A plant-specific syntaxin-6 protein contributes to the intracytoplasmic route for the begomovirus CabLCV.

Because of limited free diffusion in the cytoplasm, viruses must use active transport mechanisms to move intracellularly. Nevertheless, how the plant single-stranded DNA begomoviruses hijack the host intracytoplasmic transport machinery to move from the nucleus to the plasmodesmata remains enigmatic. Here, we identified nuclear shuttle protein (NSP)-interacting proteins from Arabidopsis (Arabidopsis thaliana) by probing a protein microarray and demonstrated that the cabbage leaf curl virus NSP, a facilitator of the nucleocytoplasmic trafficking of viral (v)DNA, interacts in planta with an endosomal vesicle-localized, plant-specific syntaxin-6 protein, designated NSP-interacting syntaxin domain-containing protein (NISP). NISP displays a proviral function, unlike the syntaxin-6 paralog AT2G18860 that failed to interact with NSP. Consistent with these findings, nisp-1 mutant plants were less susceptible to begomovirus infection, a phenotype reversed by NISP complementation. NISP-overexpressing lines accumulated higher levels of vDNA than wild-type. Furthermore, NISP interacted with an NSP-interacting GTPase (NIG) involved in NSP-vDNA nucleocytoplasmic translocation. The NISP-NIG interaction was enhanced by NSP. We also showed that endosomal NISP associates with vDNA. NISP may function as a docking site for recruiting NIG and NSP into endosomes, providing a mechanism for the intracytoplasmic translocation of the NSP-vDNA complex toward and from the cell periphery.

Repeat variants for the SbMATE transporter protect sorghum roots from aluminum toxicity by transcriptional interplay in cis and trans.

Acidic soils, where aluminum (Al) toxicity is a major agricultural constraint, are globally widespread and are prevalent in developing countries. In sorghum, the root citrate transporter SbMATE confers Al tolerance by protecting root apices from toxic Al3+, but can exhibit reduced expression when introgressed into different lines. We show that allele-specific SbMATE transactivation occurs and is caused by factors located away from SbMATE Using expression-QTL mapping and expression genome-wide association mapping, we establish that SbMATE transcription is controlled in a bipartite fashion, primarily in cis but also in trans Multiallelic promoter transactivation and ChIP analyses demonstrated that intermolecular effects on SbMATE expression arise from a WRKY and a zinc finger-DHHC transcription factor (TF) that bind to and trans-activate the SbMATE promoter. A haplotype analysis in sorghum RILs indicates that the TFs influence SbMATE expression and Al tolerance. Variation in SbMATE expression likely results from changes in tandemly repeated cis sequences flanking a transposable element (a miniature inverted repeat transposable element) insertion in the SbMATE promoter, which are recognized by the Al3+-responsive TFs. According to our model, repeat expansion in Al-tolerant genotypes increases TF recruitment and, hence, SbMATE expression, which is, in turn, lower in Al-sensitive genetic backgrounds as a result of lower TF expression and fewer binding sites. We thus show that even dominant cis regulation of an agronomically important gene can be subjected to precise intermolecular fine-tuning. These concerted cis/trans interactions, which allow the plant to sense and respond to environmental cues, such as Al3+ toxicity, can now be used to increase yields and food security on acidic soils.

Cardiac Tamponade - a Cause of Sudden Death in a Premature Newborn.

BACKGROUND: Cardiac tamponade is a potential complication in neonates with central venous catheters (CVC). Cardiac tamponade may be due to infection, a CVC related complication, or parental nutrition (PN) effusion. CASE REPORT: This is a preterm (30 weeks gestational age), very low birth weight male, admitted to the Neonatal Intensive Care Unit, requiring nasal continuous positive airway pressure. PN was provided via an umbilical venous catheter. An unexpected cardiac arrest occurred on the third day of life with an unsuccessful resuscitation. Autopsy revealed pericardial effusion composed of PN fluid with cardiac tamponade as the cause of death. CONCLUSION: Cardiac tamponade due to total PN effusion in the premature neonate may be fatal. The mechanism of the epicardial/pericardial effusion is not known.

Redox Modification of the Iron-Sulfur Glutaredoxin GRXS17 Activates Holdase Activity and Protects Plants from Heat Stress.

Heat stress induces misfolding and aggregation of proteins unless they are guarded by chaperone systems. Here, we examined the function of the glutaredoxin GRXS17, a member of thiol reductase families in the model plant Arabidopsis (Arabidopsis thaliana). GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin domain and three CGFS active-site motif-containing GRX domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione-dependent manner. As an Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, GRXS17 presented both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induced loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active-site cysteines in the corresponding thioredoxin domains. This oxidation led to a shift of GRXS17 to a high-molecular-weight complex and thus activated its holdase activity in vitro. Moreover, GRXS17 was specifically involved in plant tolerance to moderate high temperature and protected root meristematic cells from heat-induced cell death. Finally, GRXS17 interacted with a different set of proteins upon heat stress, possibly protecting them from heat injuries. Therefore, we propose that the Fe-S cluster enzyme GRXS17 is an essential guard that protects proteins against moderate heat stress, likely through a redox-dependent chaperone activity. We reveal the mechanism of an Fe-S cluster-dependent activity shift that converts the holoenzyme GRXS17 into a holdase, thereby preventing damage caused by heat stress.

Synthetic Ingenols Maximize Protein Kinase C-Induced HIV-1 Latency Reversal.

Antiretroviral therapy (ART) does not cure HIV-1 infection due to the persistence of proviruses in long-lived resting T cells. Strategies targeting these latently infected cells will be necessary to eradicate HIV-1 in infected individuals. Protein kinase C (PKC) activation is an effective mechanism to reactivate latent proviruses and allows for recognition and clearance of infected cells by the immune system. Several ingenol compounds, naturally occurring PKC agonists, have been described to have potent latency reversal activity. We sought to optimize this activity by synthesizing a library of novel ingenols via esterification of the C-3 hydroxyl group of the ingenol core, which itself is inactive for latency reversal. Newly synthesized ingenol derivatives were evaluated for latency reversal activity, cellular activation, and cytotoxicity alongside commercially available ingenols (ingenol-3,20-dibenzoate, ingenol 3-hexanoate, and ingenol-3-angelate) in HIV latency cell lines and resting CD4+ T cells from aviremic participants. Among the synthetic ingenols that we produced, we identified several compounds that demonstrate high efficacy and represent promising leads as latency reversal agents for HIV-1 eradication.

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

Reliability and validity of the EASYCare-2010 Standard to assess elderly people in Portuguese Primary Health Care.

INTRODUCTION: The EASYCare is a multidimensional assessment tool for older people, which corresponds to the concerns and priorities of older people in relation to their needs, health, and quality of life. The EASYCare instrument has been used in many countries worldwide. Lack of reliability evidence has recently been raised by researchers. This study aimed to test the validity and reliability of the EASYCare-2010 instrument in community-dwelling Portuguese older people attended in Primary Health Care centres. METHODS: The sample for this transversal study (N=244) was collected from Portuguese Primary Health Care Centers. Categorical Principal Component Analysis was used to assess the underlying dimensions of EASYCare-2010. Construct validity was evaluated through correlation with the World Health Organization Quality of Life Assessment Instrument-Short Form. RESULTS: A two-factor model (labelled "mobility and activities of daily life", and "general well-being and safety") was found. The EASYCare-2010 instrument showed acceptable levels for internal consistency (≥0.70). The EASYCare-2010 factors were correlated with measures of quality of life. Results showed that in most polytomous items, some of the more extreme categories were not considered at all or only by a residual number of participants. CONCLUSION: EASY Care -2010 version is a valid and reliable instrument for holistic assessment of the older people attended in Primary Health Care centres in Portugal.

Janus kinase inhibition suppresses PKC-induced cytokine release without affecting HIV-1 latency reversal ex vivo.

BACKGROUND: Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation. DESIGN AND METHODS: We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants. RESULTS: We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo. CONCLUSION: The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.

Ex Vivo Bioactivity and HIV-1 Latency Reversal by Ingenol Dibenzoate and Panobinostat in Resting CD4(+) T Cells from Aviremic Patients.

The human immunodeficiency virus type 1 (HIV-1) latent reservoir in resting CD4(+) T cells represents a major barrier to viral eradication. Small compounds capable of latency reversal have not demonstrated uniform responses across in vitro HIV-1 latency cell models. Characterizing compounds that demonstrate latency-reversing activity in resting CD4(+) T cells from aviremic patients ex vivo will help inform pilot clinical trials aimed at HIV-1 eradication. We have optimized a rapid ex vivo assay using resting CD4(+) T cells from aviremic HIV-1(+) patients to evaluate both the bioactivity and latency-reversing potential of candidate latency-reversing agents (LRAs). Using this assay, we characterize the properties of two candidate compounds from promising LRA classes, ingenol 3,20-dibenzoate (a protein kinase C agonist) and panobinostat (a histone deacetylase inhibitor), in cells from HIV-1(+) antiretroviral therapy (ART)-treated aviremic participants, including the effects on cellular activation and cytotoxicity. Ingenol induced viral release at levels similar to those of the positive control (CD3/28 receptor stimulation) in cells from a majority of participants and represents an exciting LRA candidate, as it combines a robust viral reactivation potential with a low toxicity profile. At concentrations that blocked histone deacetylation, panobinostat displayed a wide range of potency among participant samples and consistently induced significant levels of apoptosis. The protein kinase C agonist ingenol 3,20-dibenzoate demonstrated significant promise in a rapid ex vivo assay using resting CD4(+) T cells from treated HIV-1-positive patients to measure latent HIV-1 reactivation.

Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs.

The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.

Recent advances in photodynamic therapy combined with chemotherapy for cervical cancer: a systematic review.

INTRODUCTION: Despite the evidence that photodynamic therapy (PDT) associated with chemotherapy presents great potential to overcome the limitations of monotherapy, little is known about the current status of this combination against cervical cancer. This systematic review aimed to address the currently available advances in combining PDT and chemotherapy in different research models and clinical trials of cervical cancer. METHODS: We conducted a systematic review based on PRISMA Statement and Open Science Framework review protocol using PubMed, Web of Science, Embase, Scopus, LILACS, and Cochrane databases. We selected original articles focusing on 'Uterine Cervical Neoplasms' and 'Photochemotherapy and Chemotherapy' published in the last 10 years. The risk of bias in the studies was assessed using the CONSORT and SYRCLE tools. RESULTS: Twenty-three original articles were included, focusing on HeLa cells, derived from endocervical adenocarcinoma and on combinations of several chemotherapeutics. Most of the combinations used modern drug delivery systems for improved simultaneous delivery and presented promising results with increased cytotoxicity compared to monotherapy. CONCLUSION: Despite the scarcity of animal studies and the absence of clinical studies, the combination of chemotherapy with PDT presents a potential option for cervical cancer therapy requiring additional studies. OSF REGISTRATION: https://doi.org/10.17605/OSF.IO/WPHN5 [Figure: see text].

Drosophila Nhe2 overexpression induces autophagic cell death.

Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via overexpression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.

Single-molecule RNA-FISH analysis reveals stochasticity in reactivation of latent HIV-1 regulated by Nuclear Orphan Receptors NR4A and cMYC.

HIV-1 eradication strategies require complete reactivation of HIV-1 latent cells by Latency Reversing Agents (LRA). Current methods lack effectiveness due to incomplete proviral reactivation. We employed a single-molecule RNA-FISH (smRNA-FISH) and FISH-Quant analysis and found that proviral reactivation is highly variable from cell-to-cell, stochastic, and occurs in bursts and waves, with different kinetics in response to diverse LRAs. Approximately 1-5% of latent cells exhibited stochastic reactivation without LRAs. Through single-cell RNA-seq analysis, we identified NR4A3 and cMYC as extrinsic factors associated with stochastic HIV-1 reactivation. Concomitant with HIV-1 reactivation cMYC was downregulated and NR4A3 was upregulated in both latent cell lines and primary CD4+ T-cells from aviremic patients. By inhibiting cMYC using SN-38, an active metabolite of irinotecan, we induced NR4A3 and HIV-1 expression. Our results suggest that inherent stochasticity in proviral reactivation contributes to cell-to-cell variability, which could potentially be modulated by drugs targeting cMYC and NR4A3.

Brazilian Version of the Yale Food Addiction Scale for Individuals with Severe Obesity.

PURPOSE: Adapting and validating the Portuguese version of Br-YFAS 2.0-Obes to allow it to be used by the Brazilian candidates for bariatric surgery. MATERIALS AND METHODS: This study included 329 individuals with body mass indexes (BMI) ≥ 30 kg/m2, candidates for bariatric surgery at a reference hospital in Brazil. They were given a questionnaire that identified sociodemographic data, and the YFAS 2.0 scale, Portuguese version (BR-YFAS2.0-Obes), was applied to assess their food dependence levels. The Food Craving Questionnaire - Trait: The FCQ-T-reduced was subsequently used for a correlation analysis. RESULTS: The patients' average BMI was 41.6 ± 8.8 kg/m2. Br-YFAS2.0-Obes presented an average of 4.9 ± 3.1 for the FA diagnostic criteria. The resulting values of the Comparative Fit Index, Tucker Lewis Index, and Standardized Root Mean Square Residual were 0.990, 0.986, and 0.074, respectively. The internal consistency analysis of the 11 domains presented a Kuder-Richardson α of 0.82. The convergent validity, obtained through an analysis of the Pearson correlation coefficient, was r = 0.43 (p < 0.001). It was found that an increase in the number of Br-YFAS 2.0-Obes symptoms is associated with an increase in the FCQ-T-r mean. CONCLUSION: Much like the YFAS 2.0 in other languages, the BR-YFAS 2.0-Obes presented adequate convergent validity, reliability, and one-factor structure results, which makes it suitable for Brazilian candidates for bariatric surgery or any individual who is within BMI > = 30 kg/m2.

Moving targets: Mechanisms regulating siRNA production and DNA methylation during plant development.

DNA methylation is a conserved modification that must be precisely regulated during development to facilitate its roles in silencing transposable elements and regulating gene expression. In plants, DNA methylation changes during reproduction are widely documented and, in many cases, the underlying mechanisms are well understood. In somatic tissues, the diversity of methylation patterns are only recently emerging but they are often associated with the RNA-directed DNA methylation (RdDM) pathway. Here, we discuss advances in our understanding of how the locus-specific targeting and tissue-specific expression of RdDM proteins regulate methylation patterns, how the targeting of methylation at loci with imperfect homology expands the purview of RdDM, and how natural variation within RdDM factors impacts DNA methylation patterns.

CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing.

Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

HIV-1-induced type I IFNs promote viral latency in macrophages.

Macrophages chronically infected with HIV-1 serve as a reservoir that contributes to HIV-1 persistence during antiretroviral therapy; however, the mechanisms governing the establishment and maintenance of this virus reservoir have not been fully elucidated. Here, we show that HIV-1 enters a state reminiscent of latency in monocyte-derived macrophages (MDMs), characterized by integrated proviral DNA with decreased viral transcription. This quiescent state is associated with decreased NF-κB p65, RNA polymerase II, and p-TEFb recruitment to the HIV-1 promoter as well as maintenance of promoter chromatin in a transcriptionally nonpermissive state. MDM transition to viral latency is mediated by type I IFN signaling, as inhibiting type I IFN signaling or blocking type 1 IFN prevents the establishment of latent infection. Knockdown studies demonstrate that the innate immune signaling molecule mitochondrial antiviral signaling protein (MAVS) is required for the transition to latency. Finally, we demonstrate a role for the viral accessory protein Vpr in the establishment of HIV-1 latency in macrophages. Our data indicate that HIV-1-induced type I IFN production is responsible for the establishment of viral latency in MDMs and identify possible therapeutic targets for the prevention or elimination of this important HIV-1 reservoir.

The CLASSY family controls tissue-specific DNA methylation patterns in Arabidopsis.

DNA methylation shapes the epigenetic landscape of the genome, plays critical roles in regulating gene expression, and ensures transposon silencing. As is evidenced by the numerous defects associated with aberrant DNA methylation landscapes, establishing proper tissue-specific methylation patterns is critical. Yet, how such differences arise remains a largely open question in both plants and animals. Here we demonstrate that CLASSY1-4 (CLSY1-4), four locus-specific regulators of DNA methylation, also control tissue-specific methylation patterns, with the most striking pattern observed in ovules where CLSY3 and CLSY4 control DNA methylation at loci with a highly conserved DNA motif. On a more global scale, we demonstrate that specific clsy mutants are sufficient to shift the epigenetic landscape between tissues. Together, these findings reveal substantial epigenetic diversity between tissues and assign these changes to specific CLSY proteins, elucidating how locus-specific targeting combined with tissue-specific expression enables the CLSYs to generate epigenetic diversity during plant development.

Perioperative administration of recombinant activated factor VII in a Glanzmann's thrombasthenia patient with platelet refractoriness: case report.

Glanzmann's Trombasthenia (GT) is a genetic disorder, that develops with a tendency toward bleeding and is characterized by the absence or decrease in platelet aggregation. Surgical bleeding may be difficult to control. Platelet transfusion is the main treatment, albeit refractoriness can occur. We describe the case of a patient with GT and platelet refractoriness, who was submitted to radical prostatectomy and dental extraction. The perioperative treatment with apheresis platelet concentrate and activated recombinant factor seven allowed the procedures to be performed uneventfully. We discuss the complexity of the case and the treatment option.

Intracranial rebleeding post spinal anesthesia in pregnant patient with undiagnosed chronic subdural hematoma: case report.

Intracranial hematoma after spinal anesthesia is a rare complication. It generally presents with posture-dependent headache that becomes persistent. We describe the case of patient submitted to spinal anesthesia for cesarean section who presented a non-posture-dependent headache, resistant to clinical treatment, that progressively worsened and with symptoms of intracranial hypertension. The patient had a history of head trauma without symptoms. The CT-scan revealed a chronic bilateral parietal hematoma with a recent bleeding component, treated surgically. We concluded that spinal puncture led to chronic hematoma to rebleed. We have reported the case to draw attention to the importance of investigating atypical headache after spinal anesthesia.