Densità di popolazione e SARS-CoV-2: uno studio epidemiologico di urban health.

Despite SARS-CoV-2 transmission being a complex phenomenon, greater population density seems to be a risk factor. The aim of this study was to analyze through an epidemiologic urban health approach the relationship between population density and SARS-CoV-2 incidence using data which are comparable with regard to testing strategies. All 10,300 SARS-CoV-2 confirmed cases between October and December 2020 were included. We conducted separate analysis by gender standardizing and stratifying by age and month. In the Province Capital (p.d.=765 inhabitants/km2), standardized SARS-CoV-2 incidence rate was higher than the expected, both in men (SIR=1.17, 95%CI=1.12;1.22, p<0.0001) and women (SIR=1.20, 95%CI=1.15;1.25, p<0.0001). In municipalities with p.d. >200 inhabitants/km2, standardized SARS-CoV-2 incidence rate was similar to the expected (p>0.05). In municipalities with p.d. <200 inhabitants/km2, standardized SARS-CoV-2 incidence rate was lower than the expected, both in men (SIR=0.85, 95%CI=0.81;0.90, p<0.0001) and women (SIR=0.84, 95%CI=0.80;0.88, p<0.0001). Stratified analysis by months with likelihood ratio test showed heterogeneity of the p.d. effect in men and women (p<0.05). SARS-CoV-2 incidence rate seemed to be higher in most densely populated areas, both in men and women. Our results confirmed the great importance of restrictive measures as well as the importance of limiting the epidemic wave in the initial stages and could help guide pandemic management strategies according to urban context and population density.

Potential of Stem Cell-Based Therapy for Ischemic Stroke.

Ischemic stroke is one of the major health problems worldwide. The only FDA approved anti-thrombotic drug for acute ischemic stroke is the tissue plasminogen activator. Several studies have been devoted to assessing the therapeutic potential of different types of stem cells such as neural stem cells (NSCs), mesenchymal stem cells, embryonic stem cells, and human induced pluripotent stem cell-derived NSCs as treatments for ischemic stroke. The results of these studies are intriguing but many of them have presented conflicting results. Additionally, the mechanism(s) by which engrafted stem/progenitor cells exert their actions are to a large extent unknown. In this review, we will provide a synopsis of different preclinical and clinical studies related to the use of stem cell-based stroke therapy, and explore possible beneficial/detrimental outcomes associated with the use of different types of stem cells. Due to limited/short time window implemented in most of the recorded clinical trials about the use of stem cells as potential therapeutic intervention for stroke, further clinical trials evaluating the efficacy of the intervention in a longer time window after cellular engraftments are still needed.

Surface functionalization of acrylic based photocrosslinkable resin for 3D printing applications.

The limited number of resins, available for stereolithography applications, is one of the key drivers in research applied to rapid prototyping. In this work an acrylic photocrosslinkable resin based on methyl methacrylate (MMA), butyl methacrylate (BMA) and poly(ethylene glycol) dimethacrylate (PEGDA) was developed with different composition and characterized in terms of mechanical, thermal and biological behaviour. Two different systems have been developed using different amount of reagent. The influence of every components have been evaluated on the final characteristic of the resin in order to optimize the final composition for applications in bone tissue engineering. The crosslinked materials showed good mechanical properties and thermal stabilities and moreover cytotoxicity test confirms good biocompatibility with no cytotoxic effect on cells metabolism. Moreover two different treatments have been proposed, using fetal bovine serum (FBS) and methanol (MeOH), in order to improve cell recognition of the surfaces. Samples threatened with MeOH allow cell adhesion and survival, promoting spreading, elongation and fusion of C2C12 muscle myoblast cells.

Schnitzler syndrome: validation and applicability of diagnostic criteria in real-life patients.

BACKGROUND: Schnitzler syndrome is characterized by an urticarial rash, a monoclonal gammopathy, and clinical, histological, and biological signs of neutrophil-mediated inflammation. The aim of this study was to assess the applicability and validity of the existing diagnostic criteria in real-life patients. METHODS: This multicentric study was conducted between 2009 and 2014 in 14 hospitals in which patients with Schnitzler syndrome or controls with related disorders were followed up. We compared the sensitivities and specificities and calculated the positive and negative predictive values of the Lipsker and of the Strasbourg criteria for the patients with Schnitzler syndrome and for the controls. We included 42 patients with Schnitzler syndrome, 12 with adult-onset Still's disease, 7 with cryopyrin-associated periodic disease, 9 with Waldenström disease, and 10 with chronic spontaneous urticaria. RESULTS: All patients with Schnitzler syndrome met the Lipsker criteria. According to the Strasbourg criteria, 34 patients had definite Schnitzler syndrome, five had probable Schnitzler syndrome, and three did not meet the criteria. One control met the Lipsker criteria and had probable Schnitzler syndrome according to the Strasbourg criteria. Sensitivity and specificity of the Lipsker criteria were 100% and 97%, respectively. For the Strasbourg criteria, sensitivity for definite and probable diagnosis was 81% and 93%, respectively, with a corresponding specificity of 100% and 97%. CONCLUSION: Diagnostic criteria currently in use to diagnose Schnitzler syndrome are reliable. More investigations must be done to attest their efficiency in patients with recent-onset manifestations.

DOT1L-mediated H3K79me2 modification critically regulates gene expression during cardiomyocyte differentiation.

Epigenetic changes on DNA and chromatin are implicated in cell differentiation and organogenesis. For the heart, distinct histone methylation profiles were recently linked to stage-specific gene expression programs during cardiac differentiation in vitro. However, the enzymes catalyzing these modifications and the genes regulated by them remain poorly defined. We therefore decided to identify the epigenetic enzymes that are potentially involved in cardiomyogenesis by analyzing the expression profile of the 85 genes encoding the epigenetic-related proteins in mouse cardiomyocytes (CMs), and then study how they affect gene expression during differentiation and maturation of this cell type. We show here with gene expression screening of epigenetic enzymes that the highly expressed H3 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) drives a transitional pattern of di-methylation on H3 lysine 79 (H3K79) in CMs at different stages of differentiation in vitro and in vivo. Through a genome-wide chromatin-immunoprecipitation DNA-sequencing approach, we found H3K79me2 enriched at genes expressed during cardiac differentiation. Moreover, knockdown of Dot1L affected the expression of H3K79me2-enriched genes. Our results demonstrate that histone methylation, and in particular DOT1L-mediated H3K79me2 modification, drives cardiomyogenesis through the definition of a specific transcriptional landscape.

Self-assembly of polydimethylsiloxane structures from 2D to 3D for bio-hybrid actuation.

This work aims to demonstrate the feasibility of a novel approach for the development of 3D self-assembled polydimethylsiloxane structures, to be used as engineered flexible matrices for bio-hybrid actuation. We described the fabrication of engineered bilayers, organized in a 3D architecture by means of a stress-induced rolling membrane technique. Such structures were provided with ad hoc surface topographies, for both cell alignment and cell survival after membrane rolling. We reported the results of advanced finite element model simulations, predicting the system behavior in terms of overall contraction, induced by the contractile activity of muscle cells seeded on the membrane. Then, we tested in vitro the structure with primary cardiomyocytes to evaluate the real bio-actuator contraction, thus validating the simulation results. At a later stage, we provided the samples with a stable fibronectin coating, by covalently binding the protein on the polymer surface, thus enabling long-term cultures with C2C12 skeletal muscle cells, a more controllable cell type. These tests revealed cell viability and alignment on the rolled structures, but also the ability of cells to differentiate and to form multinucleated and oriented myotubes on the polymer surface, also supported by a fibroblast feeder layer. Our results highlighted the possibility of developing 3D rolled PDMS structures, characterized by different mechanical properties, as novel bio-hybrid actuators.

"Real world" outcome of lenalidomide plus dexamethasone in the setting of recurrent and refractory multiple myeloma: extended follow-up of a retrospective multicenter study by the "rete ematologica pugliese".

This current retrospective multicenter analysis represents, to our knowledge, the first Italian study evaluating the efficacy and toxicity profile of "lenalidomide plus dexamethasone" as salvage therapy in patients with recurrent-refractory MM in the real life contest. Our study included patients who are usually excluded from clinical trials because of unfavorable baseline characteristics. Median OS was significantly longer in patients receiving "lenalidomide plus dexamethasone" for more than 12 months compared with those who had received "lenalidomide plus dexamethasone" for a shorter interval (P<0.0001). Median OS was not affected by best response achieved (P 0.4) and age (P 0.3). Quality of response did not correlate with number of previous lines of therapy (P 0.77) and age. Higher ORRs were recorded in the patients group with relapsed MM compared to those with refractory disease, but this difference was not statistically significant (P 0.38).

Selection for milk coagulation properties predicted by Fourier transform infrared spectroscopy in the Italian Holstein-Friesian breed.

Milk coagulation is based on a series of physicochemical changes at the casein micelle level, resulting in formation of a gel. Milk coagulation properties (MCP) are relevant for cheese quality and yield, important factors for the dairy industry. They are also evaluated in herd bulk milk to reward or penalize producers of Protected Designation of Origin cheeses. The economic importance of improving MCP justifies the need to account for this trait in the selection process. A pilot study was carried out to determine the feasibility of including MCP in the selection schemes of the Italian Holstein. The MCP were predicted in 1,055 individual milk samples collected in 16 herds (66 ± 24 cows per herd) located in Brescia province (northeastern Italy) by means of Fourier transform infrared (FTIR) spectroscopy. The coefficient of determination of prediction models indicated moderate predictions for milk rennet coagulation time (RCT=0.65) and curd firmness (a30=0.68), and poor predictions for curd-firming time (k20=0.49), whereas the range error ratio (8.9, 6.9, and 9.5 for RCT, k20, and a30, respectively) indicated good practical utility of the predictive models for all parameters. Milk proteins were genotyped and casein haplotypes (αS1-, ß-, αS2-, and κ-casein) were reconstructed. Data from 51 half-sib families (19.9 ± 16.4 daughters per sire) were analyzed by an animal model to estimate (1) the genetic parameters of predicted RCT, k20, and a30; (2) the breeding values for these predicted clotting variables; and (3) the effect of milk protein genotypes and casein haplotypes on predicted MCP (pMCP). This is the first study to estimate both genetic parameters and breeding values of pMCP, together with the effects of milk protein genotypes and casein haplotypes, that also considered k20, probably the most important parameter for the dairy industry (because it indicates the time for the beginning of curd-cutting). Heritability of predicted RCT (0.26) and k20 (0.31) were close to the average heritability described in literature, whereas the heritability of a30 was higher (0.52 vs. 0.27). The effects of milk proteins were statistically significant and similar to those obtained on measured MCP. In particular, haplotypes including uncommon variants showed positive (B-I-A-B) or negative (B-A(1)-A-E) effects. Based on these findings, FTIR spectroscopy-pMCP is proposed as a potential selection criterion for the Italian Holstein.

PlGF-MMP9-engineered iPS cells supported on a PEG-fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium.

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)-fibrinogen scaffold. When assessing optimal stiffness of the PEG-fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness.

Schnitzler's syndrome: diagnosis, treatment, and follow-up.

Schnitzler's syndrome is characterized by recurrent urticarial rash and monoclonal gammopathy, associated with clinical and biological signs of inflammation and a long-term risk of AA amyloidosis and overt lymphoproliferation. An extensive literature review was performed, and the following questions were addressed during an expert meeting: In whom should Schnitzler's syndrome be suspected? How should the diagnosis of Schnitzler's syndrome be established? How should a patient with Schnitzler's syndrome be treated? How should a patient with Schnitzler's syndrome be followed up?. A diagnosis of Schnitzler's syndrome is considered definite in any patient with two obligate criteria: a recurrent urticarial rash and a monoclonal IgM gammopathy, and two of the following minor criteria: recurrent fever, objective signs of abnormal bone remodeling, elevated CRP level or leukocytosis, and a neutrophilic infiltrate on skin biopsy. It is considered probable, if only 1 minor criterion is present. In patients with monoclonal IgG gammopathies, diagnosis is definite if three minor criteria are present and possible if two are present. First-line treatment in patients with significant alteration of quality of life or persistent elevation of markers of inflammation should be anakinra. Follow-up should include clinical evaluation, CBC and CRP every 3 months and MGUS as usually recommended.

Post-natal cardiomyocytes can generate iPS cells with an enhanced capacity toward cardiomyogenic re-differentation.

Adult mammalian cells can be reprogrammed to a pluripotent state by forcing the expression of a few embryonic transcription factors. The resulting induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers. It is well known that post-natal cardiomyocytes (CMs) lack the capacity to proliferate. Here, we report that neonatal CMs can be reprogrammed to generate iPS cells that express embryonic-specific markers and feature gene-expression profiles similar to those of mouse embryonic stem (mES) cell and cardiac fibroblast (CF)-derived iPS cell populations. CM-derived iPS cells are able to generate chimeric mice and, moreover, re-differentiate toward CMs more efficiently then either CF-derived iPS cells or mES cells. The increased differentiation capacity is possibly related to CM-derived iPS cells retaining an epigenetic memory of the phenotype of their founder cell. CM-derived iPS cells may thus lead to new information on differentiation processes underlying cardiac differentiation and proliferation.

Monitoring of genetic diversity in the endangered Martina Franca donkey population.

The Martina Franca (MF) donkey, an ancient native breed of Apulia, was mostly famous for mule production. The breed was at serious risk of extinction in the 1980s following the decrease in demand for draft animals because they were increasingly replaced by agricultural machinery. Much has been done in the last few decades to safeguard the existing donkey breeds, but the situation remains critical. Successful implementation of conservation measures includes an evaluation of the present degree of breed endangerment, so the aim of this work was to analyze the demographic and genetic parameters of this breed to suggest effective conservation strategies. With a current breed register counting less than 500 recorded animals, the pedigree data set included 1,658 MF donkeys born between 1929 and 2006. Analyses were carried out on the whole data set as well as on a smaller one consisting of 422 living animals. Demographic and genetic variability parameters were evaluated using the ENDOG (v4.6) software. The pedigree completeness level was evaluated as well as the generation length, which was calculated for each of the 4 gametic pathways. This information was obtained from animal birth date records together with those of their fathers and mothers. The effective number of founders (f(e)), the effective number of ancestors (f(a)), the founder genome (f(g)), individual inbreeding (F), average relatedness (AR), and the rate of inbreeding per generation were analyzed to describe the genetic variability of the population. Because pedigree depth and completeness were appropriate, especially regarding the current population, the parameters defining genetic variability, namely, f(e), f(a), f(g), F, and AR, could be reliably estimated. Analysis of these parameters highlighted the endangerment status of the MF donkey. Our special concern was with the increased percentage of males and females exhibiting increased AR values. Moreover, the effective size of the current population, 48.08, is slightly less than the range of the minimum effective size, and the rates of inbreeding per generation found in the current MF population exceed the maximum recommended level of 1%. Such a scenario heightens concerns over the endangered status of the MF breed and calls for proper conservation measures and breeding strategies, such as selecting individuals for mating when relationships are below 12.5%.

Myeloma cells suppress osteoblasts through sclerostin secretion.

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of ß-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

Short communication: milk protein genetic variation and casein haplotype structure in the Original Pinzgauer cattle.

Milk protein genetic polymorphisms are often used for characterizing domesticated mammalian species and breeds, and for studying associations with economic traits. The aim of this work was to analyze milk protein genetic variation in the Original Pinzgauer, a dual-purpose (dairy and beef) cattle breed of European origin that was influenced in the past by human movements from different regions as well as by crossbreeding with Red Holstein. A total of 485 milk samples from Original Pinzgauer from Austria (n=275) and Germany (n=210) were typed at milk proteins alpha(S1)-casein, beta-casein, kappa-casein, alpha-lactalbumin, and beta-lactoglobulin by isoelectrofocusing to analyze the genetic variation affecting the protein amino acid charge. The Original Pinzgauer breed is characterized by a rather high genetic variation affecting the amino acid charge of milk proteins, with a total of 15 alleles, 12 of which were found at a frequency >0.05. The most polymorphic protein was beta-casein with 4 alleles detected. The prevalent alleles were CSN1S1*B, CSN2*A(2), CSN1S2*A, CSN3*A, LGB*A, and LAA*B. A relatively high frequency of CSN1S2*B (0.202 in the whole data set) was found, mainly occurring within the C-A(2)-B-A haplotype (in the order CSN1S1-CSN2-CSN1S2-CSN3), which seems to be peculiar to the Original Pinzgauer, possibly because the survival of an ancestral haplotype or the introgression of Bos indicus.

Soluble decoy receptor 3 modulates the survival and formation of osteoclasts from multiple myeloma bone disease patients.

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is known to be involved in cell survival and osteoclast (OC) formation. In this study, we show that malignant plasma cells and T lymphocytes from multiple myeloma (MM) bone disease patients, as well as Karpas 909, a human myeloma cell line, directly produce DcR3. By interacting with FasL, this molecule could inhibit OC apoptosis. In fact, the use of a neutralizing anti-DcR3 antibody induces a reduction of cell viability with a consequent increase of apoptotic cell number, the activation of caspase-8 and -3, and DNA fragmentation. Furthermore, we show that DcR3 supports OC formation in samples from MM patients through the upregulation of RANKL and TNFalpha by T lymphocytes and only TNFalpha by CD14+ cells. In conclusion, our data provide the first evidence of the expression of DcR3 in MM, and the involvement of this molecule in supporting the survival and formation of OCs from MM bone disease patients. The production of DcR3 by T lymphocytes confers these cells a role in the pathogenesis of bone disease associated with MM.

ASPicDB: a database resource for alternative splicing analysis.

MOTIVATION: Alternative splicing has recently emerged as a key mechanism responsible for the expansion of transcriptome and proteome complexity in human and other organisms. Although several online resources devoted to alternative splicing analysis are available they may suffer from limitations related both to the computational methodologies adopted and to the extent of the annotations they provide that prevent the full exploitation of the available data. Furthermore, current resources provide limited query and download facilities. RESULTS: ASPicDB is a database designed to provide access to reliable annotations of the alternative splicing pattern of human genes and to the functional annotation of predicted splicing isoforms. Splice-site detection and full-length transcript modeling have been carried out by a genome-wide application of the ASPic algorithm, based on the multiple alignments of gene-related transcripts (typically a Unigene cluster) to the genomic sequence, a strategy that greatly improves prediction accuracy compared to methods based on independent and progressive alignments. Enhanced query and download facilities for annotations and sequences allow users to select and extract specific sets of data related to genes, transcripts and introns fulfilling a combination of user-defined criteria. Several tabular and graphical views of the results are presented, providing a comprehensive assessment of the functional implication of alternative splicing in the gene set under investigation. ASPicDB, which is regularly updated on a monthly basis, also includes information on tissue-specific splicing patterns of normal and cancer cells, based on available EST sequences and their library source annotation. AVAILABILITY: www.caspur.it/ASPicDB

Short communication: Carora cattle show high variability in alpha(s1)-casein.

The objective of this study was to analyze the genetic variability of milk proteins of the Carora, a shorthorned Bos taurus cattle breed in Venezuela and in other Southern American countries that is primarily used for milk production. A total of 184 individual milk samples were collected from Carora cattle in 5 herds in Venezuela. The milk protein genes alpha(s1)-casein (CN) (CSN1S1), beta-CN (CSN2), kappa-CN (CSN3), and beta-lactoglobulin (LGB) were typed at the protein level by isoelectrofocusing. It was necessary to further analyze CSN1S1 at the DNA level by a PCR-based method to distinguish CSN1S1*G from B. Increased variation was found in particular at the CSN1S1 gene, where 4 variants were identified. The predominant variant was CSN1S1*B (frequency = 0.8). The second most common CSN1S1 variant was CSN1S1*G (0.101), followed by CSN1S1*C (0.082). Moreover, a new isoelectrofocusing pattern was identified, which may result from a novel CSN1S1 variant, named CSN1S1*I, migrating at an intermediate position between CSN1S1*B and CSN1S1*C. Six cows carried the variant at the heterozygous condition. For the other loci, predominance of CSN2*A2 (0.764), CSN3*B (0.609), and LGB*B (0.592) was observed. Haplotype frequencies (AF) at the CSN1S1-CSN2-CSN3 complex were also estimated by taking association into account. Only 7 haplotypes showed AF values >0.05, accounting for a cumulative frequency of 0.944. The predominant haplotype was B-A2-B (frequency = 0.418), followed by B-A2-A (0.213). The occurrence of the G variant is at a rather high frequency, which is of interest for selection within the Carora breed because of the negative association of this variant with the synthesis of the specific protein. From a cheese-making point of view, this variant is associated with improved milk-clotting parameters but is negatively associated with cheese ripening. Thus, milk protein typing should be routinely carried out in the breed, with particular emphasis on using a DNA test to detect the CSN1S*G variant. The CSN1S*G allele is likely to have descended from the Brown Swiss, which contributed to the Carora breed and also carries this allele.