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Marine heatwaves (MHWs) induce significant impacts on marine ecosystems. There is a growing need for knowledge about extreme climate events to better inform decision-makers on future climate-related risks. Here we present a unique observational dataset of MHW macroevents and their characteristics over the southern Europe and western Asian (SEWA) basins, named the SEWA-MHW dataset (https://doi.org//10.5281/zenodo.7153255; Bonino et al., 2022). The SEWA-MHW dataset is derived from the European Space Agency Sea Surface Temperature Climate Change Initiative (ESA SST CCI) v2 dataset, and it covers the 1981–2016 period. The methodological framework used to build the SEWA-MHW dataset is the novelty of this work. First, the MHWs detected in each grid point of the ESA CCI SST dataset are relative to a time-varying baseline climatology. Since intrinsic fluctuation and anthropogenic warming are redefining the mean climate, the baseline considers both the trend and the time-varying seasonal cycle. Second, using a connected component analysis, MHWs connected in space and time are aggregated in order to obtain macroevents. Basically, a macroevent-based dataset is obtained from a grid cell-based dataset without losing high-resolution (i.e., grid cell) information. The SEWA-MHW dataset can be used for many scientific applications. For example, we identified phases of the well-known MHW of summer 2003, and taking advantage of statistical clustering methods, we clustered the largest macroevents in SEWA basins based on shared metrics and characteristics.

The evaluation of drinking behaviors can help in limiting high-risk situation, such as driving under the influence (DUI). We investigate ethyl glucuronide in hair (hEtG) levels to evaluate alcohol consumption behavior in subjects followed up after having been charged for DUI of psychoactive substances and/or alcohol. We performed a retrospective observational cohort study on 4328 subjects over 18 years old who underwent hEtG analysis in the period 2015-2019 in the Italian Province of Pavia. hEtG level was used as a proxy for the alcohol consumption behavior. Effects of age, sex, and district on alcohol drinking behavior were investigated with an ordinal logit model. A state sequence analysis was used to study people's alcohol consumption behavior over time. hEtG was found ≥7.0 pg/mg in 22.2% of the drivers (of which 7% has an hEtG ≥30.0 pg/mg). Among positive cases, a prevalence of males (96.3%) aged 35-44 (32.6%), coming from main city and hinterland (38.2%), was observed. The propensity to drink was higher for males (odds ratio [OR] ≈ 2.28, p 0.001) and for subject coming from the district devoted to the cultivation of vineyards. Young age classes have a reduced drinking risk if compared to the drivers over 55 years old (p 0.001). A general decreasing trend over time in hEtG values was observed. Being male, age ≥ 55 years, and coming from rural areas are potential risk factors related to alcohol drinking habits among drivers. Ethyl glucuronide in hair test in the driving license reissuing protocol contributed to decrease alcohol misuse behaviors.

The collection of liquid biological matrices onto paper cards (dried matrix spots [DMS]) is becoming an alternative sampling strategy. The stability over time of molecules of interest for therapeutic, sport drug monitoring, and forensic toxicology on DMS has been recently investigated representing a reliable alternative to conventional analytical techniques. When a tampering of a urine sample in drug monitoring or doping control cases is suspected, it could be relevant to know whether genetic profiles useful for individual identification could be generated from urine samples spotted onto paper (dried urine spot [DUS]). To understand the influence of sex, storage conditions, and time on the quality and quantity of the DNA, five female and ten male urine samples were dispensed onto Whatman 903 paper and sampled after different storage conditions over time, from 1 to 12 weeks. Direct PCR was performed starting from 2-mm punches collected from each spot amplifying a panel of markers useful for individual identification. The female DUS stored in different conditions produced genetic profiles fully matching the reference samples. The same result was obtained for the male DUS but using urine 30X concentrated by centrifugation instead of the original samples. Our data show that this approach is valid for genetic individual identification of urine samples spotted onto paper cards up to 12 weeks after deposition and could be easily incorporated in anti-doping or drug screening protocols to help on the suspicion of evidence tampering or to solve questions on the reliability of samples collection.

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

Marine Heat Waves (MHWs) induce significant impacts on marine ecosystems. There is a growing need for knowledge about extreme climate events to better inform decision-makers on future climate-related risks. Here we present a unique observational dataset of MHWs macro events and their characteristics over the Southern Europe and Western Asian (SEWA) basins, named SEWA-MHWs dataset. The SEWA-MHWs dataset is derived from the European Space Agency (ESA) Climate Change Initiative (CCI) Sea Surface Temperature (SST) v2 dataset and it covers the 1981–2016 period. The methodological framework used to build the SEWA-MHWs dataset is the novelty of this work. Firstly, the MHWs detected in each grid point of the ESA CCI SST dataset are relative to a time-varying baseline climatology. Since intrinsic fluctuation and anthropogenic warming are redefining the mean climate, the baseline considers both the trend and time-varying seasonal cycle. Secondly, using a connected component analysis, MHWs connected in space and time are aggregated in order to obtain macro events. Basically, a macro event-based dataset is obtained from a pixel-based dataset without losing high resolution (i. e. pixel) information. SEWA-MHWs dataset can be used for many scientific applications. For example, we identified phases of the well-know MHW of summer 2003 and, taking advantage of statistical clustering methods, we identified the largest macro events in SEWA basins and we clustered them based on shared metrics and characteristics.

A number of neurodegenerative conditions are associated with the formation of cytosolic inclusions of TDP-43 within neurons. We expressed full-length TDP-43 in a motoneuron/neuroblastoma hybrid cell line (NSC-34) and exploited the high-resolution power of stimulated emission depletion microscopy to monitor the changes of nuclear and cytoplasmic TDP-43 levels and the formation of various size classes of cytoplasmic TDP-43 aggregates with time. Concomitantly, we monitored oxidative stress and mitochondrial impairment using the MitoSOX and MTT reduction assays, respectively. Using a quantitative biology approach, we attributed neuronal dysfunction associated with cytoplasmic deposition component to the formation of the largest inclusions, independently of stress granules. This is in contrast to other neurodegenerative diseases where toxicity is attributed to small oligomers. Using specific inhibitors, markers, and electron microscopy, the proteasome and autophagy were found to target mainly the largest deleterious inclusions, but their efficiency soon decreases without full recovery of neuronal viability.

Italy and the United States are two of the countries most affected by SARS-CoV-2 (COVID-19), with more than 240,760 confirmed cases in Italy and 2,699,658 in the United States (as of July 2, 2020). The current COVID-19 pandemic has led to substantial changes in many fields of medicine, specifically in the forensic discipline. Medicolegal activities related to conducting autopsies have been largely affected by the COVID-19 pandemic. Postmortem examinations are generally discouraged by government regulations due to the risk of spreading the disease further through the handling and dissection of bodies from patients who succumbed to COVID-19 infection. There is a paucity of data regarding the persistence of SARS-CoV-2 in bodies, as well as concerning the reliability of swabbing methods in human remains. On the other hand, the autopsy is an essential tool to provide necessary information about the pathophysiology of the disease that presents useful clinical and epidemiological insights. On this basis, we aim to address issues concerning general medical examiner/coroner organization, comparing the Italian and American systems. We also discuss the pivotal roles of forensic pathologists in informing infectious disease surveillance. Finally, we focus on the impact of COVID-19 emergency on medicolegal practices in Italy and the United States, as well as the responses of the forensic scientific community to the emerging concerns related to the pandemic. We believe that stronger efforts by authorities are necessary to facilitate completing postmortem examinations, as data derived from such assessments are expected to be paramount to improving patient management and disease prevention.

PurposeWhatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results.MethodsTwenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography–mass spectrometry methods.ResultsWe were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent.ConclusionsThis study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood.

Background: The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis. Objective: A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases. Methods: 100 µL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode. Results: 25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine. Conclusion: The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.

In spite of the growing awareness on medical students’ mental health in recent years, epidemiologic literature about the topic is scarce, particularly for what concerns suicide. Here, we collected cases of suicides among medical students enrolled at a local University in Italy and compared it to the general population of the same age group. Our sample was collected using records of suicides in the city of Pavia, Italy between the years of 2014 and 2019. This record was cross-referenced with enrolment numbers to the Medical Faculty of the city in order to select cases that involved medical students A time-window of 6 years was chosen for the evaluation. The odds ratio of suicide in medical students in comparison to the general population was of 14.58 (p value at the exact Poisson test

Fluvoxamine is a selective serotonin reuptake inhibitor, with a half-life of about 30 hours, that is commonly prescribed in the treatment of depression and obsessive and compulsive disorders. Though its more favorable adverse effect profile in comparison to tricyclic antidepressants, overdosages could lead to severe central nervous system depression. We hereby report the case of a 48-year-old woman with psychiatric disorders, who died in the Protected Community where she lived. An autopsy, during which multiorgan congestion and aspiration of gastric content were found, was performed 9 days after the death. Femoral and cardiac blood, urine and bile were collected for toxicological analysis. GC–MS, LC–MS-MS and LC–HRMS screenings were performed on blood samples. The analysis allowed to identify the following drugs: fluvoxamine, clotiapine, 7-aminoclonazepam, propranolol, gabapentin and haloperidol. Quantification of the detected drugs in blood was performed by means of a validated LC–MS-MS analytical procedure, and the following results were achieved: fluvoxamine (2.20 mg/L), gabapentin (41.00 mg/L), 7-aminoclonazepam (0.24 mg/L), clotiapine (0.07 mg/L), haloperidol (

Multifactorial diseases affecting musculoskeletal tissues are characterized by the interactions between multiple tissues, such as muscle and nerves in neuromuscular diseases, or multiple cellular components in a tissue, as in the case of bone tumors, interacting with bone cells. For these diseases also the influence of different biophysical and biochemical stimuli, such as mechanical overload and inflammatory molecules in osteoarthritis, play a key role. To investigate these complex phenomena, organ-on-a-chip systems have been developed, taking into account specific disease characteristics such as being directly derived from patients, the presence of specifically mutated cells, or a combination of relevant biophysical and/or biochemical stimuli. Depending on the envisaged application, different issues remain to be addressed. In particular, improving automation and output sensors are key for drug screening applications, while refining model microarchitecture to enhance physiological fidelity is needed for more basic science studies.

Since the association of SARS-Cov-2 infection with Nervous System (NS) manifestations, we performed RNA-sequencing analysis in Frontal Cortex of COVID-19 positive or negative individuals and affected or not by Dementia individuals. We examined gene expression differences in individuals with COVID-19 and Dementia compared to Dementia only patients by collecting transcript counts in each sample and performing Differential Expression analysis. We found eleven genes satisfying our significance criteria, all of them being protein coding genes. These data are suitable for integration with supplemental samples and for analysis according to different individuals' classification. Also, differential expression evaluation may be implemented with other scientific purposes, such as research of unannotated genes, mRNA splicing and genes isoforms. The analysis of Differential Expressed genes in COVID-19 positive patients compared to non-COVID-19 patients is published in: S. Gagliardi, E.T. Poloni, C. Pandini, M. Garofalo, F. Dragoni, V. Medici, A. Davin, S.D. Visona, M. Moretti, D. Sproviero, O. Pansarasa, A. Guaita, M. Ceroni, L. Tronconi, C. Cereda, Detection of SARS-CoV-2 genome and whole transcriptome sequencing in frontal cortex of COVID-19 patients., Brain. Behav. Immun. (2021). https://doi.org/10.1016/j.bbi.2021.05.012.

This protocol describes a comprehensive practical guide for the biofabrication of 3D in vitro models of vascularized and mineralized bone Minitissues. These models give the possibility to study the contribution of physical and biochemical parameters on bone vascularization, as well as the osteoblast/osteoclast mediated matrix remodeling. Based on the specific pathophysiological processes to be investigated, the 3D bone Minitissues allow to select the most suitable cell composition, by coculturing up to four cell types, and to customize the material properties of the hydrogel matrix. Considering their versatility, these 3D bone Minitissues could be relevant for the recapitulation of bone pathologies such as bone tumors and metastases and could be and used as screening platforms to test antimetastatic drugs.

Microfluidics allows for recapitulating organotypic environments in miniaturized cell culture platforms. This ability paves the way to the investigation of complex biological processes in a relevant milieu. Here we describe the protocols to generate an organotypic model including a vascularized compartment mimicking the synovial membrane and designed for the study of monocyte extravasation during osteoarthritis.

This protocol describes the biofabrication of 3D millimeter-scale human muscle units, embedding non-planar muscle fibers wrapped by fibroblasts-rich endomysium and intertwined with microvascular networks. Suspended muscle fibers are formed through the self-assembly of human myoblasts within cylindrical cavities generated in a sacrificial gelatin template cast in a 3D printed frame. Following myotube differentiation, muscle fibers are embedded in a 3D matrix containing endothelial cells and muscle-derived fibroblasts. The cellular complexity of the environment is instrumental to drive fibroblast migration towards muscle fibers and to induce the organ-specific differentiation of endothelial cells. This advanced 3D muscle model can be applied to analyze the biological mechanisms underlying specific muscle diseases which involve a complex remodeling of the muscle environment (e.g., muscular dystrophies and fibrosis) whereby the pathological interplay among different cell populations drives the onset and progression of the disease.

This protocol describes the biofabrication of 3D millimeter-scale human muscle units, embedding non-planar muscle fibers wrapped by fibroblasts-rich endomysium and intertwined with microvascular networks. Suspended muscle fibers are formed through the self-assembly of human myoblasts within cylindrical cavities generated in a sacrificial gelatin template cast in a 3D printed frame. Following myotube differentiation, muscle fibers are embedded in a 3D matrix containing endothelial cells and muscle-derived fibroblasts. The cellular complexity of the environment is instrumental to drive fibroblast migration towards muscle fibers and to induce the organ-specific differentiation of endothelial cells. This advanced 3D muscle model can be applied to analyze the biological mechanisms underlying specific muscle diseases which involve a complex remodeling of the muscle environment (e.g., muscular dystrophies and fibrosis) whereby the pathological interplay among different cell populations drives the onset and progression of the disease.

This protocol describes a comprehensive practical guide for the biofabrication of 3D in vitro models of vascularized and mineralized bone Minitissues. These models give the possibility to study the contribution of physical and biochemical parameters on bone vascularization, as well as the osteoblast/osteoclast mediated matrix remodeling. Based on the specific pathophysiological processes to be investigated, the 3D bone Minitissues allow to select the most suitable cell composition, by coculturing up to four cell types, and to customize the material properties of the hydrogel matrix. Considering their versatility, these 3D bone Minitissues could be relevant for the recapitulation of bone pathologies such as bone tumors and metastases and could be and used as screening platforms to test antimetastatic drugs.

Microfluidics allows for recapitulating organotypic environments in miniaturized cell culture platforms. This ability paves the way to the investigation of complex biological processes in a relevant milieu. Here we describe the protocols to generate an organotypic model including a vascularized compartment mimicking the synovial membrane and designed for the study of monocyte extravasation during osteoarthritis.

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. Graphical Abstract 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com).

Dried urine spots (DUS) represent a potential alternative sample storage for forensic toxicological analysis. The aim of the current study was to develop and validate a liquid chromatographic tandem mass spectrometric procedure for the detection and quantitative determination of cannabinoids and metabolites in DUS. A two-step extraction was performed on DUS and urine samples. An LC-MS/MS system was operated in multiple reaction monitoring and positive polarization mode. The method was checked for sensitivity, specificity, linearity, accuracy, precision, recovery, matrix effects and carryover. The method was applied to 70 urine samples collected from healthy volunteers and drug addicts undergoing withdrawal treatment. The method was successfully developed for DUS. LODs lower than 2.0 ng/mL were obtained for all the monitored substances. All the validation parameters fulfilled the acceptance criteria either for DUS or urine. Among the real samples, 45 cases provided positive results for at least one compound. A good quali-quantitative agreement was obtained between DUS and urine. A good stability of THC, THCCOOH and THCCOOH-gluc was observed after a 24 h storage, in contrast to previously published results. DUS seems to provide a good alternative storage condition for urine that should be checked for the presence of cannabinoids and metabolites.

The late early Eocene to middle Eocene ~51–45 Million years ago (Ma) time interval in the middle bathyal, pelagic/hemipelagic succession of the western Tethys Possagno section in the Carcoselle quarry (Southern Alps, northeastern Italy), contains several episodes of negative carbon isotope excursions (CIEs) and concomitant dissolution of carbonates. Comparison with previously published carbon isotope records from deep-sea successions allows the identification of long-term trends and short-term events in our record, which provides a sound chemostratigraphic basis for correlation against the robust bio- and magnetostratigraphic scheme available for the studied succession and additional evidence of the global significance of the long-term trend and superposed perturbations tracked changes in δ13C values. Spectral analysis indicates that CIEs and associated lithological cycles are paced by orbital forcing, similar to what previously observed on the same interval in other deep-sea successions. The identification of astronomically forced geochemical cycles allows us to develop an orbitally tuned age model allowing to test the astrochronology of the ~56.0 Ma to ~47.5 Ma time interval.

A multi-analyte method for detection and quantification of 16 synthetic cathinones (known also as “bath salts”) in human hair has been developed and fully validated using liquid chromatography–tandem mass spectrometry system. About 20 mg of hair samples, previously washed and homogenized, were ultrasonicated with 1 mL HCl 0.1 M solution. Samples were then extracted using a solid phase extraction procedure (SPE), taken to dryness and reconstituted in 100 μL mobile phase. Finally, they were directly injected into a liquid chromatographic system, coupled with tandem mass spectrometer detector. The validation criteria parameters were satisfactory according with the international guidelines. A LOQ of 5 pg/mg was obtained for 4-fluoromethcathinone (4-FMC), buphedrone, ethcathinone, methcathinone, mephedrone and naphyrone, while the method proved to be more sensitive for 4-methylethcathinone (4-MEC), methedrone, alpha-pyrrolidinopentiophenone (α-PVP), alpha-pyrrolidinohexiophenone (α-PHP), methylenedioxypyrovalerone (MDPV), butylone, ethylone, 3,4-dimethylmethcathinone (3,4—DMMC), pentedrone and pentylone, reaching a LOQ of 1 pg/mg. Potential use of bath salts was investigated in postmortem cases of young subjects previously tested positive at least to one traditional drug of abuse. Two samples out of 17 cases analyzed provided positive results for synthetic cathinones. One sample has been divided in two segments of 2.5 cm length each. Both segments were positive for 8 different cathinone derivatives, namely: 3,4-DMMC, 4-FMC, 4-MEC, α-PHP, α-PVP, methcathinone, methedrone and pentedrone. The second case provided positive results for ethcathinone.

Tissue-engineered decellularized matrices can progress clinical replacement of full-thickness ruptures or tendon defects. This study develops and validates a custom-made automated bioreactor, called oscillating stretch-perfusion bioreactor (OSPB), consisting of multiple, independent culture chambers able to combine a bidirectional perfusion with a programmable, uniaxial strain to functionalize cell-seeded decellularized tendons. Decellularized tendon matrices were seeded on their surfaces and within the tendon fibers with mesenchymal stem cells. Then, they were subjected to a bidirectional perfusion and programmed stretching cycles of 15–30–60 min on–off two times per day for 7 days of culture. In vitro analyses showed viable cells, homogenously distributed on the surface of the constructs. More importantly, cell-seeded decellularized tendon grafts undergoing cyclic load in our bioreactor had a superior production and organization of newly formed collagen matrix compared to static cultured constructs. The coherency and local alignment of the new collagen deposition within the inner injected channels quantitatively supported histological findings. The designed OSPB could be considered a unique, cost-effective system able to involve multiple independently controlled chambers in terms of biological and mechanical protocols. This system allows parallel processing of several customized tendon constructs to be used as grafts to enhance the surgical repair of large tendon defects. Electronic supplementary material The online version of this article (10.1007/s10439-019-02257-6) contains supplementary material, which is available to authorized users.

The actual role of SARS‐CoV‐2 in brain damage remains controversial due to lack of matched controls. We aim to highlight to what extent is neuropathology determined by SARS‐CoV‐2 or by pre‐existing conditions. Findings of 9 Coronavirus disease 2019 (COVID‐19) cases and 6 matched non‐COVID controls (mean age 79 y/o) were compared. Brains were analyzed through immunohistochemistry to detect SARS‐CoV‐2, lymphocytes, astrocytes, endothelium, and microglia. A semi‐quantitative scoring was applied to grade microglial activation. Thal‐Braak stages and the presence of small vessel disease were determined in all cases. COVID‐19 cases had a relatively short clinical course (0–32 days; mean: 10 days), and did not undergo mechanical ventilation. Five patients with neurocognitive disorder had delirium. All COVID‐19 cases showed non‐SARS‐CoV‐2‐specific changes including hypoxic‐agonal alterations, and a variable degree of neurodegeneration and/or pre‐existent SVD. The neuroinflammatory picture was dominated by ameboid CD68 positive microglia, while only scant lymphocytic presence and very few traces of SARS‐CoV‐2 were detected. Microglial activation in the brainstem was significantly greater in COVID‐19 cases (p = 0.046). Instead, microglial hyperactivation in the frontal cortex and hippocampus was clearly associated to AD pathology (p = 0.001), regardless of the SARS‐CoV‐2 infection. In COVID‐19 cases complicated by delirium (all with neurocognitive disorders), there was a significant enhancement of microglia in the hippocampus (p = 0.048). Although higher in cases with both Alzheimer's pathology and COVID‐19, cortical neuroinflammation is not related to COVID‐19 per se but mostly to pre‐existing neurodegeneration. COVID‐19 brains seem to manifest a boosting of innate immunity with microglial reinforcement, and adaptive immunity suppression with low number of brain lymphocytes probably related to systemic lymphopenia. Thus, no neuropathological evidence of SARS‐CoV‐2‐specific encephalitis is detectable. The microglial hyperactivation in the brainstem, and in the hippocampus of COVID‐19 patients with delirium, appears as a specific topographical phenomenon, and probably represents the neuropathological basis of the “COVID‐19 encephalopathic syndrome” in the elderly., The neuropathological alterations strictly attributable to SARS‐CoV‐2 infection are overall modest, predominantly caused by innate immunity activation, without evidence of SARS‐CoV‐2‐specific encephalitis. All COVID‐19 cases presented remarkable activation of ameboid CD68‐positive microglia, forming perivascular infiltrates and parenchymal nodules, mostly in the brainstem. Although higher in cases with both Alzheimer's pathology and COVID‐19, cortical neuroinflammation is not related to COVID‐19 per se but mostly to pre‐existing neurodegeneration. Cases with dementia appear more prone to a strong microglial response causing delirium.

Significance This work reports the development of a physiologically relevant human alveolar lung-on-a-chip model, composed of a three-dimensional (3D) porous hydrogel made of gelatin methacryloyl (GelMA) featuring an inverse opal structure, bonded to a compartmentalized chip device that provides air–liquid interface and cyclic breathing motions. Significantly, this GelMA structure has a high similarity to native human alveolar sacs in that they both possess sac-like pores and interconnecting windows between the sacs, in addition to a stiffness similar to the native human distal lung. We showed through multiscale analyses that our 3D GelMA inverse opal structure was better able to maintain the functions of primary human alveolar epithelial cells in a more in vivo-like manner compared with planar models., Here, we present a physiologically relevant model of the human pulmonary alveoli. This alveolar lung-on-a-chip platform is composed of a three-dimensional porous hydrogel made of gelatin methacryloyl with an inverse opal structure, bonded to a compartmentalized polydimethylsiloxane chip. The inverse opal hydrogel structure features well-defined, interconnected pores with high similarity to human alveolar sacs. By populating the sacs with primary human alveolar epithelial cells, functional epithelial monolayers are readily formed. Cyclic strain is integrated into the device to allow biomimetic breathing events of the alveolar lung, which, in addition, makes it possible to investigate pathological effects such as those incurred by cigarette smoking and severe acute respiratory syndrome coronavirus 2 pseudoviral infection. Our study demonstrates a unique method for reconstitution of the functional human pulmonary alveoli in vitro, which is anticipated to pave the way for investigating relevant physiological and pathological events in the human distal lung.

Heart‐on‐chip is an unprecedented technology for recapitulating key biochemical and biophysical cues in cardiac pathophysiology. Several designs have been proposed to improve its ability to mimic the native tissue and establish it as a reliable research platform. However, despite mimicking one of most vascularized organs, reliable strategies to deliver oxygen and substrates to densely packed constructs of metabolically demanding cells remain unsettled. Herein, we describe a new heart‐on‐chip platform with precise fluid control, integrating an on‐chip peristaltic pump, allowing automated and fine control over flow on channels flanking a 3D cardiac culture. The application of distinct flow rates impacted on temporal dynamics of microtissue structural and transcriptional maturation, improving functional performance. Moreover, a widespread transcriptional response was observed, suggesting flow‐mediated activation of critical pathways of cardiomyocyte structural and functional maturation and inhibition of cardiomyocyte hypoxic injury. In conclusion, the present design represents an important advance in bringing engineered cardiac microtissues closer to the native heart, overcoming traditional bulky off‐chip fluid handling systems, improving microtissue performance, and matching oxygen and energy substrate requirements of metabolically active constructs, avoiding cellular hypoxia. Distinct flow patterns differently impact on microtissue performance and gene expression program., A heart‐on‐chip platform integrating an automated peristaltic pumping system is developed as an accurate tool to study the influence of perfusion on cardiac tissue maturation. The study focus on the impact of flow rate on the temporal dynamics of cell morphology, cellular organization, contractile behavior, together with a comprehensive analysis of relevant transcriptional profile.

SARS-Cov-2 infection is frequently associated with Nervous System manifestations. However, it is not clear how SARS-CoV-2 can cause neurological dysfunctions and which molecular processes are affected in the brain. In this work, we examined the frontal cortex tissue of patients who died of COVID-19 for the presence of SARS-CoV-2, comparing qRT-PCR with ddPCR. We also investigated the transcriptomic profile of frontal cortex from COVID-19 patients and matched controls by RNA-seq analysis to characterize the transcriptional signature. Our data showed that SARS-CoV-2 could be detected by ddPCR in 8 (88%) of 9 examined samples while by qRT-PCR in one case only (11%). Transcriptomic analysis revealed that 11 genes (10 mRNAs and 1 lncRNA) were differential expressed when frontal cortex of COVID-19 patients were compared to controls. These genes fall into categories including hypoxia, hemoglobin-stabilizing protein, hydrogen peroxide processes. This work demonstrated that the quantity of viral RNA in frontal cortex is minimal and it can be detected only with a very sensitive method (ddPCR). Thus, it is likely that SARS-CoV-2 does not actively infect and replicate in the brain; its topography within encephalic structures remains uncertain. Moreover, COVID-19 may have a role on brain gene expression, since we observed an important downregulation of genes associated to hypoxia inducting factor system (HIF) that may inhibit the capacity of defense system during infection and oxigen deprivation, showing that hypoxia, well known multi organ condition associated to COVID-19, also marked the brain.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic condition with soft tissue progressive ossification, leading to severe disability. We describe a 27-years-old female affected by FOP who died after a fall. An autopsy was performed. Upper and lower extremities resulted in fixed flexion, with kyphoscoliosis of the spine and chest wall deformity. Moreover, a cranial fracture was pointed out. At histology, atypical abundance of corpora amylacea in gray matter was observed. In a sample of macroscopically non-affected muscular tissue, small areas with necrosis of myocytes and hyperplasia of fibroblasts were seen in light microscopy, with intracellular inorganic dystrophic inclusions in transmission electron microscopy. Thyroid gland histology showed diffuse lymphocytic infiltration. Postmortem examination of FOP patients provided precious information about involvement of other tissues, suggesting an initial and widespread inflammatory/dystrophic phase, to be further investigated, because it might reveal new insights about a FOP mutation cascade.

Correction for 'A microphysiological early metastatic niche on a chip reveals how heterotypic cell interactions and inhibition of integrin subunit β

Identification of cancer stemness genes is crucial to understanding the underlying biology of therapy resistance, relapse, and metastasis. Ewing sarcoma (EwS) is the second most common bone tumor in children and adolescents. It is a highly aggressive cancer associated with a dismal survival rate (EWSR1-ETS). Thus, EwS constitutes an ideal model to study how perturbation of a transcriptional network by a dominant oncogene can mediate metastasis, even though canonical metastasis-associated genes are not mutated.Here, through the implementation of an integrative systems biology approach, we identified transcription factor 7 like 1 (TCF7L1, alias TCF3) as a prognostically-relevant and EWSR1-ETS suppressed determinant of metastasis in EwS. We demonstrated that conditional TCF7L1 re-expression significantly reduces EwS single-cell migration, invasion and anchorage-independent growth in 3D assays in vitro, and tumorigenesis in vivo mediated by its DNA binding domain. In primary EwS tumors as well as in functional orthotopic in vivo models, low TCF7L1 expression was associated with pro-metastatic gene signatures and a much higher migratory and metastatic capacity of EwS cells, which correlated with poor outcome of EwS patients.Collectively, our findings establish TCF7L1 as a major regulator of metastasis in EwS, which may be utilized as a prognostic biomarker and open inroads to future therapeutic intervention.

During metastatic progression multiple players establish competitive mechanisms, whereby cancer cells (CCs) are exposed to both pro- and anti-metastatic stimuli. The early metastatic niche (EMN) is a transient microenvironment which forms in the circulation during CC dissemination. EMN is characterized by the crosstalk among CCs, platelets, leukocytes and endothelial cells (ECs), increasing CC ability to extravasate and colonize secondary tissues. To better understand this complex crosstalk, we designed a human "EMN-on-a-chip" which involves the presence of blood cells as compared to standard metastases-on-chip models, hence providing a microenvironment more similar to the in vivo situation. We showed that CC transendothelial migration (TEM) was significantly increased in the presence of neutrophils and platelets in the EMN-on-a-chip compared to CC alone. Moreover, exploiting the EMN-on-chip in combination with multi-culture experiments, we showed that platelets increased the expression of epithelial to mesenchymal transition (EMT) markers in CCs and that the addition of a clinically approved antiplatelet drug (eptifibatide, inhibiting integrin β

In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved extracellular matrix component that has proven to play an important role in supporting axonal guiding and peripheral nerve regeneration. Up to now, the known decellularized techniques are time and effort consuming. The present study, performed on rat sciatic nerves, aims at investigating a novel nerve decellularization protocol able to combine an effective decellularization in short time with a good preservation of the extracellular matrix component. To do this, a decellularization protocol proven to be efficient for tendons (DN-P1) was compared with a decellularization protocol specifically developed for nerves (DN-P2). The outcomes of both the decellularization protocols were assessed by a series of in vitro evaluations, including qualitative and quantitative histological and immunohistochemical analyses, DNA quantification, SEM and TEM ultrastructural analyses, mechanical testing, and viability assay. The overall results showed that DN-P1 could provide promising results if tested in vivo, as the in vitro characterization demonstrated that DN-P1 conserved a better ultrastructure and ECM components compared to DN-P2. Most importantly, DN-P1 was shown to be highly biocompatible, supporting a greater number of viable metabolically active cells., Ministry of Health, Italy, Fondazione Cassa di Risparmio di Torino (Turin, Italy) 2017.AI190.U219 RF: 2016.2388, Spanish "Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) FIS PI17-0393 FIS PI20-0318, Fondo Europeo de Desarrollo Regional ERDF-FEDER European Union, Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía, España P18-RT-5059, Programa Operativo FEDER Andalucía 2014-2020, Universidad de Granada, Junta de Andalucía, España A-CTS-498-UGR18, ERDF-FEDER, the European Union

Cell seeding on 3D scaffolds is a very delicate step in tissue engineering applications, influencing the outcome of the subsequent culture phase, and determining the results of the entire experiment. Thus, it is crucial to maximize its efficiency. To this purpose, a detailed study of the influence of the geometry of the scaffold fibers on dynamic seeding efficiency is presented. 3D printing technology was used to realize polylactic acid porous scaffolds, formed by fibers with a non-circular cross-sectional geometry, named multilobed to highlight the presence of niches and ridges. An oscillating perfusion bioreactor was used to perform bidirectional dynamic seeding of MG63 cells. The fiber shape influences the fluid dynamic parameters of the flow, affecting values of fluid velocity and wall shear stress. The path followed by cells through the scaffold fibers is also affected and results in a larger number of adhered cells in multilobed scaffolds compared to scaffolds with standard pseudo cylindrical fibers. Geometrical and fluid dynamic features can also have an influence on the morphology of adhered cells. The obtained results suggest that the reciprocal influence of geometrical and fluid dynamic features and their combined effect on cell trajectories should be considered to improve the dynamic seeding efficiency when designing scaffold architecture.

Simple Summary Ionizing radiation is used as a treatment for cancer, but it also affects the endothelial cells that make up the microvasculature. In-vitro models can be used to study the detrimental effect of irradiation on those cells. This systematic review analyzed the literature models, highlighting the critical components of the production, irradiation, and analysis of radiobiological in-vitro models for microvascular endothelial cell damage. Based on those data, we suggest future directions, including advanced in-vitro models to recapitulate microenvironment features. We pinpoint essential information to be included for the good characterization of the experiments, especially in terms of the dose delivered by ionizing radiation. Abstract Ionizing radiation (IR) is used in radiotherapy as a treatment to destroy cancer. Such treatment also affects other tissues, resulting in the so-called normal tissue complications. Endothelial cells (ECs) composing the microvasculature have essential roles in the microenvironment’s homeostasis (ME). Thus, detrimental effects induced by irradiation on ECs can influence both the tumor and healthy tissue. In-vitro models can be advantageous to study these phenomena. In this systematic review, we analyzed in-vitro models of ECs subjected to IR. We highlighted the critical issues involved in the production, irradiation, and analysis of such radiobiological in-vitro models to study microvascular endothelial cells damage. For each step, we analyzed common methodologies and critical points required to obtain a reliable model. We identified the generation of a 3D environment for model production and the inclusion of heterogeneous cell populations for a reliable ME recapitulation. Additionally, we highlighted how essential information on the irradiation scheme, crucial to correlate better observed in vitro effects to the clinical scenario, are often neglected in the analyzed studies, limiting the translation of achieved results.

During metastatic progression multiple players establish competitive mechanisms, whereby cancer cells (CCs) are exposed to both pro- and anti-metastatic stimuli. The early metastatic niche (EMN) is a transient microenvironment which forms in the circulation during CC dissemination. EMN is characterized by the crosstalk among CCs, platelets, leukocytes and endothelial cells (ECs), increasing CC ability to extravasate and colonize secondary tissues. To better understand this complex crosstalk, we designed a human "EMN-on-a-chip" which involves the presence of blood cells as compared to standard metastases-on-chip models, hence providing a microenvironment more similar to the in vivo situation. We showed that CC transendothelial migration (TEM) was significantly increased in the presence of neutrophils and platelets in the EMN-on-a-chip compared to CC alone. Moreover, exploiting the EMN-on-chip in combination with multi-culture experiments, we showed that platelets increased the expression of epithelial to mesenchymal transition (EMT) markers in CCs and that the addition of a clinically approved antiplatelet drug (eptifibatide, inhibiting integrin beta3) impaired platelet aggregation and decreased CC expression of EMT markers. Inhibition of integrin beta3 in the co-culture system modulated the activation of the Src-FAK-VE-cadherin signaling axis and partially restored the architecture of inter-endothelial junctions by limiting VE-cadherinY658 phosphorylation and its nuclear localization. These observations correlate with the decreased CC TEM observed in the presence of integrin beta3 inhibitor. Our EMN-on-a-chip can be easily implemented for drug repurposing studies and to investigate new candidate molecules counteracting CC extravasation.

Background Understanding the molecular mechanisms of metastatic dissemination, the leading cause of death in cancer patients, is required to develop novel, effective therapies. Extravasation, an essential rate-limiting process in the metastatic cascade, includes three tightly coordinated steps: cancer cell adhesion to the endothelium, trans -endothelial migration, and early invasion into the secondary site. Focal adhesion proteins, including Tln1 and FAK, regulate the cytoskeleton dynamics: dysregulation of these proteins is often associated with metastatic progression and poor prognosis. Methods Here, we studied the previously unexplored role of these targets in each extravasation step using engineered 3D in vitro models, which recapitulate the physiological vascular niche experienced by cancer cells during hematogenous metastasis. Results Human breast cancer and fibrosarcoma cell lines respond to Cdk5/Tln1/FAK axis perturbation, impairing their metastatic potential. Vascular breaching requires actin polymerization-dependent invadopodia formation. Invadopodia generation requires the structural function of FAK and Tln1 rather than their activation through phosphorylation. Our data support that the inhibition of FAKS732 phosphorylation delocalizes ERK from the nucleus, decreasing ERK phosphorylated form. These findings indicate the critical role of these proteins in driving trans-endothelial migration. In fact, both knock-down experiments and chemical inhibition of FAK dramatically reduces lung colonization in vivo and TEM in microfluidic setting. Altogether, these data indicate that engineered 3D in vitro models coupled to in vivo models, genetic, biochemical, and imaging tools represent a powerful weapon to increase our understanding of metastatic progression. Conclusions These findings point to the need for further analyses of previously overlooked phosphorylation sites of FAK, such as the serine 732, and foster the development of new effective antimetastatic treatments targeting late events of the metastatic cascade.

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.