Functional analysis of 3'-UTR hairpins supports a two-tiered model for posttranscriptional regulation of MAT2A by METTL16.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events, so cells need to carefully control SAM levels. MAT2A encodes the only SAM synthetase expressed in the majority of human cells, and its 3'-UTR has six conserved regulatory hairpins (hp1-6) that can be methylated by the N6-methyladenosine methyltransferase METTL16. Hp1 begins 8 nt from the stop codon, whereas hp2-6 are clustered further downstream (∼800 nt). These hairpins have been proposed to regulate MAT2A mRNA levels in response to intracellular SAM levels by regulating intron detention of the last intron of MAT2A and by modulating the stability of the fully spliced mRNA. However, a dissection of these two posttranscriptional mechanisms has not been previously reported. Using a modular reporter system, we show that hp1 functions primarily when the detained intron is included in the reporter and when that intron has a suboptimal polypyrimidine tract. In contrast, the hp2-6 cluster modulates mRNA stability independent of the detained intron, although hp1 may make a minor contribution to the regulation of decay as well. Taken with previously published reports, these data support a two-tiered model for MAT2A posttranscriptional regulation by METTL16 through its interactions with hp1 and hp2-6. In the upstream tier, hp1 and METTL16 control MAT2A intron detention, whereas the second tier involves METTL16-dependent methylation of hp2-6 to control MAT2A mRNA stability. Thus, cells use a similar set of molecular factors to achieve considerable complexity in the posttranscriptional regulation of SAM homeostasis.

The PNUTS-PP1 complex acts as an intrinsic barrier to herpesvirus KSHV gene expression and replication.

Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-PP1 complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion enhances global KSHV gene expression and overall viral replication. Mechanistically, PNUTS requires PP1 interaction, binds viral RNAs downstream of polyadenylation sites, and restricts transcription readthrough of viral genes. Surprisingly, PNUTS also represses productive elongation at the 5´ ends of the KSHV reporter and the KSHV T1.4 RNA. From these data, we conclude that PNUTS' activity constitutes an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.

Epidemiological and molecular characterization of a novel adenovirus of squirrel monkeys after fatal infection during immunosuppression.

Adenoviruses are a frequent cause of acute upper respiratory tract infections that can also cause disseminated disease in immunosuppressed patients. We identified a novel adenovirus, squirrel monkey adenovirus 1 (SqMAdV-1), as the cause of fatal infection in an immunocompromised squirrel monkey (Saimiri boliviensis) at the Keeling Center for Comparative Medicine and Research (KCCMR). Sequencing of SqMAdV-1 revealed that it is most closely related (80.4 % pairwise nucleotide identity) to the titi monkey (Plecturocebus cupreus) adenovirus (TMAdV). Although identified in the titi monkey, TMAdV is highly lethal in these monkeys, and they are not thought to be the natural host. While SqMAdV-1 is similar to other primate adenoviruses in size and genomic characteristics, a nucleotide polymorphism at the expected stop codon of the DNA polymerase gene results in a 126 amino acid extension at the carboxy terminus, a feature not previously observed among other primate adenoviruses. PCR testing and partial sequencing of 95 archived faecal samples from other squirrel monkeys (Saimiri boliviensis and Saimiri sciureus) housed at the KCCMR revealed the presence of three distinct, and apparently endemic species of adenoviruses. A grouping of ten squirrel monkey adenovirus variants has high similarity to SqMAdV-1. A single adenovirus variant (designated SqMAdV-3), detected in five monkeys, has similarity to tufted capuchin (Sapajus apella) adenoviruses. The largest group of adenovirus variants detected (designated SqMAdV-2.0-2.16) has very high similarity (93-99 %) to the TMAdV, suggesting that squirrel monkeys may be the natural host of the TMAdV.

Kaposi's Sarcoma-Associated Herpesvirus Fine-Tunes the Temporal Expression of Late Genes by Manipulating a Host RNA Quality Control Pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic nuclear DNA virus that expresses its genes using the host cell transcription and RNA processing machinery. As a result, KSHV transcripts are subject to degradation by at least two host-mediated nuclear RNA decay pathways, the PABPN1- and poly(A) polymerase α/γ (PAPα/γ)-mediated RNA decay (PPD) pathway and an ARS2-dependent decay pathway. Here, we present global analyses of viral transcript levels to further understand the roles of these decay pathways in KSHV gene expression. Consistent with our recent report that the KSHV ORF57 protein increases viral transcript stability by impeding ARS2-dependent decay, ARS2 knockdown has only modest effects on viral gene expression 24 h after lytic reactivation of wild-type virus. In contrast, inactivation of PPD has more widespread effects, including premature accumulation of late transcripts. The upregulation of late transcripts does not require the primary late-gene-specific viral transactivation factor, suggesting that cryptic transcription produces the transcripts that then succumb to PPD. Remarkably, PPD inactivation has no effect on late transcripts at their proper time of expression. We show that this time-dependent PPD evasion by late transcripts requires the host factor nuclear RNAi-defective 2 (NRDE2), which has previously been reported to protect cellular RNAs by sequestering decay factors. From these studies, we conclude that KSHV uses PPD to fine-tune the temporal expression of its genes by preventing their premature accumulation.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposi's sarcoma and other lymphoproliferative disorders. Nuclear expression of KSHV genes results in exposure to at least two host-mediated nuclear RNA decay pathways, the PABPN1- and PAPα/γ-mediated RNA decay (PPD) pathway and an ARS2-mediated decay pathway. Perhaps unsurprisingly, we previously found that KSHV uses specific mechanisms to protect its transcripts from ARS2-mediated decay. In contrast, here we show that PPD is required to dampen the expression of viral late transcripts that are prematurely transcribed, presumably due to cryptic transcription early in infection. At the proper time for their expression, KSHV late transcripts evade PPD through the activity of the host factor NRDE2. We conclude that KSHV fine-tunes the temporal expression of its genes by modulating PPD activity. Thus, the virus both protects from and exploits the host nuclear RNA decay machinery for proper expression of its genes.

Kaposi's sarcoma-associated herpesvirus ORF57 protein protects viral transcripts from specific nuclear RNA decay pathways by preventing hMTR4 recruitment.

Nuclear RNAs are subject to a number of RNA decay pathways that serve quality control and regulatory functions. As a result, any virus that expresses its genes in the nucleus must have evolved mechanisms that avoid these pathways, but the how viruses evade nuclear RNA decay remains largely unknown. The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (Mta) protein is required for the nuclear stability of viral transcripts. In the absence of ORF57, we show that viral transcripts are subject to degradation by two specific nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) in which decay factors are recruited through poly(A) tails, and an ARS2-mediated RNA decay pathway dependent on the 5' RNA cap. In transcription pulse chase assays, ORF57 appears to act primarily by inhibiting the ARS2-mediated RNA decay pathway. In the context of viral infection in cultured cells, inactivation of both decay pathways by RNAi is necessary for the restoration of ORF57-dependent viral genes produced from an ORF57-null bacmid. Mechanistically, we demonstrate that ORF57 protects viral transcripts by preventing the recruitment of the exosome co-factor hMTR4. In addition, our data suggest that ORF57 recruitment of ALYREF inhibits hMTR4 association with some viral RNAs, whereas other KSHV transcripts are stabilized by ORF57 in an ALYREF-independent fashion. In conclusion, our studies show that KSHV RNAs are subject to nuclear degradation by two specific host pathways, PPD and ARS2-mediated decay, and ORF57 protects viral transcripts from decay by inhibiting hMTR4 recruitment.

Niche adaptation and viral transmission of human papillomaviruses from archaic hominins to modern humans.

Recent discoveries on the origins of modern humans from multiple archaic hominin populations and the diversity of human papillomaviruses (HPVs) suggest a complex scenario of virus-host evolution. To evaluate the origin of HPV pathogenesis, we estimated the phylogeny, timing, and dispersal of HPV16 variants using a Bayesian Markov Chain Monte Carlo framework. To increase precision, we identified and characterized non-human primate papillomaviruses from New and Old World monkeys to set molecular clock models. We demonstrate specific host niche adaptation of primate papillomaviruses with subsequent coevolution with their primate hosts for at least 40 million years. Analyses of 212 HPV16 complete genomes and 3582 partial sequences estimated ancient divergence of HPV16 variants (between A and BCD lineages) from their most recent common ancestors around half a million years ago, roughly coinciding with the timing of the split between archaic Neanderthals and modern Homo sapiens, and nearly three times longer than divergence times of modern Homo sapiens. HPV16 A lineage variants were significantly underrepresented in present African populations, whereas the A sublineages were highly prevalent in European (A1-3) and Asian (A4) populations, indicative of viral sexual transmission from Neanderthals to modern non-African humans through multiple interbreeding events in the past 80 thousand years. Remarkably, the human leukocyte antigen B*07:02 and C*07:02 alleles associated with increased risk in cervix cancer represent introgressed regions from Neanderthals in present-day Eurasians. The archaic hominin-host-switch model was also supported by other HPV variants. Niche adaptation and virus-host codivergence appear to influence the pathogenesis of papillomaviruses.

Metformin Induces Cell Cycle Arrest, Reduced Proliferation, Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients.

BACKGROUND: Several epidemiological studies in diabetic patients have demonstrated a protective effect of metformin to the development of several types of cancer. The underlying mechanisms of such phenomenon is related to the effect of metformin on cell proliferation among which, mTOR, AMPK and other targets have been identified. However, little is known about the role that metformin treatment have on other cell types such as keratinocytes and whether exposure to metformin of these cells might have serious repercussions in wound healing delay and in the development of complications in diabetic patients with foot ulcers or in their exacerbation. MATERIAL AND METHODS: HaCaT Cells were exposed to various concentrations of metformin and cell viability was evaluated by a Resazurin assay; Proliferation was also evaluated with a colony formation assay and with CFSE dilution assay by flow cytometry. Cell cycle was also evaluated by flow cytometry by PI staining. An animal model of wound healing was used to evaluate the effect of metformin in wound closure. Also, an analysis of patients receiving metformin treatment was performed to determine the effect of metformin treatment on the outcome and wound area. Statistical analysis was performed on SPSS v. 18 and GraphPad software v.5. RESULTS: Metformin treatment significantly reduces cell proliferation; colony formation and alterations of the cell cycle are observed also in the metformin treated cells, particularly in the S phase. There is a significant increase in the area of the wound of the metformin treated animals at different time points (P<0.05). There is also a significant increase in the size and wound area of the patients with diabetic foot ulcers at the time of hospitalization. A protective effect of metformin was observed for amputation, probably associated with the anti inflammatory effects reported of metformin. CONCLUSIONS: Metformin treatment reduces cell proliferation and reduces wound healing in an animal model and affects clinical outcomes in diabetic foot ulcer patients. Chronic use of this drug should be further investigated to provide evidence of their security in association with DFU.

F-box and leucine-rich repeat protein 5 (FBXL5): sensing intracellular iron and oxygen.

Though essential for many vital biological processes, excess iron results in the formation of damaging reactive oxygen species (ROS). Therefore, iron metabolism must be tightly regulated. F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, regulates cellular and systemic iron homeostasis by facilitating iron regulatory protein 2 (IRP2) degradation. FBXL5 possesses an N-terminal hemerythrin (Hr)-like domain that mediates its own differential stability by switching between two different conformations to communicate cellular iron availability. In addition, the FBXL5-Hr domain also senses O2 availability, albeit by a distinct mechanism. Mice lacking FBXL5 fail to sense intracellular iron levels and die in utero due to iron overload and exposure to damaging levels of oxidative stress. By closely monitoring intracellular levels of iron and oxygen, FBLX5 prevents the formation of conditions that favor ROS formation. These findings suggest that FBXL5 is essential for the maintenance of iron homeostasis and is a key sensor of bioavailable iron. Here, we describe the iron and oxygen sensing mechanisms of the FBXL5 Hr-like domain and its role in mediating ROS biology.

F-box and leucine-rich repeat protein 5 (FBXL5) is required for maintenance of cellular and systemic iron homeostasis.

Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.

Hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5) communicates cellular iron and oxygen availability by distinct mechanisms.

Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells.

Structural and molecular characterization of iron-sensing hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5).

Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.

Activation of the HIF prolyl hydroxylase by the iron chaperones PCBP1 and PCBP2.

Mammalian cells express dozens of iron-containing proteins, yet little is known about the mechanism of metal ligand incorporation. Human poly (rC) binding protein 1 (PCBP1) is an iron chaperone that binds iron and delivers it to ferritin, a cytosolic iron storage protein. We have identified the iron-dependent prolyl hydroxylases (PHDs) and asparaginyl hydroxylase (FIH1) that modify hypoxia-inducible factor α (HIFα) as targets of PCBP1. Depletion of PCBP1 or PCBP2 in cells led to loss of PHD activity, manifested by reduced prolyl hydroxylation of HIF1α, impaired degradation of HIF1α through the VHL/proteasome pathway, and accumulation of active HIF1 transcription factor. PHD activity was restored in vitro by addition of excess Fe(II), or purified Fe-PCBP1, and PCBP1 bound to PHD2 and FIH1 in vivo. These data indicated that PCBP1 was required for iron incorporation into PHD and suggest a broad role for PCBP1 and 2 in delivering iron to cytosolic nonheme iron enzymes.

An E3 ligase possessing an iron-responsive hemerythrin domain is a regulator of iron homeostasis.

Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of genes responsible for iron uptake, release, use, and storage through the actions of the iron regulatory proteins IRP1 and IRP2. However, the manner in which iron levels are sensed to affect IRP2 activity is poorly understood. We found that an E3 ubiquitin ligase complex containing the FBXL5 protein targets IRP2 for proteasomal degradation. The stability of FBXL5 itself was regulated, accumulating under iron- and oxygen-replete conditions and degraded upon iron depletion. FBXL5 contains an iron- and oxygen-binding hemerythrin domain that acted as a ligand-dependent regulatory switch mediating FBXL5's differential stability. These observations suggest a mechanistic link between iron sensing via the FBXL5 hemerythrin domain, IRP2 regulation, and cellular responses to maintain mammalian iron homeostasis.

Análisis de 1,000 histerectomías: simplificaciones técnicas y reflexiones, hospitales del ISSSTE / Analysis of 1,000 hysterectomies. Technical simplifications and reflexions, ISSSTE's hospitals

Se informa acerca de 1,000 histerectomías realizadas por el autor en un lapso de 20 años en cuatro hospitales del ISSSTE. Se analizaron algunos parámetros clínicopatológicos, morbi-mortalidad, etc. Se informa de los resultados obtenidos utilizando simplificaciones en las técnicas quirúrgicas. Los síntomas más frecuentes fueron trastornos menstruales, y el diagnóstico más frecuente fue la miomatosis con (61.2 por ciento), la media de edad entre los 35-45 años. Todas las histerectomías fueron totales, 92.6 por ciento intrafasciales y 74.8 por ciento abdominales, Llamó la atención que 7.2 por ciento de las pacientes presentaron síndrome de congestión pélvica post oclusión tubaria y la asociación de adenomiosis. La mortalidad global fue de 12.2 por ciento predominando la del tipo urosepsis. La mortalidad se presentó en un caso por mioma intraligamentario gigante y coagulopatía por consumo. Se utilizó sangre sólo en 4.2 por ciento. Se piensa que la simplificación quirúrgica aquí enunciada, permite ofrecer mayor seguridad, menor morbilidad, mínimo sangrado y tiempo quirúrgico-anestésico breve. Además se realizó un análisis crítico del uso y abuso de este tipo de intervención quirúrgica

Factores ambientales que afectan el comportamiento de primates en habitad especiales / Annual Reports 1991 / Environmental factors affecting the behavior of primates in special habitats

It was observed the effects of captivity on the reproductive competence of monkeys, to measure the degree of adaptation to a confined situation. These observation were made on Cebus, Aotus, and Saimiri monkeys, from two different monkeys colonies, facing a project of establishment a breeding monkey colony at the IICS. It was observed the behavior of the monkeys concerning the aggressivness against the other monkeys in the cage and themselves, the organization of social groups, and its hierarchy. It was evidenced the importance of the tryning of the staff dealing with the monkeys, as factor influencing the behavior of the animals. It was observed the importance of the organization of the colony, concerning the surroundings of the cages, ventilation, room temperature, illumination, presence of personnel, location of other cages, as factors exerting a strong influence in the behavior of the monkeys