systems within the cytoplasm. No systematic strategy currently exists to infer large-scale physical properties of a cell from its many molecular components. This is a significant obstacle to understanding biophysical processes such as cell adhesion and migration. Here, we develop a data-driven biophysical modeling approach to learn the mechanical behavior of adherent cells. We first train neural networks to predict forces generated by adherent cells from images of cytoskeletal proteins. Strikingly, experimental images of a single focal adhesion protein, such as zyxin, are sufficient to predict forces and generalize to unseen biological regimes. This protein field alone contains enough information to yield accurate predictions even if forces themselves are generated by many interacting proteins. We next develop two approaches - one explicitly constrained by physics, the other more agnostic - that help construct data-driven continuum models of cellular forces using this single focal adhesion field. Both strategies consistently reveal that cellular forces are encoded by two different length scales in adhesion protein distributions. Beyond adherent cell mechanics, our work serves as a case study for how to integrate neural networks in the construction of predictive phenomenological models in cell biology, even when little knowledge of the underlying microscopic mechanisms exist.

Quantifying Strain-Sensing Protein Recruitment During Stress Fiber Repair.

A family of proteins have been identified that recognize damaged, strained actin filaments in stress fibers. These proteins are often referred to as strain- or force-sensing and trigger downstream signaling mechanisms that can facilitate repair at these strain sites. Here we describe a method using high-resolution microscopy to screen and quantify the mechanosensitive recruitment of proteins to these stress fiber strain sites. Strain sites are induced using spatially controlled illumination of UV light to locally damage actin stress fibers. Recruitment of potential strain-sensing proteins can then either be compared to (Blanchoin, Physiol Rev 94, 235-263, 2014) a known control (e.g., zyxin-GFP) or (Hoffman, Mol Biol Cell 23, 1846-1859, 2012) the pre-damaged stress fiber protein distribution. With this method, strain-sensing proteins and their dynamic association with stress fiber strain sites can be reproducibly measured and compared.

Microbial Community Successional Changes in a Full-Scale Mesophilic Anaerobic Digester from the Start-Up to the Steady-State Conditions.

Anaerobic digestion is a widely used technology for sewage sludge stabilization and biogas production. Although the structure and composition of the microbial communities responsible for the process in full-scale anaerobic digesters have been investigated, little is known about the microbial successional dynamics during the start-up phase and the response to variations occurring in such systems under real operating conditions. In this study, bacterial and archaeal population dynamics of a full-scale mesophilic digester treating activated sludge were investigated for the first time from the start-up, performed without adding external inoculum, to steady-state operation. High-throughput 16S rRNA gene sequencing was used to describe the microbiome evolution. The large majority of the reads were affiliated to fermentative bacteria. Bacteroidetes increased over time, reaching 22% of the total sequences. Furthermore, Methanosaeta represented the most abundant methanogenic component. The specific quantitative data generated by real-time PCR indicated an enrichment of bacteria and methanogens once the steady state was reached. The analysis allowed evaluation of the microbial components more susceptible to the shift from aerobic to anaerobic conditions and estimation of the microbial components growing or declining in the system. Additionally, activated sludge was investigated to evaluate the microbial core selected by the WWTP operative conditions.

Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA.

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin, while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate that activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin's mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton's mechanical state.

Effectiveness of Different Analytical Methods for the Characterization of Propolis: A Case of Study in Northern Italy.

: Propolis is used as folk medicine due to its spectrum of alleged biological and pharmaceutical properties and it is a complex matrix not still totally characterized. Two batches of propolis coming from two different environments (plains of Po Valley and the hilly Ligurian-Piedmont Apennines) of Northern Italy were characterized using different analytical methods: Spectrophotometric analysis of phenols, flavones and flavonols, and DPPH radical scavenging activity, HPLC, NMR, HSPME and GC-MS and HPLC-MS Orbitrap. Balsam and moisture content were also considered. No statistical differences were found at the spectrophotometric analysis; balsam content did not vary significantly. The most interesting findings were in the VOCs composition, with the Po Valley samples containing compounds of the resins from leaf buds of Populus nigra L. The hills (Appennines) samples were indeed characterize by the presence of phenolic glycerides already found in mountain environments. HPLC-Q-Exactive-Orbitrap®-MS analysis is crucial in appropriate recognition of evaluate number of metabolites, but also NMR itself could give more detailed information especially when isomeric compounds should be identified. It is necessary a standardized evaluation to protect and valorize this production and more research on propolis characterization using different analytical techniques.

An emerging link between LIM domain proteins and nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that partake in several biological processes including development, reproduction and metabolism. Over the last decade, evidence has accumulated that group 2, 3 and 4 LIM domain proteins, primarily known for their roles in actin cytoskeleton organization, also partake in gene transcription regulation. They shuttle between the cytoplasm and the nucleus, amongst other as a consequence of triggering cells with ligands of nuclear receptors. LIM domain proteins act as important coregulators of nuclear receptor-mediated gene transcription, in which they can either function as coactivators or corepressors. In establishing interactions with nuclear receptors, the LIM domains are important, yet pleiotropy of LIM domain proteins and nuclear receptors frequently occurs. LIM domain protein-nuclear receptor complexes function in diverse physiological processes. Their association is, however, often linked to diseases including cancer.

The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions.

The focal adhesion protein testin is a modular scaffold and tumour suppressor that consists of an N-terminal cysteine rich (CR) domain, a PET domain of unknown function and three C-terminal LIM domains. Testin has been proposed to have an open and a closed conformation based on the observation that its N-terminal half and C-terminal half directly interact. Here we extend the testin conformational model by demonstrating that testin can also form an antiparallel homodimer. In support of this extended model we determined that the testin region (amino acids 52-233) harbouring the PET domain interacts with the C-terminal LIM1-2 domains in vitro and in cells, and assign a critical role to tyrosine 288 in this interaction.

Expanding the Interactome of TES by Exploiting TES Modules with Different Subcellular Localizations.

The multimodular nature of many eukaryotic proteins underlies their temporal or spatial engagement in a range of protein cocomplexes. Using the multimodule protein testin (TES), we here report a proteomics approach to increase insight in cocomplex diversity. The LIM-domain containing and tumor suppressor protein TES is present at different actin cytoskeleton adhesion structures in cells and influences cell migration, adhesion and spreading. TES module accessibility has been proposed to vary due to conformational switching and variants of TES lacking specific domains target to different subcellular locations. By applying iMixPro AP-MS ("intelligent Mixing of Proteomes"-affinity purification-mass spectrometry) to a set of tagged-TES modular variants, we identified proteins residing in module-specific cocomplexes. The obtained distinct module-specific interactomes combine to a global TES interactome that becomes more extensive and richer in information. Applying pathway analysis to the module interactomes revealed expected actin-related canonical pathways and also less expected pathways. We validated two new TES cocomplex partners: TGFB1I1 and a short form of the glucocorticoid receptor. TES and TGFB1I1 are shown to oppositely affect cell spreading providing biological validity for their copresence in complexes since they act in similar processes.

A multi-modal approach to training in laparoscopic colorectal surgery accelerates proficiency gain.

BACKGROUND: How to efficiently train and transfer skills in laparoscopic colorectal surgery is unclear. Errors are rarely avoidable during learning but may incur patient morbidity. Multi-modality training with a modular operative approach provides proficiency-based structured task-specific training in a sequential manner, fragmenting complex laparoscopic colorectal procedures by difficulty allowing more than one trainee to gain experience irrespective of prior experience. This study assessed multi-modality training and its effect on proficiency gain in laparoscopic colorectal fellows. METHODS: A prospective study of 750 consecutive laparoscopic colon and rectal resection training cases assessing proficiency gain using a modified direct observation of procedural skills (DOPS) (behaviors-assessment) and weighted global modular attainment score (GMAS) (maneuvers-assessment) was carried out. Two mentors delivered training in a standardized format from 2008. Consequential intra-operative errors (requiring a corrective maneuver to permit further progression of the operation) were recorded. Eight Laparoscopic Fellows were assessed in six-month periods over 4 years. Primary outcome was proficiency gain measured by cumulative sum (CUSUM) analysis with boot-strapping comparing weighted GMAS and modified DOPS assessment. Morbidity (Clavien-Dindo classification), and consequential errors were submitted to similar analysis to assess significant variations during the training period. RESULTS: Fellows were trained on over 100 laparoscopic colorectal resections in a six Fellowship month period. Proficiency gain was identifiable in the DOPS and GMAS with 32 (99 % CI 25-37) and 39 (99 % CI 32-44) cases, respectively. Two- versus single-mentor training improved proficiency gain 35 (99 % CI 30-43) versus 55 (99 % CI 50-60). Overall consequential error rate and major morbidity rate (CD III-IV) were stable over time at 25 and 8.7 %, respectively. CONCLUSIONS: Multi-modality training with modular operative training and technique standardization shortens the time to proficiency gain with low morbidity accepting an intra-operative consequential error rate of 25 %.

SOX2, OTX2 and PAX6 analysis in subjects with anophthalmia and microphthalmia.

Anophthalmia (A) and microphthalmia (M) are rare developmental anomalies that have significant effects on visual activity. In fraction of A/M subjects, single genetic defects have been identified as causative. In this study we analysed 65 Italian A/M patients, 21 of whom are syndromic, for mutations in SOX2, OTX2 and PAX6 genes. In syndromic patients the presence of genome imbalances through array CGH was also investigated. No mutations were found for OTX2 and PAX6 genes. Three causative SOX2 mutations were found in subjects with syndromic A. In a subject with syndromic signs and monolateral M, two de novo 6.26 Mb and 1.37 Mb deletions in 4q13.2q13.3 have been identified. A SOX2 missense (p.Ala161Ser) mutation was found in 1 out of 39 a subject with non-syndromic monolateral M. Alanine at position 161 is conserved along phylogeny and the p.Ala161Ser mutation is estimated pathogenic by in silico analysis. However, this mutation was also present in the unaffected patient's daughter.

Ecotoxicological evaluation of industrial port of Venice (Italy) sediment samples after a decontamination treatment.

This work assesses the ecotoxicological effects of polluted sediment after a decontamination treatment process using a new sediment washing technique. Sediment samples were collected from four sites in Marghera Port industrial channels (Venice, Italy). Ecotoxicological evaluations were performed with Vibrio fischeri and Crassostrea gigas bioassays. Whole sediment and elutriate were deemed as the most suitable environmental matrices for this study. Toxicity scores developed in the Lagoon of Venice for V. fischeri on whole sediment and for C. gigas on elutriate were considered for the final ranking of samples. Ecotoxicological results showed that the treated sediment samples presented both acute and sub-chronic toxicities, which were mainly attributed to the presence of some remaining chemicals such as metals and polyaromatic hydrocarbons. The acute toxicity ranged from low to medium, while the sub-chronic one from absent to very high, suggesting that treated sediments could not be reused in direct contact with seawater.