Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in ...indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists – International Society of Forensic Genetics (Ge.F.I. – ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of −1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.
•A collaborative exercise was carried out between 2020 and 2021 within ISFG-Ge.F.I.•The exercise was focused on a 13-locus multiplex Cannabis sativa STR kit.•The outcome was that typing success percentage varied from 69.2% to 100%.•Some training on this new Forensic Plant STRs will be necessary.
Force measurements with atomic force microscopy (AFM) in liquid are subjected to the hydrodynamic drag force artifact (F d) due to viscous friction of the cantilever with the liquid. This artifact ...may be especially relevant in microrheological studies of soft samples. Common approaches estimate F d at a certain distance above the sample and subtract its value from the contact force measured on the sample. However, this procedure can underestimate F d at contact. The aim of this work was to assess the effect of the hydrodynamic drag in microrheological AFM measurements of soft samples in liquid at low Reynolds numbers (Re < 1). Drag forces of water on rectangular and V-shaped cantilevers were measured in noncontact when subjecting the substrate to low-amplitude (35 nm) sinusoidal oscillations at different frequencies (1−200 Hz) and tip−substrate distances (h) (0.2−3 μm). F d increased proportionally with the relative velocity (v). Moreover, the drag factor b(h) defined as F d/v rose when the cantilever approached the substrate. Thus, the hydrodynamic drag exhibited locally a pure viscous behavior. Drag factor dependence on distance was well-fitted (r 2 ≈ 0.95) by the scaled spherical model b(h) = 6πηa eff 2/(h + h eff), where η is the viscosity of the liquid, a eff is the effective radius of the cantilever, and h eff is the effective height of the tip. Drag factor at contact was estimated as b(0) = 1.38 × 10-6 (rectangular) and 1.55 × 10-6 Ns/m (V-shaped) by extrapolating b(h) to h = 0. Drag factor measured at 2 μm underestimated b(0) by 30−50%. Thus, correction of the hydrodynamic artifact with drag factor measured a few micrometers above the surface could result in substantial errors in AFM microrheological measurements of soft samples. Our results suggest that drag artifact in contact microrheological measurements under low Re can be accurately estimated by b(0). Precise correction of drag artifact could lead to an improvement in scan speed in contact AFM imaging and in pulling speed in force spectroscopy studies.
The engraftment ability of mesenchymal cells was investigated in 26 patients receiving allogeneic transplantation from HLA-identical siblings with reduced-intensity conditioning (RIC). The stem cell ...source was bone marrow (BM) in eight patients and G-CSF-mobilized peripheral blood hematopoietic cells in 18 cases. A total of 32 patients engrafted very quickly and the chimerism evaluation (both on myeloid and on lymphoid subsets) showed that they were full donor by day 60. At the time of the study they were in complete hematological remission and displayed a full donor hematopoiesis. Two patients showed early disease progression while one did not engraft. Forty-eight out-marrow samples harvested from the 26 patients generated a marrow stromal layer adequate for the chimerism evaluation. Monocyte-macrophage contamination of marrow stromal layers was always reduced below 2% by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. The chimerism evaluation was performed by PCR analysis of STRs microsatellites and the amelogenin locus, by using capillary electrophoresis (CE) and by FISH analysis in case of the sex mismatch. In eight patients, a partial donor origin of stromal cells was shown (7-86% cells of donor). The source of hematopoietic cells was BM in three patients and mobilized peripheral blood in the other five.
Spinal muscular atrophy is a rare, autosomal recessive, neuromuscular disease caused by biallelic loss of the survival motor neuron 1 (SMN1) gene, resulting in motor neuron dysfunction. In this ...STR1VE-EU study, we aimed to evaluate the safety and efficacy of onasemnogene abeparvovec gene replacement therapy in infants with spinal muscular atrophy type 1, using broader eligibility criteria than those used in STR1VE-US.
STR1VE-EU was a multicentre, single-arm, single-dose, open-label phase 3 trial done at nine sites (hospitals and universities) in Italy (n=4), the UK (n=2), Belgium (n=2), and France (n=1). We enrolled patients younger than 6 months (180 days) with spinal muscular atrophy type 1 and the common biallelic pathogenic SMN1 exon 7–8 deletion or point mutations, and one or two copies of SMN2. Patients received a one-time intravenous infusion of onasemnogene abeparvovec (1·1 × 1014 vector genomes vg/kg). The outpatient follow-up consisted of assessments once per week starting at day 7 post-infusion for 4 weeks and then once per month until the end of the study (at age 18 months or early termination). The primary outcome was independent sitting for at least 10 s, as defined by the WHO Multicentre Growth Reference Study, at any visit up to the 18 months of age study visit, measured in the intention-to-treat population. Efficacy was compared with the Pediatric Neuromuscular Clinical Research (PNCR) natural history cohort. This trial is registered with ClinicalTrials.gov, NCT03461289 (completed).
From Aug 16, 2018, to Sept 11, 2020, 41 patients with spinal muscular atrophy were assessed for eligibility. The median age at onasemnogene abeparvovec dosing was 4·1 months (IQR 3·0–5·2). 32 (97%) of 33 patients completed the study and were included in the ITT population (one patient was excluded despite completing the study because of dosing at 181 days). 14 (44%, 97·5% CI 26–100) of 32 patients achieved the primary endpoint of functional independent sitting for at least 10 s at any visit up to the 18 months of age study visit (vs 0 of 23 untreated patients in the PNCR cohort; p<0·0001). 31 (97%, 95% CI 91–100) of 32 patients in the ITT population survived free from permanent ventilatory support at 14 months compared with six (26%, 8–44) of 23 patients in the PNCR natural history cohort (p<0·0001). 32 (97%) of 33 patients had at least one adverse event and six (18%) had adverse events that were considered serious and related to onasemnogene abeparvovec. The most common adverse events were pyrexia (22 67% of 33), upper respiratory infection (11 33%), and increased alanine aminotransferase (nine 27%). One death, unrelated to the study drug, occurred from hypoxic-ischaemic brain damage because of a respiratory tract infection during the study.
STR1VE-EU showed efficacy of onasemnogene abeparvovec in infants with symptomatic spinal muscular atrophy type 1. No new safety signals were identified, but further studies are needed to show long-term safety. The benefit–risk profile of onasemnogene abeparvovec seems favourable for this patient population, including those with severe disease at baseline.
Novartis Gene Therapies.
Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus (
G
*(
ω)) of human ...alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1–100
Hz) and at different loading forces (0.1–0.9
nN) with atomic force microscopy.
G
*(
ω) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic viscous drag. Both cell types displayed similar viscoelastic properties. The storage modulus
G′(
ω) increased with frequency following a power law with exponent ∼0.2. The loss modulus
G″(
ω) was ∼2/3 lower and increased similarly to
G′(
ω) up to ∼10
Hz, but exhibited a steeper rise at higher frequencies. The cells showed a weak force dependence of
G′(
ω) and
G″(
ω).
G
*(
ω) conformed to the power-law model with a structural damping coefficient of ∼0.3, indicating a coupling of elastic and dissipative processes within the cell. Power-law behavior implies a continuum distribution of stress relaxation time constants. This complex dynamics is consistent with the rheology of soft glassy materials close to a glass transition, thereby suggesting that structural disorder and metastability may be fundamental features of cell architecture.
Our aim was to describe the molecular epidemiology and antimicrobial resistance determinants of isolates of Neisseria gonorrhoeae with decreased susceptibility and resistance to extended-spectrum ...cephalosporins (ESCs) in Argentina in 2011-16.
Gonococcal isolates (n=158) with decreased susceptibility and resistance to ESCs collected in 2011-16 across Argentina were subjected to WGS and antimicrobial susceptibility testing for six antimicrobials.
In total, 50% of the isolates were resistant to cefixime, 1.9% were resistant to ceftriaxone, 37.3% were resistant to azithromycin and 63.9% of the isolates showed an MDR phenotype. Resistance and decreased susceptibility to ESCs was mainly associated with isolates possessing the mosaic penA-34.001, in combination with an mtrR promoter A deletion, and PorB1b amino acid substitutions G120K/A121N. Phylogenetic analysis revealed two main clades of circulating strains, which were associated with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 and closely related STs, and characterized by a high prevalence rate, wide geographical distribution and temporal persistence.
N. gonorrhoeae isolates with decreased susceptibility and resistance to ESCs in Argentina have emerged and rapidly spread mainly due to two clonal expansions after importation of one or two strains, which are associated with the international MDR NG-MAST ST1407 clone. The identification of the geographical dissemination and characteristics of these predominant clones may help to focus action plans and public health policies to control the spread of ESC resistance in Argentina. Dual antimicrobial therapy (ceftriaxone plus azithromycin) for gonorrhoea needs to be considered in Argentina.
Single-nucleotide polymorphisms of Y chromosome (Y-SNPs) are a class of markers of interest in forensic investigations, because many of them show regional specificity, providing useful information ...about the geographic origin of a subject or evidence under investigation. A first multiplex with 7 SNPs (M35, M89, M9, M170, M172, M45, M173), which occur in the basal branches of the phylogenetic tree and are able to assign a subject to known most frequent European haplogroups, was designed. SNP genotyping was accomplished by hot-start PCR with primers amplifying fragments between 96 and 136 nucleotides, minisequencing, and capillary electrophoresis of extension products. Ninety seven subjects of known geographic provenance were studied, of which 68 from Europe. Of these, 57 had mutations found more frequently in European haplogroups and 11 more frequent in Asian populations. Subjects from non-European countries were also examined and had haplogroups common in their regions of provenance. Experiments with low molecular weight DNA gave positive amplification from 1 ng of DNA for all seven SNPs.
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