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The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5′-GGTTAT-3′) is located within the 5′-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in β-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify β-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in β-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of β-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment.

Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.

Background Patients with glioblastoma (GBM) have a dramatically poor prognosis. The recent REGOMA trial suggested an overall survival (OS) benefit of regorafenib in recurrent GBM patients. Considering the extreme genetic heterogeneity of GBMs, we aimed to identify molecular biomarkers predictive of differential response to the drug. Methods Total RNA was extracted from tumor samples of patients enrolled in the REGOMA trial. Genome-wide transcriptome and micro (mi)RNA profiles were associated with patients’ OS and progression-free survival. Results In the first step, a set of 11 gene transcripts (HIF1A, CTSK, SLC2A1, KLHL12, CDKN1A, CA12, WDR1, CD53, CBR4, NIFK-AS1, RAB30-DT) and 10 miRNAs (miR-93-5p, miR-203a-3p, miR-17-5p, let-7c-3p, miR-101-3p, miR-3607-3p, miR-6516-3p, miR-301a-3p, miR-23b-3p, miR-222-3p) was filtered by comparing survival between regorafenib and lomustine arms. In the second step, a mini-signature of 2 gene transcripts (HIF1A, CDKN1A) and 3 miRNAs (miR-3607-3p, miR-301a-3p, miR-93-5p) identified a subgroup of patients showing prolonged survival after regorafenib administration (median OS range, 10.6–20.8 mo). Conclusions The study provides evidence that a signature based on the expression of 5 biomarkers could help identify a subgroup of GBM patients exhibiting a striking survival advantage when treated with regorafenib. Although the presented results must be confirmed in larger replication cohorts, the study highlights potential biomarker options to help guide the clinical decision among regorafenib and other treatments in patients with relapsing GBM.

Aims The role of a Acinetobacter johnsonii strain, isolated from a soil sample, in the biotransformation of bile acids (BAs) was already described but the enzymes responsible for these transformations were only partially purified and molecularly characterized. Methods and Results This study describes the use of hybrid de novo assemblies, that combine long-read Oxford Nanopore and short-read Illumina sequencing strategies, to reconstruct the entire genome of A. johnsonii ICE_NC strain and to identify the coding region for a 12α-hydroxysteroid dehydrogenase (12α-HSDH), involved in BAs metabolism. The de novo assembly of the A. johnsonii ICE_NC genome was generated using Canu and Unicycler, both strategies yielded a circular chromosome of about 3.6 Mb and one 117 kb long plasmid. Gene annotation was performed on the final assemblies and the gene for 12α-HSDH was detected on the plasmid. Conclusions Our findings illustrate the added value of long read sequencing in addressing the challenges of whole genome characterization and plasmid reconstruction in bacteria. These approaches also allowed the identification of the A. johnsonii ICE_NC gene for the 12α-HSDH enzyme, whose activity was confirmed at the biochemical level. Significance and impact or the study At present, this is the first report on the characterization of a 12α-HSDH gene in an A. johnsonii strain able to biotransform cholic acid into ursodeoxycholic acid, a promising therapeutic agent for several diseases.

Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common β thalassemia mutations in the Mediterranean area (β+IVSI-110 and β039), maternally and/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of β+IVSI-110 and β039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.

Circulating miRNAs are increasingly studied and proposed as tumor markers with the aim of investigating their role in monitoring the response to therapy as well as the natural evolution of primary or secondary brain tumors. This study aimed to evaluate the modulation of the expression of three miRNAs, miR-21, miR-222 and miR-124-3p, in the serum exosomes of patients with high-grade gliomas (HGGs) and brain metastases (BMs) to verify their usefulness in the differential diagnosis of brain masses; then, it focused on their variations following the surgical and/or radiosurgical treatment of the BMs. A total of 105 patients with BMs from primary lung or breast cancer, or melanoma underwent neurosurgery or radiosurgery treatment, and 91 patients with HGGs were enrolled, along with 30 healthy controls. A significant increase in miR-21 expression in serum exosomes was observed in both HGGs and BMs compared with healthy controls; on the other hand, miR-124-3p was significantly decreased in BMs, and it was increased in HGGs. After the surgical or radiosurgical treatment of patients with BMs, a significant reduction in miR-21 was noted with both types of treatments. This study identified a signature of exosomal miRNAs that could be useful as a noninvasive complementary analysis both in the differential diagnosis of BMs from glial tumors and in providing information on tumor evolution over time.

Differences between sexes contribute to variation in the levels of fasting glucose and insulin. Epidemiological studies established a higher prevalence of impaired fasting glucose in men and impaired glucose tolerance in women, however, the genetic component underlying this phenomenon is not established. We assess sex-dimorphic (73,089/50,404 women and 67,506/47,806 men) and sex-combined (151,188/105,056 individuals) fasting glucose/fasting insulin genetic effects via genome-wide association study meta-analyses in individuals of European descent without diabetes. Here we report sex dimorphism in allelic effects on fasting insulin at IRS1 and ZNF12 loci, the latter showing higher RNA expression in whole blood in women compared to men. We also observe sex-homogeneous effects on fasting glucose at seven novel loci. Fasting insulin in women shows stronger genetic correlations than in men with waist-to-hip ratio and anorexia nervosa. Furthermore, waist-to-hip ratio is causally related to insulin resistance in women, but not in men. These results position dissection of metabolic and glycemic health sex dimorphism as a steppingstone for understanding differences in genetic effects between women and men in related phenotypes., Sex differences in fasting glucose and insulin have been identified, but the genetic loci underlying these differences have not. Here, the authors perform a meta-analysis of genome-wide association studies to detect sex-specific and sex-dimorphic loci associated with fasting glucose and insulin.

Homeostatic control of blood glucose requires different physiological responses in the fasting and post-prandial states. We reasoned that glucose measurements under non-standardised conditions (random glucose; RG) may capture diverse glucoregulatory processes more effectively than previous genome-wide association studies (GWAS) of fasting glycaemia or after standardised glucose loads. Through GWAS meta-analysis of RG in 493,036 individuals without diabetes of diverse ethnicities we identified 128 associated loci represented by 162 distinct signals, including 14 with sex-dimorphic effects, 9 discovered through trans-ethnic analysis, and 70 novel signals for glycaemic traits. Novel RG loci were particularly enriched in expression in the ileum and colon, indicating a prominent role for the gastrointestinal tract in the control of blood glucose. Functional studies and molecular dynamics simulations of coding variants of GLP1R, a well-established type 2 diabetes treatment target, provided a genetic framework for optimal selection of GLP-1R agonist therapy. We also provided new evidence from Mendelian randomisation that lung function is modulated by blood glucose and that pulmonary dysfunction is a diabetes complication. Thus, our approach based on RG GWAS provided wide-ranging insights into the biology of glucose regulation, diabetes complications and the potential for treatment stratification.

In insects, tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through an RNAi approach, topically delivered HhTAR1 dsRNA induced a 50% downregulation in gene expression after 24 h in H. halys 2nd instar nymphs. An innovative behavioural assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared with controls. These results provide critical information concerning the role of TAR1 in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work opens the way for further investigation on innovative methods for controlling H. halys.

Simple Summary The burial of corpses may interfere with the succession of sarcosaprophagous fauna and forensic evaluation of post-mortem interval. For the first time in Italy, an experimental study was conducted on arthropods associated with buried pig carcasses in a rural area near Cosenza (Southern Italy). One carcass was left above the ground and five were buried: one of the buried ones was periodically exhumed to evaluate the effects of disturbance on decay processes and on arthropod fauna, and the other four were exhumed only once at given time intervals. The results revealed differences in taxa and colonization of arthropod fauna in the above ground and periodically exhumed carcasses. No arthropod colonization was detected in the carcasses exhumed only once, showing that a burial at about 25 cm depth could be sufficient to prevent colonization by sarcosaprophagous taxa. Abstract Burial could be used by criminals to conceal the bodies of victims, interfering with the succession of sarcosaprophagous fauna and with the evaluation of post-mortem interval. In Italy, no experimental investigation on arthropods associated with buried remains has been conducted to date. A first experimental study on arthropods associated with buried carcasses was carried out in a rural area of Arcavacata di Rende (Cosenza), Southern Italy, from November 2017 to May 2018. Six pig carcasses (Sus scrofa Linnaeus) were used, five of which were buried in 60-cm deep pits, leaving about 25-cm of soil above each carcass, and one was left above ground. One of the buried carcasses was periodically exhumed to evaluate the effects of disturbance on decay processes and on arthropod fauna. The other four carcasses were exhumed only once, respectively after 43, 82, 133, and 171 days. As expected, the decay rate was different among carcasses. Differences in taxa and colonization of arthropod fauna were also detected in the above ground and periodically exhumed carcasses. In carcasses exhumed only once, no arthropod colonization was detected. The results showed that a burial at about 25 cm depth could be sufficient to prevent colonization by sarcosaprophagous taxa and these data could be relevant in forensic cases involving buried corpses.

During oviposition, ectoparasitoid wasps not only inject their eggs but also a complex mixture of proteins and peptides (venom) in order to regulate the host physiology to benefit their progeny. Although several endoparasitoid venom proteins have been identified, little is known about the components of ectoparasitoid venom. To characterize the protein composition of Torymus sinensis Kamijo (Hymenoptera: Torymidae) venom, we used an integrated transcriptomic and proteomic approach and identified 143 venom proteins. Moreover, focusing on venom gland transcriptome, we selected additional 52 transcripts encoding putative venom proteins. As in other parasitoid venoms, hydrolases, including proteases, phosphatases, esterases, and nucleases, constitute the most abundant families in T. sinensis venom, followed by protease inhibitors. These proteins are potentially involved in the complex parasitic syndrome, with different effects on the immune system, physiological processes and development of the host, and contribute to provide nutrients to the parasitoid progeny. Although additional in vivo studies are needed, initial findings offer important information about venom factors and their putative host effects, which are essential to ensure the success of parasitism.

The original version of this Article contained an error in Fig. 2, in which panels a and b were inadvertently swapped. This has now been corrected in the PDF and HTML versions of the Article.

Simple Summary Historical pharmacies are cultural and scientific assets, and 482 of them have been catalogued thus far in Italy. The Navarra-Bragliani Pharmacy in Ferrara has valuable wood furniture dating back to the 18th century. In order to monitor and preserve it, a study on biodeteriogenic insects was carried out for the first time on the furnishing of this pharmacy, collecting insect samples and examining them by stereomicroscopy and scanning electron microscopy. The presence of three species of Coleoptera was detected—the first one specialised to attack wood materials, the second one polyphagous, and the third one feeding on materials of animal origin. In addition to their use in the preservation of the precious furnishing of the Navarra-Bragliani Pharmacy, the results of this study could be useful for similar studies on other ancient pharmacies, in order to protect these valuable cultural and scientific assets. Abstract Historical pharmacies are valuable artistic, cultural, and scientific assets. In Italy, 482 historical pharmacies have been catalogued thus far, among which is the Navarra-Bragliani Pharmacy in Ferrara, whose wood furniture dates back to the 18th century. A study on insects causing biodeterioration was carried out for the first time on this valuable asset in order to monitor and preserve it. Insect samples were collected during surveys and examined by stereomicroscopy and scanning electron microscopy, using taxonomical keys for identification. The results revealed the presence of three species of Coleoptera—the first one specialised to attack wood materials, the second one polyphagous, and the third one feeding on materials of animal origin. The data obtained in this investigation may be useful for the preservation of the precious furnishing of the historical Navarra-Bragliani Pharmacy for performing similar studies on other ancient pharmacies aimed to protect these valuable cultural and scientific assets.

In insects, the tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. The RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy (SEM) investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through a RNAi approach, topically delivered HhTAR1 dsRNA induced a 50 % gene downregulation after 24 h in H. halys 2nd instar nymphs. An innovative behavioral assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared to control. These results provide critical information concerning the TAR1 role in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work paves the way for further investigation on innovative methods for controlling H. halys.

A relevant species in waste management but also in forensic, medical, and veterinary sciences is the black soldier fly, Hermetia illucens (Linnaeus; Diptera: Stratiomyidae). An ultrastructural study by scanning electron microscopy (SEM) was conducted for the first time on maxillary palps of both sexes, describing in detail the morphology and distribution of sensilla and microtrichia. The maxillary palps, composed of two segments, show sexual dimorphism in length and shape. In both sexes, the first segment is covered only by microtrichia, but the second one is divided into two parts: the proximal one, covered only by microtrichia, and the distal one containing both microtrichia and sensory structures. These structures include two types of sensory pits and one of chaetic sensilla. Due to sexual dimorphism in palp size, females have a higher number of sensory pits. The sexual dimorphism of palps and the presence and role of sensilla in H. illucens was discussed in comparison to other species of the family Stratiomyidae and of other Diptera. This study may represent a base for further investigations on mouthpart structures of this species, involved in key physiological activities, such as feeding, mating and oviposition.

The first case of myiasis caused by Lucilia caesar (L.) (Diptera: Calliphoridae) in a wild boar, Sus scrofa L. (Artiodactyla: Suidae) is described. The myiasis occurred in October 2019 in Southern Italy and the identification of the agent was based on adult male morphology. The wild boar had a wound on its right side, near the neck, which was largely infested by larvae. The ecology, distribution and current literature status about cases of animal myiasis by this species is also included.

Musca domestica Linnaeus (Diptera: Muscidae), a relevant synanthropic pest, is the most frequent dipteran species in farms and waste landfills. Insect Growth Regulators (IGR), insecticides with species-specific toxicity and low health and environmental impact, are known to act mostly on larval stages but may induce sterility in adults. We investigated the effects of diofenolan, an IGR analogue of juvenile hormone, on M. domestica, with special attention to female reproductive ability (egg-laying and hatching), and ovarian and ovariole morphology, using optical and transmission electron microscopy. We also tested the interactions between diofenolan and the activity of corpora allata, endocrine gland producing juvenile hormone required for ovarian development, by morphological and ultrastructural investigations. The results show that diofenolan negatively affects the reproductive ability of M. domestica, causing extensive morphological alterations in ovaries and ovarioles. In treated females, ovarioles showed nine different morphotypes that could be arranged into three “transformation paths”, and these alterations were able to reduce egg-laying. The effects of diofenolan on corpora allata, investigated at the optical and ultrastructural level in M. domestica, showed extensive alterations of the nuclei, cytoplasms, and mitochondria, strongly suggesting a rapid transition of the gland from inactivity to degeneration. The sterilizing effects of diofenolan in M. domestica are very interesting for integrated pest management programs within sustainable defence strategies against this relevant and annoying pest.

Postmenopausal osteoporosis (PO) has a strong genetic component. Presently, the published evidence on the association between the main single-nucleotide polymorphisms (SNPs) of the receptor activator of nuclear factor-kb ligand (RANKL), osteoprotegerin (OPG) and vitamin D receptor (VDR) and bone mass density (BMD) are scarce, mostly considering Italian population. This study sought to determine whether OPG (rs2073618), RANKL (rs9525641) and the VDR (rs2228570) SNPs were associated with BMD in a sample of 139 North-Italian postmenopausal women. The allelic distribution of rs9525641 in women with PO or osteopenia (OP + OPE group) differed from controls (p 0.05), suggesting that this allele might confer a greater susceptibility to bone resorption. Concerning rs2228570, CC genotype was associated with OP + OPE women, with a worst total hip BMD. Notably, the combined genotype RANK (CT)-VDR (TT) was significantly associated to spine BMD (p 0.05). In conclusion, this pilot study showed that rs9525641 and rs2228570 polymorphisms might contribute, separately or in combination, in determining BMD phenotype in selected postmenopausal populations.

Plague, a highly infective disease caused by Yersinia pestis (Proteobacteria: Enterobacteriales), ravaged Europe from 1347 over the course of more than 450 years. During the Italian Plague (1629–1631), the disease was rampaging in the entire Northern Italy down to Tuscany, but the city of Ferrara was relatively spared, in spite that the economic activities were maintained with highly affected cities, such as Milan, through the relevant salt commerce. The aim of the study is to evaluate the hygiene rules that were effective in preventing the spread of the plague in Ferrara in 1630, by examining historical documents and reports. According to these documents, a kind of empirical “integrated disease management” was carried out, using remedies including compounds with bactericidal, anti-parasite and repellent activity, and by technical strategies including avoidance of possible plague carriers. The anti-plague remedies and technical strategies used in ancient Ferrara are critically analysed using a multidisciplinary approach (pharmaceutic, medical, epidemiologic and entomological) and compared to current prevention protocols.

The report describes a case of urogenital myiasis in a puppy,Canis lupus familiaris(Carnivora: Canidae) caused byWohlfahrtia magnifica(Diptera: Sarcophagidae) in Calabria, southern Italy. This species is an obligatory agent of myiasis in human and other warm-blooded vertebrates. The puppy was healthy and was not living near farm animals, usual hosts of this flesh fly. An overview of cases of human and animal myiasis caused byW. magnificain Italy and of data and specimens documented in entomology museum collections is also reported.

Surname distribution can be a useful tool for studying the genetic structure of a human population. In South America, the Uruguay population has traditionally been considered to be of European ancestry, despite its trihybrid origin, as proved through genetics. The aim of this study was to investigate the structure of the Uruguayan population, resulting from population movements and surname drift in the country. The distribution of the surnames of 2,501,774 people on the electoral register was studied in the nineteen departments of Uruguay. Multivariate approaches were used to estimate isonymic parameters. Isolation by Distance was measured by correlating isonymic and geographic distances. In the study sample, the most frequent surnames were consistently Spanish, reflecting the fact that the first immigration waves occurred before Uruguayan independence. Only a few surnames of Native origin were recorded. The effective surname number (α) for the entire country was 302, and the average for departments was 235.8 ± 19. Inbreeding estimates were lower in the south-west of the country and in the densely populated Montevideo area. Isonymic distances between departments were significantly correlated with linear geographic distance (p < 0.001) indicating continuously increasing surname distances up to 400 km. Surnames form clusters related to geographic regions affected by different historical processes. The isonymic structure of Uruguay shows a radiation towards the east and north, with short-range migration playing a major role, while the contribution of drift, considering the small variance of α, appears to be minor.

Type 2 diabetes (T2D) affects the health of millions of people worldwide. The identification of genetic determinants associated with changes in glycemia over time might illuminate biological features that precede the development of T2D. Here we conducted a genome-wide association study of longitudinal fasting glucose changes in up to 13,807 non-diabetic individuals of European descent from nine cohorts. Fasting glucose change over time was defined as the slope of the line defined by multiple fasting glucose measurements obtained over up to 14 years of observation. We tested for associations of genetic variants with inverse-normal transformed fasting glucose change over time adjusting for age at baseline, sex, and principal components of genetic variation. We found no genome-wide significant association (P −8) with fasting glucose change over time. Seven loci previously associated with T2D, fasting glucose or HbA1c were nominally (P

Objective The in vitro evaluation of SPF is still a problem due to the lack of repeatability and correlation between the in vitro and in vivo data and many authors are currently working to develop an internationally harmonized method.(1) Very recently, the use of several “adjuvant” ingredients such as boosters, antioxidants, immuno-modulators, solvents and film forming ingredients have further complicated the pattern for product developers, that should frequently run in vivo test. The aim of this study is to understand if a simple and cheap in vitro method could be optimized in order to provide both statistically repeatable and predictive SPF measurement. Methods In vitro SPF assessments were carried out on 75 commercial products. The SPF was measured according to two laboratory methods (A and B), using different substrates (PMMA and surgical tape Transpore™), quantity of product, spectrophotometers. In order to evaluate if a standard technique of spreading could lead to a statistically reliable result, we applied different spreading pressure (100 g and 200 g). Furthermore, we investigate if other parameters characterizing the product (SPF Category, Filter and Texture) might represent statically significant variables affecting the measures. We then compared the results obtained from in vitro SPF measure of 11 products to in vivo SPF, in order to assess the predictivity of in vitro methods. Results Several problems were encountered in confirming the weakness of the in vitro procedures. Pressure, SPF Category, Filter and Texture did not affect significantly the results. Overall best results were obtained with the B2 method that in terms of repeatability and predictivity provided statistically better results. Method A with Transpore™ tape showed better in vitro-in vivo correlation than Method B with PMMA plates. Conclusion In our investigation we demonstrated that it is possible for a single laboratory to optimize internal methods and protocols to achieve repeatable and predictive in vitro results, but it is extremely difficult to develop methods reproducible and equally reliable in different laboratories, probably due to “external variables” (eg. environmental, operator), which are difficult to control. This article is protected by copyright. All rights reserved.

In order to develop potential anticancer agents stimulating apoptosis, novel 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives have been synthetized. The original structures of geldanamycin and radicicol, which are known natural heat shock protein (HSP) inhibitors, were deeply modified because both of them exhibit several drawbacks, such as poor solubility, hepatotoxicity, intrinsic chemical instability or deprivation of the in vivo activity. This novel class of synthetic compounds containing the isoxazole nucleus exhibited potent and selective inhibition of HSP90 in previous studies. Biological assays (focusing on in vitro antiproliferative effects and pro-apoptotic activity) in human erythroleukemic K562 cells (as a model system referring to tumor cells grown in suspension), glioblastoma U251-MG and glioblastoma temozolomide (TMZ)-resistant T98G cell lines (two model systems referring to tumor cells grown attached to the flask), were performed. Almost all isoxazole derivatives demonstrated significant antiproliferative and pro-apoptotic activities, showing induction of both early and late apoptosis of K562 cells. Different effects were observed on the glioma U251-MG and T98G cells, depending on the structure of the analogues. Antiproliferative and pro-apoptotic activities in K562 cells were associated with the activation of the erythroid differentiation program. The present study demonstrated that 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives should be considered for in vivo studies focusing on the development of anticancer drugs acting, at least partially, via activation of apoptosis.

Objective The aim of this study was to use high-resolution whole metagenomic shotgun sequencing to characterize the subgingival microbiome of patients with/without type 2 Diabetes Mellitus and with/without periodontitis. Design Twelve subjects, falling into one of the four study groups based on the presence/absence of poorly controlled type 2 Diabetes Mellitus and moderate-severe periodontitis, were selected. For each eligible subject, subgingival plaque samples were collected at 4 sites, all representative of the periodontal condition of the individual (i.e., non-bleeding sulci in subjects without a history of periodontitis, bleeding pockets in patients with moderate-severe periodontitis). The subgingival microbiome was evaluated using high-resolution whole metagenomic shotgun sequencing. Results The results showed that: (i) the presence of type 2 Diabetes Mellitus and/or periodontitis were associated with a tendency of the subgingival microbiome to decrease in richness and diversity; (ii) the presence of type 2 Diabetes Mellitus was not associated with significant differences in the relative abundance of one or more species in patients either with or without periodontitis; (iii) the presence of periodontitis was associated with a significantly higher relative abundance of Anaerolineaceae bacterium oral taxon 439 in type 2 Diabetes Mellitus patients. Conclusions Whole metagenomic shotgun sequencing of the subgingival microbiome was extremely effective in the detection of low-abundant taxon. Our results point out a significantly higher relative abundance of Anaerolineaceae bacterium oral taxon 439 in patients with moderate to severe periodontitis vs patients without history of periodontitis, which was maintained when the comparison was restricted to type 2 diabetics.

Myiasis is an infestation caused by larvae of Diptera in humans and other vertebrates. In domestic cats, Felis silvestris catus L. (Carnivora: Felidae), four dipteran families have been reported as agents of obligatory and facultative myiasis: Oestridae, Calliphoridae, Sarcophagidae and Muscidae. Among agents of obligatory myiasis, the most frequent genus is Cuterebra Clark (Oestridae) and the most frequent species is Cochliomyia hominivorax (Coquerel) (Calliphoridae). Among the agents of facultative myiasis, the most frequent species is Lucilia sericata (Meigen) (Calliphoridae). A survey of myiasis in cats reported in literature shows that the cases are distributed worldwide and linked to the geographical range of the dipteran species. Factors favouring the occurrence of myiasis in cats are prowling in infested areas, poor hygiene conditions due to diseases and/or neglect, and wounds inflicted during territorial or reproductive competition. The aim of the review is to provide an extended survey of literature on myiasis in cats, as general information and possible development of guidelines for veterinarians, entomologists and other researchers interested in the field.