A comparison of high-content screening versus manual analysis to assay the effects of mesenchymal stem cell-conditioned medium on neurite outgrowth in vitro.

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells

Dynamic wavefront shaping with an acousto-optic lens for laser scanning microscopy

Acousto-optic deflectors (AODs) arranged in series and driven with linearly chirped frequencies can rapidly focus and tilt optical wavefronts, enabling high-speed 3D random access microscopy. Non-linearly chirped acoustic drive frequencies can also be used to shape the optical wavefront allowing a range of higher-order aberrations to be generated. However, to date, wavefront shaping with AODs has been achieved by using single laser pulses for strobed illumination to 'freeze' the moving acoustic wavefront, limiting voxel acquisition rates. Here we show that dynamic wavefront shaping can be achieved by applying non-linear drive frequencies to a pair of AODs with counter-propagating acoustic waves, which comprise a cylindrical acousto-optic lens (AOL). Using a cylindrical AOL we demonstrate high-speed continuous axial line scanning and the first experimental AOL-based correction of a cylindrical lens aberration at 30 kHz, accurate to 1/35th of a wave at 800 nm. Furthermore, we develop a model to show how spherical aberration, which is the major aberration in AOL-based remote-focusing systems, can be partially or fully corrected with AOLs consisting of four or six AODs, respectively

Random-access scanning microscopy for 3D imaging in awake behaving animals

Understanding how neural circuits process information requires rapid measurements of activity from identified neurons distributed in 3D space. Here we describe an acousto-optic lens two-photon microscope that performs high-speed focusing and line scanning within a volume spanning hundreds of micrometers. We demonstrate its random-access functionality by selectively imaging cerebellar interneurons sparsely distributed in 3D space and by simultaneously recording from the soma, proximal and distal dendrites of neocortical pyramidal cells in awake behaving mice

Transmission of SARS-CoV-2 by children and young people in households and schools: a meta-analysis of population-based and contact-tracing studies

Background: The role of children and young people (CYP) in transmission of SARS-CoV-2 in household and educational settings remains unclear. We undertook a systematic review and meta-analysis of contact-tracing and population-based studies at low risk of bias. / Methods: We searched 4 electronic databases on 28 July 2021 for contact-tracing studies and population-based studies informative about transmission of SARS-CoV-2 from 0-19 year olds in household or educational settings. We excluded studies at high risk of bias, including from under-ascertainment of asymptomatic infections. We undertook multilevel random effects meta-analyses of secondary attack rates (SAR: contact-tracing studies) and school infection prevalence, and used meta-regression to examine the impact of community SARS-CoV-2 incidence on school infection prevalence. / Findings: 4529 abstracts were reviewed, resulting in 37 included studies (16 contact-tracing; 19 population studies; 2 mixed studies). The pooled relative transmissibility of CYP compared with adults was 0.92 (0.68, 1.26) in adjusted household studies. The pooled SAR from CYP was lower (p=0.002) in school studies 0.7% (0.2, 2.7) than household studies (7.6% (3.6, 15.9) . There was no difference in SAR from CYP to child or adult contacts. School population studies showed some evidence of clustering in classes within schools. School infection prevalence was associated with contemporary community 14-day incidence (OR 1.003 (1.001, 1.004), p<0.001). / Interpretation: We found no difference in transmission of SARS-CoV-2 from CYP compared with adults within household settings. SAR were markedly lower in school compared with household settings, suggesting that household transmission is more important than school transmission in this pandemic. School infection prevalence was associated with community infection incidence, supporting hypotheses that school infections broadly reflect community infections. These findings are important for guiding policy decisions on shielding, vaccination school and operations during the pandemic

Transmission of SARS-CoV-2 by children and young people in households and schools: A meta-analysis of population-based and contact-tracing studies.

BACKGROUND: The role of children and young people (CYP) in transmission of SARS-CoV-2 in household and educational settings remains unclear. We undertook a systematic review and meta-analysis of contact-tracing and population-based studies at low risk of bias. METHODS: We searched 4 electronic databases on 28 July 2021 for contact-tracing studies and population-based studies informative about transmission of SARS-CoV-2 from 0 to 19 year olds in household or educational settings. We excluded studies at high risk of bias, including from under-ascertainment of asymptomatic infections. We undertook multilevel random effects meta-analyses of secondary attack rates (SAR: contact-tracing studies) and school infection prevalence, and used meta-regression to examine the impact of community SARS-CoV-2 incidence on school infection prevalence. FINDINGS: 4529 abstracts were reviewed, resulting in 37 included studies (16 contact-tracing; 19 population studies; 2 mixed studies). The pooled relative transmissibility of CYP compared with adults was 0.92 (0.68, 1.26) in adjusted household studies. The pooled SAR from CYP was lower (p = 0.002) in school studies 0.7% (0.2, 2.7) than household studies (7.6% (3.6, 15.9) . There was no difference in SAR from CYP to child or adult contacts. School population studies showed some evidence of clustering in classes within schools. School infection prevalence was associated with contemporary community 14-day incidence (OR 1.003 (1.001, 1.004), p<0.001). INTERPRETATION: We found no difference in transmission of SARS-CoV-2 from CYP compared with adults within household settings. SAR were markedly lower in school compared with household settings, suggesting that household transmission is more important than school transmission in this pandemic. School infection prevalence was associated with community infection incidence, supporting hypotheses that school infections broadly reflect community infections. These findings are important for guiding policy decisions on shielding, vaccination school and operations during the pandemic

The ICAM-3/LFA-1 interaction is critical for epidermal Langerhans cell alloantigen presentation to CD4 + T cells

Intercellular adhesion molecule (ICAM)-3 is a recently described member of the immunoglobulin superfamily and, as such, is closely related to ICAM-1 and ICAM-2. All three ICAMS are cognate for the counter-receptor lymphocyte function associated antigen-1 (LFA-L CD11a/CD18). Unlike ICAM-1 and ICAM-2. ICAM-3 is constitutively expressed at high levels on resting leucocytes. We investigated the expression and function of ICAM-3 in normal skin ( n = 5), as well as its expression in psoriasis ( n = 4). atopic eczema ( n = 4), allergic (rhus) contact dermatitis ( n =3). and cutaneous T-cell lymphoma (CTCL. n =2). Five-micrometre cryostat sections of skin were stained using monoclonal antibodies to ICAM-3 and A well characterized immunoperoxidase technique. In normal skin. ICAM-3 was expressed by all cutaneous leucocytes hut most striking was the strong expression of ICAM-3 by Langerhans cells within both epidermis and dermis. This observation was confirmed by double-labelling with CD1a and negative staining with an IgG1 isotype control. In psoriasis, atopic eczema, allergic contact dermatitis, and CTCL. ICAM-3 was co-expressed on all CD1a + cells, although, in psoriasis, the intensity of ICAM-3 expression was reduced. Functional blocking experiments were performed to determine whether the observed ICAM-3 expression on Langerhans cells was functionally important in antigen presentation. CD4 + T cells were prepared from peripheral blood and 10 5 CD4 + T cells combined with 10 5 epidermal cells harvested from keratome biopsies of normal skin of an individual allogeneic to the T-cell donor. Addition of 50 Μg anti-ICAM-3 to the co-culture resulted in a consistent (50%) reduction in degree of alloantigen presentation by Langerhans cells to T cells. Inhibition was 77% of that produced by the addition of anti-LFA-1. These data indicate that ICAM-3 is constitutively expressed by Langerhans cells and is a major ligand for LFA-1 on CD4 + T cells during their response to Langerhans cells. Because fresh Langerhans ceils constitutively express little ICAM-1. whereas ICAM-3 is constitutively expressed at high levels, it would appear that 1CAM-3 is the dominant functional ICAM on in situ Langerhans cells in the normal epidermis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73969/1/j.1365-2133.1995.tb06911.x.pd

Bacterial microevolution and the Pangenome

The comparison of multiple genome sequences sampled from a bacterial population reveals considerable diversity in both the core and the accessory parts of the pangenome. This diversity can be analysed in terms of microevolutionary events that took place since the genomes shared a common ancestor, especially deletion, duplication, and recombination. We review the basic modelling ingredients used implicitly or explicitly when performing such a pangenome analysis. In particular, we describe a basic neutral phylogenetic framework of bacterial pangenome microevolution, which is not incompatible with evaluating the role of natural selection. We survey the different ways in which pangenome data is summarised in order to be included in microevolutionary models, as well as the main methodological approaches that have been proposed to reconstruct pangenome microevolutionary history

Human Cytomegalovirus: detection of congenital and perinatal infection in Argentina

BACKGROUND: Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection. METHODS: The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells. Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology. RESULTS: The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia, hepatosplenomegaly and enzymatic alteration were predominant, and 4 patients were HIV positive. The primers used to amplify the gB region had a PCR positivity rate of 100%, whereas those that amplified IE and LA regions had a PCR positivity rate of 54% and 61% respectively, in CMV isolates. Amplification by PCR of urine samples (with no previous DNA extraction), using primers for the gB region, detected 34/61 positive samples. Out of the 33 samples from patients with congenital infection, 24 (73%) were positive. When nPCR was used in these samples, all were positive, whereas in the remaining 28 patients, two negative cases were found. Cytomegalovirus DNA detection in 11 samples was also carried out in DBS: 7 DBS samples were positive and 4 were negative. CONCLUSIONS: Primers directed to the gB fragment region were the best choice for the detection of CMV DNA in positive isolates. In congenital infections, direct PCR in urine was positive in a high percentage (73%) of samples; however, in patients grouped as with perinatal infection only 36% of the cases were positive. With n-PCR, total sample positivity reached 97%. PCR technique performed in DBS allowed identifying congenital infection in four patients and to be confirmed in 3. These results show the value of nPCR for the detection of all cases of CMV infection. The assay offers the advantage that it may be performed within the normal working day and provides reliable results in a much shorter time frame than that required for either traditional tissue culture or the shell-viral assay