Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration.

In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.

Dynamics of Major and Trace Elements in Water-Soil-Tree Interaction: Translocation in Pyrus malus in Chihuahua, Mexico Using ICP-OES and Its Health Risk Implications.

The transference of metals from water irrigation and soil to plants is a possible pathway of contamination for the trophic chain. This research is focused on the distribution of 16 analytes in the water-soil-tree (Pyrus malus) interaction in an agricultural region in the state of Chihuahua in Mexico from August 2019 (first sampling) to August 2020 (second sampling). The apple variety under investigation was Golden Delicious; it was found that the trace elements of As (0.18-0.34 mg·kg-1) and Cd (0.11-0.14 mg·kg-1) in the apple were above the corresponding permissible limit, according to FAO/WHO, and Cr (0.08-0.86 mg·kg-1) was below the limit. Furthermore, the health risk implications were estimated by the Hazard Quotients (HQ) and carcinogenic risk (CR). For carcinogenic risk, As, Cd, and Cr exceeded the risk limit (CR > 10-4). This investigation as well provides a link for similar research around the globe. Major and trace elements detection was performed with the Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) technique, along with a prior homogenization of samples and microwave acid digestion. To obtain the statistical behavior, an analysis of variance and correlation was performed.

Human influenza virus challenge identifies cellular correlates of protection for oral vaccination.

Developing new influenza vaccines with improved performance and easier administration routes hinges on defining correlates of protection. Vaccine-elicited cellular correlates of protection for influenza in humans have not yet been demonstrated. A phase-2 double-blind randomized placebo and active (inactivated influenza vaccine) controlled study provides evidence that a human-adenovirus-5-based oral influenza vaccine tablet (VXA-A1.1) can protect from H1N1 virus challenge in humans. Mass cytometry characterization of vaccine-elicited cellular immune responses identified shared and vaccine-type-specific responses across B and T cells. For VXA-A1.1, the abundance of hemagglutinin-specific plasmablasts and plasmablasts positive for integrin α4ß7, phosphorylated STAT5, or lacking expression of CD62L at day 8 were significantly correlated with protection from developing viral shedding following virus challenge at day 90 and contributed to an effective machine learning model of protection. These findings reveal the characteristics of vaccine-elicited cellular correlates of protection for an oral influenza vaccine.

Author Correction: Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis.

Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.

Distribution of Chemical Species in the Water-Soil-Plant (Carya illinoiensis) System near a Mineralization Area in Chihuahua, Mexico-Health Risk Implications.

The aim of this study was to quantify major and trace elements in the water, soil, and plants (Carya illionensis) in an agricultural area; and to determine the health risks associated with the walnuts ingestion by calculating the risk quotient. Samples of water, soil, tree leaves, and walnuts were collected; in total, 135 samples were analyzed. Physicochemical parameters were obtained in irrigation water and soil samples. Elemental measurements were performed in an ICP, -OES and -MS. In addition, the distribution coefficient (soil⁻water), transfer factor (soil⁻plant), and hazard quotient were evaluated. In the irrigation water, As, Cr, and Pb, showed concentrations above the maximum allowable limits. Likewise, high concentrations of As, Cr, Pb, and Sb were found in tree leave samples, indicating a possible tendency of hyperaccumulation of those elements. Furthermore, Cr concentrations in walnuts were high by far than the reference value (FAO/WHO). A possible competition between chemical congeners were detected from transfer factors. Although, Sb concentrations in walnuts were also high, and no legislation for it in fruits exists. The hazard risk quotient for Sb did indicate a potential health risk. Finally, it is important to consider that the health risk increases when exposure through consumption takes place over a prolonged period of time, even in low concentrations.

Decoupling of tumor-initiating activity from stable immunophenotype in HoxA9-Meis1-driven AML.

Increasing evidence suggests tumors are maintained by cancer stem cells; however, their nature remains controversial. In a HoxA9-Meis1 (H9M) model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy--stem/progenitor cells (Lin(-)kit(+)) and committed progenitors of the myeloid (Gr1(+)kit(+)) and lymphoid lineages (Lym(+)kit(+)). These distinct tumor-initiating cells (TICs) clonally recapitulated the immunophenotypic spectrum of the original tumor in vivo (including cells with a less-differentiated immunophenotype) and shared signaling networks, such that in vivo pharmacologic targeting of conserved TIC survival pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, this suggests that in certain malignancies tumor-initiation activity (or "cancer stemness") can represent a cellular state that exists independently of distinct immunophenotypic definition.

Structural linkage between ligand discrimination and receptor activation by type I interferons.

Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.

Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.

Fluorescent cell barcoding for multiplex flow cytometry.

Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10- to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed.

Phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples.

Phospho-specific flow cytometry, or phospho flow, measures the phosphorylation state of intracellular proteins at the single cell level. Many phosphorylation events can be analyzed simultaneously in each cell, along with cell surface markers, enabling complex biochemical signaling networks to be resolved in heterogeneous cell populations. The method has been applied to many diverse areas of biology, including the characterization of signaling pathways in normal immune responses to antigenic stimulation and microbial challenge, alteration of signaling networks that occur in cancer and autoimmune diseases, and high-throughput, high-content drug discovery. In this chapter, we provide detailed experimental protocols for performing phospho flow in cell lines, Ficoll-purified peripheral blood mononuclear cells, and whole blood. These protocols are applicable to both human and murine samples. We also provide methods for the validation of surface marker antibodies for use in phospho flow. Finally, we discuss data analysis methods, in particular, how to quantify changes in phosphorylation and how to visualize the large data sets that can result from experiments in primary cells.

Increased host neuronal survival and motor function in BMT Parkinsonian mice: involvement of immunosuppression.

We examined the potential of bone marrow transplantation (BMT) to rescue dopaminergic neurons in a mouse model of Parkinson's disease (PD). A BMT from mice transgenic for green fluorescent protein (GFP(+)) given either before or after administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) led to the accumulation of transplanted adult GFP(+) bone-marrow-derived cells (BMDC) in the substantia nigra, where dopaminergic neurodegeneration occurs in PD. Post-BMT, mice exposed to MPTP had substantially greater numbers of endogenous tyrosine hydroxylase-positive neuronal cell bodies in the substantia nigra and increased dopamine transporter-positive projections into the striatum compared to controls. Moreover, motor function was restored to normal within 1 month post-MPTP in BMT-treated mice assayed by a rotarod behavioral test. The effect of BMT on PD was indirect, as no evidence of BMDC fusion with or transdifferentiation into dopaminergic neurons was observed. BMDC activated by BMT or associated factors could play a trophic role in rescuing damaged cells. Alternatively, the beneficial effects of BMT are due to immunosuppression reflected by a reduction in the proportion of T-cells and a reduction of T-cell proliferation in BMT mice. These findings highlight that when immunosuppression is required for transplantation studies, the amelioration of symptoms may not be due to the transplant itself. Further, they suggest that the immune system plays a role in the development of characteristics typical of PD.

[Causality and hospital perinatal mortality trend at the Mexican Institute of Social Security, 1998-2002]. / Causalidad y tendencia de la mortalidad perinatal hospitalaria en el Instituto Mexicano del Seguro Social, 1998-2002.

OBJECTIVE: To analyze levels, trend and causes of hospital perinatal mortality at the Instituto Mexicano del Seguro Social (IMSS) during the 5 years from 1998 to 2002 to identify magnitude and related factors in our population and discuss some technical bases and epidemiologic aspects for planning strategies to contribute to its reduction. MATERIAL AND METHODS: Descriptive study on the death certificates of 39,994 cases of perinatal deaths distributed among 19,108 fetal deaths of 28 weeks and more of gestation and 20,886 neonatal deaths 7 to days of life that occurred in IMSS hospitals during the reference period. With this information and the data on total births, dead or live from the official information system of our Institution, we established annual rates for the entire IMSS, including administrative regions and zones of medical services. We also generated 5-year cumulated numbers for frequencies and rates of perinatal death causes in the IMSS, using the codes of Tenth Revision of the International Diseases Classification. RESULTS: Hospital perinatal mortality at the IMSS showed a reduction from 1998 (13 per 1,000 births) to 2002 (11.4 per 1,000 births). That trend was observed in the four administrative regions and in the majority of number of medical services zones, but with many differences in levels. It was similar in proportions of fetal (47.8%) and neonatal deaths (52.2%). Two thirds of fetal mortality was linked to maternal complications during pregnancy and labor. A similar proportion of neonatal deaths was due to premature birth and its complications.

Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant.

Heterokaryons are the product of cell fusion without subsequent nuclear or chromosome loss. Decades of research using Sendai-virus or polyethylene glycol (PEG)-mediated fusion in tissue culture showed that the terminally differentiated state of a cell could be altered. But whether stable non-dividing heterokaryons could occur in animals has remained unclear. Here, we show that green fluorescent protein (GFP)-positive bone-marrow-derived cells (BMDCs) contribute to adult mouse Purkinje neurons through cell fusion. The formation of heterokaryons increases in a linear manner over 1.5 years and seems to be stable. The dominant Purkinje neurons caused the BMDC nuclei within the resulting heterokaryons to enlarge, exhibit dispersed chromatin and activate a Purkinje neuron-specific transgene, L7-GFP. The observed reprogrammed heterokaryons that form in brain may provide insights into gene regulation associated with cell-fate plasticity.

Manifestaciones otorrinolaringológicas en pacientes con síndrome de inmunodeficiencia adquirida / Otolaringological findings in patients with acquired immunodefiency syndrome

El síndrome de Immunodeficiencia Adquirida (SIDA) es de particular interés para el Otorrinolaringólogo y Cirujano de Cabeza y Cuello porque se ha estimado que entre el 40 por ciento y 80 por ciento de los pacientes con esta enfermedad presentan sintomatología o manifestaciones físicas en el área de la cabeza y el cuello. Por ello, revisamos 318 expedientes de pacientes con diagnóstico clínico, epidemiológico y serológico de SIDA vistos en el Hospital General de México entre mayo de 1993 y diciembre de 1994. Encontramos patología en cabeza y cuello en 169 (54.14 por ciento); de estos, 113 fueron del sexo masculino y 56 del sexo femenino. La cavidad oral presento patología en 87 de los pacientes (51.48 por ciento), orofaringe en 34 (20.12 por ciento), Nariz y senos paranasales en 26 (15.38 por ciento), piel de cabeza y cuello en 8 (4.73 por ciento), oído medio 7(4.14 por ciento), oído externo 4(2.37 por ciento, laringe 1 (0.59 por ciento) y un absceso lateral de cuello (0.59 por ciento)