HIV-1 competition experiments in humanized mice show that APOBEC3H imposes selective pressure and promotes virus adaptation

APOBEC3 (A3) family proteins are DNA cytosine deaminases recognized for contributing to HIV-1 restriction and mutation. Prior studies have demonstrated that A3D, A3F, and A3G enzymes elicit a robust anti-HIV-1 effect in cell cultures and in humanized mouse models. Human A3H is polymorphic and can be categorized into three phenotypes: stable, intermediate, and unstable. However, the anti-viral effect of endogenous A3H in vivo has yet to be examined. Here we utilize a hematopoietic stem cell-transplanted humanized mouse model and demonstrate that stable A3H robustly affects HIV-1 fitness in vivo. In contrast, the selection pressure mediated by intermediate A3H is relaxed. Intriguingly, viral genomic RNA sequencing reveled that HIV-1 frequently adapts to better counteract stable A3H during replication in humanized mice. Molecular phylogenetic analyses and mathematical modeling suggest that stable A3H may be a critical factor in human-to-human viral transmission. Taken together, this study provides evidence that stable variants of A3H impose selective pressure on HIV-1

Ciencias de la Biología y Agronomía

Este volumen I contiene 17 capítulos arbitrados que se ocupan de estos asuntos en Tópicos Selectos de Ciencias de la Biología y Agronomía, elegidos de entre las contribuciones, reunimos algunos investigadores y estudiantes. Se presenta un Estudio Comparativo de los Recursos Hidrológico-Forestales de la Microcuenca de la Laguna de Epatlan, Pue. (1993 a 2014); la Situación Actual de la Mancha de Asfalto en Maíz (Zea mays L.) en los Municipios de Jiquipilas y Ocozocoautla, Chiapas, México; las poblaciones sobresalientes de maíz de la raza Zapalote Chico, en la Región Istmeña de Oaxaca; Se indica el índice de área foliar de cultivo de Chile Poblano mediante dos métodos en condiciones protegidas; Esquivel, Urzúa y Ramírez exploran el efecto de la biofertilización con Azospirillum en el crecimiento y producción de Jitomate; esbozan su artículo sobre la determinación del nivel de Heterosis en híbridos de Maíz para la Comarca Lagunera; una investigación sobre la estabilización de semilla de Solanum lycopersicum durante el almacenamiento y estimulación de la germinación; acotan sobre el CTAB como una nueva opción para la detección de Huanglongbing en cítricos, plantean su evaluación sobre el aluminio y cómo afecta la vida de florero de Heliconia psittacorum; indican sobre el impacto del H-564C, como un híbrido de maíz con alta calidad de proteina para el trópico húmedo de México; presetan su investigación sobre la producción de Piña Cayena Lisa y MD2 (Ananas comosus L.) en condiciones de Loma Bonita, en Oaxaca; acotan sobre el efecto de coberteras como control biológico por conservación contra áfidos en Nogal Pecanero; esbozan sobre la caracterización de cuatro genotipos de Frijol Negro en Martínez de la Torre, Veracruz, México; presentan una caracterización hidroecológica de la microcuenca de Arroyo Prieto, Yuriría, Gto., y alternativas para su restauración ambiental; presentan su investigación sobre el efecto del hongo Beauveria bassiana sobre solubilización de fosfatos y la disponibilidad de fósforo en el suelo; plantean su investigación sobre la Germinación y regeneración in vitro de Epidendrum falcatum LINDL; esbozan su artículo sobre genotipos de frijol negro y su tolerancia a sequía terminal en Veracruz, México

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

A role for gorilla APOBEC3G in shaping lentivirus evolution including transmission to humans.

The APOBEC3 deaminases are potent inhibitors of virus replication and barriers to cross-species transmission. For simian immunodeficiency virus (SIV) to transmit to a new primate host, as happened multiple times to seed the ongoing HIV-1 epidemic, the viral infectivity factor (Vif) must be capable of neutralizing the APOBEC3 enzymes of the new host. Although much is known about current interactions of HIV-1 Vif and human APOBEC3s, the evolutionary changes in SIV Vif required for transmission from chimpanzees to gorillas and ultimately to humans are poorly understood. Here, we demonstrate that gorilla APOBEC3G is a factor with the potential to hamper SIV transmission from chimpanzees to gorillas. Gain-of-function experiments using SIVcpzPtt Vif revealed that this barrier could be overcome by a single Vif acidic amino acid substitution (M16E). Moreover, degradation of gorilla APOBEC3F is induced by Vif through a mechanism that is distinct from that of human APOBEC3F. Thus, our findings identify virus adaptations in gorillas that preceded and may have facilitated transmission to humans

Dynamics of hyper/hypo HIV-1 dissemination in human population.

<p>(<b>A</b>) A phylogenetic tree of Vif. The Vif sequences were extracted from HIV-1 sequence database (<a href="https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html" target="_blank">https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html</a>) and the phylogenetic tree was constructed as described in Materials and Methods. The branches of hyper Vif sequences (i.e., F or Y in position 39 and H in position 48) are indicated with pink. Each color surrounding the phylogenetic tree represents viral subtype (A1-K). Scale bar indicates 5.0 amino acid substitutions per site. (<b>B</b>) The percentage of hyper Vif sequences in each subtype and group. The sequences of HIV-1 Vif is classified into hyper and hypo Vif based on the amino acids positioned at 39 and 48 and the result is summarized. The numbers in parentheses represents the number of Vif sequences used. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s020" target="_blank">S10 Table</a>. (<b>C</b>) TZM-bl assay. The IMCs (1,000 ng) were cotransfected either with or without Flag-tagged A3H-II expression plasmid (50 ng) into HEK293T cells. The infectivity of released virus was determined by using TZM-bl cells, and the percentage of the value of "no A3H-II" is shown. *<i>P</i> < 0.05 versus "hyper HIV-1" by Student's <i>t</i> test. The assay was performed in triplicate. The data represents average with SD. TF, transmitted/founder; CC, chronic control. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s019" target="_blank">S9 Table</a>. (<b>D</b>) Distribution of hyper HIV-1 and individuals with stable A3H in the world. The percentages of hyper HIV-1 (pink, top) and stable A3H haplotype (green, bottom) in each region (Europe, Africa, Asia and North America; represented in bold) and country were obtained as described in Materials and Methods, and these two values are indicated by heatmap. The Vif amino acids at positions 39 and 48 are shown in logoplot, and the residues associated with hyper Vif (i.e., F or Y in position 39 and H in position 48) are represented in pink. Note that the information of the proportion of <i>A3H</i> haplotype is not available in Russia, Australia, Central America and South America. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s021" target="_blank">S11 Table</a>. (<b>E</b>) Mathematical modeling of the dissemination of hyper HIV-1 in human population. The prevalence of hyper HIV-1 in the human population with different stable A3H proportion was simulated by the mathematical model (for the detail, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#sec009" target="_blank">Materials and methods</a>). The simulated prediction is shown with purple line. Red and Black dots indicate the results from respective regions and countries (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s009" target="_blank">S9 Fig</a> & <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s022" target="_blank">S12 Table</a>).</p

Dynamics of hyper/hypo HIV-1 infection in stable A3H humanized mice.

<p>(<b>A</b>) A schematic of the <i>A3H</i> gene locus and the 5 polymorphisms in <i>A3H</i> exons 2, 3, and 4 (indicated by red vertical lines) that combine to produce 7 different haplotypes. Red arrowheads with horizontal broken lines indicate the region amplified by genotyping PCR. In the bottom panel, the 7 different A3H haplotypes based on observed protein stability or instability in previous studies [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.ref014" target="_blank">14</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.ref015" target="_blank">15</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.ref020" target="_blank">20</a>] are summarized. (<b>B</b>) (top) A schematic of the Vif protein encoded by HIV-1 used in this study. This panel shows the amino acid differences responsible for the degradation of stable A3H. (bottom) TZM-bl assay. The infectivity of released virions was determined by using TZM-bl cells. *<i>P</i> < 0.05 versus no A3H-II by Student's <i>t</i> test. The assay was performed in triplicate. The data represents average with SD. (<b>C, D</b>) HIV-1 infection in humanized mice. (<b>C</b>) A schematic of co-inoculation of hyper and hypo HIV-1s into stable A3H humanized mice. (<b>D</b>) Hyper and hypo viruses containing 2.5 ng of p24 antigen each (5 ng in total; n = 8) or RPMI1640 (n = 12; for mock infection) were inoculated into humanized mice. the amount of viral RNA in plasma (left) and the level of peripheral CD4⁺ T cells (CD45⁺ CD3⁺ CD4⁺ cells) (right) were analyzed at 0, 1, 2, 3, 5, and 6 wpi as described in Materials and Methods. The averages are shown in circles with SEMs, and the values from each mouse are shown by line. X-axes, wpi. In the left panel, horizontal broken line indicates detection limit (800 copies/ml plasma). In the right panel, *<i>P</i> < 0.05 versus mock-infected mice by Mann-Whitney U test. (<b>E</b>) Phylogenetic trees of <i>vif</i> sequence. Viral <i>vif</i> sequences in the plasma of infected mice at 6 wpi were analyzed as described in Materials and Methods. Results of each infected mouse (mice #1–8) are respectively shown. Each symbol represents identical sequence. Pink shadow indicates hyper <i>vif</i> derivatives. Scale bar represents one nucleotide substitution. Note that the 3 hypo <i>vif-</i>related sequences with the ability to counteract A3H-II (shown in <b>Fig 1F & 1G</b>) are indicated with red symbols. (<b>F, G</b>) Evaluation of anti-stable A3H activity of Vif derivatives detected in infected humanized mice. (<b>F</b>) Western blotting. The input of cell lysate was standardized to α-Tubulin (TUBA), and representative results are shown. (<b>G</b>) TZM-bl assay. The expression plasmids of the Vif derivatives were cotransfected with pNLCSFV3Δ<i>vif</i> and either with or without Flag-tagged A3H-II expression plasmid into HEK293T cells. The infectivity of released virus was determined by using TZM-bl cells, and the percentage of the value of "no A3H-II" is shown. *<i>P</i> < 0.05 versus "no Vif" by Student's <i>t</i> test. The assay was performed in triplicate. The data represents average with SD. In panels <b>F</b> and <b>G</b>, the symbols are identical to those in <b>Fig 1E</b>.</p

Immune activation and up-regulation of endogenous <i>A3H</i> expression in the human CD4⁺ T cells of infected humanized mice.

<p>(<b>A, B</b>) RNA-seq of the splenic human MNCs of infected humanized mice. (<b>A</b>) RNA-seq. Results of differentially expressed gene analysis from the splenic human MNCs of HIV-1-infected (n = 4) and mock-infected (n = 4) humanized mice are shown as a heatmap. (<b>B</b>) GSEA analysis. The procedure is described in Material and Method, and the top 50 annotations are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s018" target="_blank">S8 Table</a>. The vertical broken line indicates <i>Q</i> value = 0.05. (<b>C, D</b>) <i>A3H</i> expression in the human CD4⁺ T cells of infected mice. (<b>C</b>) Flow cytometry. Human MNCs were isolated from the spleen of HIV-1-infected (n = 6) and mock-infected (n = 6) humanized mice and analyzed the proportion of CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ cells) and activated CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁺ cells) by flow cytometry. Representative dot plots (left), the percentage of CD3⁺ CD8⁻ cells in CD45⁺ cells (middle) and the percentage of CD25⁺ cells in CD3⁺ CD8⁻ cells (right) are respectively shown. *<i>P</i> < 0.05 versus mock-infected mice by Mann-Whitney U test. In panel <b>C</b>, the numbers on each dot plot indicates the percentage of gated cells. (<b>D</b>) Real-time RT-PCR of <i>A3H</i>. Activated CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁺ cells) and non-activated CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁻ cells) of mock-infected mice (n = 6 each) and CD25⁻ CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁻ cells) of HIV-1-infected mice (n = 6) were sorted using FACSJazz (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s007" target="_blank">S7 Fig</a>). The mRNA expression level of <i>A3H</i> in each population was analyzed by real-time RT-PCR as described in Materials and Methods. The value of CD25⁻ CD4⁺ T cells of mock-infected mice is set as 1. *<i>P</i> < 0.05 versus CD25⁻ CD4⁺ T cells of mock-infected mice by Mann-Whitney U test. NS, no statistic difference. Note that CD25⁺ CD4⁺ T cells of infected mice were not available because this fraction was severely depleted (panel <b>C</b>).</p

Dynamics of hyper/hypo HIV-1 infection in intermediate A3H humanized mice.

<p>(<b>A, B</b>) HIV-1 infection in humanized mice. (<b>A</b>) A schematic of co-inoculation of hyper and hypo HIV-1s into intermediate A3H humanized mice. (<b>B</b>) Hyper and hypo viruses containing 2.5 ng of p24 antigen each (5 ng in total; n = 6) or RPMI1640 (n = 12; for mock infection) were inoculated into humanized mice. The amount of viral RNA in plasma (left) and the level of peripheral CD4⁺ T cells (CD45⁺ CD3⁺ CD4⁺ cells) (right) were analyzed at 0, 1, 2, 3, 5, and 6 wpi as described in Materials and Methods. The averages are shown in circles with SEMs, and the values from each mouse are shown by line. X-axes, wpi. In the left panel, horizontal broken line indicates detection limit (800 copies/ml plasma). In the right panel, *<i>P</i> < 0.05 versus mock-infected mice by Mann-Whitney U test. (<b>C</b>) Phylogenetic trees of <i>vif</i> sequence. Viral <i>vif</i> sequences in the plasma of infected mice at 6 wpi were analyzed as described in Materials and Methods. Results of each infected mouse (mice #9–14) are respectively shown. Blue shadow indicates hypo <i>vif</i> derivatives. Scale bar represents one nucleotide substitution.</p