The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation.

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity.

Mars' emitted energy and seasonal energy imbalance.

SignificanceThe radiant energy budget is a fundamental metric for planets. Based on the observations from multiple missions, we provide a global picture of Mars' emitted power. Furthermore, we estimate the radiant energy budget of Mars, which suggests that there are energy imbalances at the time scale of Mars' seasons. Such energy imbalances provide a new perspective to understanding the generating mechanism of dust storms. Mars' radiant energy budget is assumed to be balanced at all time scales in current models and theories, but our analyses show that the energy budget is not balanced, at least at the time scale of Mars' seasons. Therefore, current theories and models should be revisited with the newly revealed energy characteristics.

Reversed-engineered human alveolar lung-on-a-chip model.

Here, we present a physiologically relevant model of the human pulmonary alveoli. This alveolar lung-on-a-chip platform is composed of a three-dimensional porous hydrogel made of gelatin methacryloyl with an inverse opal structure, bonded to a compartmentalized polydimethylsiloxane chip. The inverse opal hydrogel structure features well-defined, interconnected pores with high similarity to human alveolar sacs. By populating the sacs with primary human alveolar epithelial cells, functional epithelial monolayers are readily formed. Cyclic strain is integrated into the device to allow biomimetic breathing events of the alveolar lung, which, in addition, makes it possible to investigate pathological effects such as those incurred by cigarette smoking and severe acute respiratory syndrome coronavirus 2 pseudoviral infection. Our study demonstrates a unique method for reconstitution of the functional human pulmonary alveoli in vitro, which is anticipated to pave the way for investigating relevant physiological and pathological events in the human distal lung.

Parasitic plant small RNA analyses unveil parasite-specific signatures of microRNA retention, loss, and gain.

Parasitism is a successful life strategy that has evolved independently in several families of vascular plants. The genera Cuscuta and Orobanche represent examples of the two profoundly different groups of parasites: one parasitizing host shoots and the other infecting host roots. In this study, we sequenced and described the overall repertoire of small RNAs from Cuscuta campestris and Orobanche aegyptiaca. We showed that C. campestris contains a number of novel microRNAs (miRNAs) in addition to a conspicuous retention of miRNAs that are typically lacking in other Solanales, while several typically conserved miRNAs seem to have become obsolete in the parasite. One new miRNA appears to be derived from a horizontal gene transfer event. The exploratory analysis of the miRNA population (exploratory due to the absence of a full genomic sequence for reference) from the root parasitic O. aegyptiaca also revealed a loss of a number of miRNAs compared to photosynthetic species from the same order. In summary, our study shows partly similar evolutionary signatures in the RNA silencing machinery in both parasites. Our data bear proof for the dynamism of this regulatory mechanism in parasitic plants.

Diffuse Sclerosing Papillary Thyroid Carcinoma: Clinicopathological Characteristics and Prognostic Implications Compared with Classic and Tall Cell Papillary Thyroid Cancer.

BACKGROUND: The clinical behaviour and oncologic outcome of diffuse sclerosing papillary thyroid carcinoma (DS-PTC) is poorly understood. The objectives of this study were to compare the clinicopathological characteristics and oncological outcomes of DS-PTC to classic PTC (cPTC) and tall cell PTC (TC-PTC). METHODS: After institutional review board approval, 86 DS-PTC, 2,080 cPTC, and 701 TC-PTC patients treated at MSKCC between 1986 and 2021 were identified. Clinicopathological characteristics were compared by using chi-square test. Kaplan-Meier and log rank were used to compare recurrence-free survival (RFS), disease-specific survival (DSS), and overall survival (OS). DS-PTC patients were propensity matched to cPTC and TC-PTC patients for further comparison. RESULTS: DS-PTC patients were younger with more advanced disease than cPTC and TC-PTC (p < 0.05). Lymphovascular invasion (LVI), extranodal extension, and positive margins were more common in DS-PTC (p < 0.02). Propensity matching confirmed more aggressive histopathological features in DS-PTC. The median number of metastatic lymph nodes was significantly greater and DS-PTC metastases were RAI avid. DS-PTC 5-year RFS was 50.4% compared with 92.4% in cPTC and 88.4% in TC-PTC (p < 0.001). Multivariate analysis confirmed DS-PTC as an independent prognostic factor of recurrence. Ten-year DSS for DS-PTC was 100% compared with 97.1% in cPTC and 91.1% in TC-PTC. Differentiated high-grade, thyroid carcinoma DS had more advanced T-stage and worse 5-year RFS than DS-PTC. CONCLUSIONS: DS-PTC presents with more advanced clinicopathological features than cPTC and TC-PTC. Large-volume nodal metastases and LVI are characteristic features. Almost half of patients develop recurrence despite aggressive initial management. Despite this, with successful salvage surgery DSS is excellent.

Ki67 proliferation index in medullary thyroid carcinoma: a comparative study of multiple counting methods and validation of image analysis and deep learning platforms.

AIMS: The International Medullary Thyroid Carcinoma Grading System, introduced in 2022, mandates evaluation of the Ki67 proliferation index to assign a histological grade for medullary thyroid carcinoma. However, manual counting remains a tedious and time-consuming task. METHODS AND RESULTS: We aimed to evaluate the performance of three other counting techniques for the Ki67 index, eyeballing by a trained experienced investigator, a machine learning-based deep learning algorithm (DeepLIIF) and an image analysis software with internal thresholding compared to the gold standard manual counting in a large cohort of 260 primarily resected medullary thyroid carcinoma. The Ki67 proliferation index generated by all three methods correlate near-perfectly with the manual Ki67 index, with kappa values ranging from 0.884 to 0.979 and interclass correlation coefficients ranging from 0.969 to 0.983. Discrepant Ki67 results were only observed in cases with borderline manual Ki67 readings, ranging from 3 to 7%. Medullary thyroid carcinomas with a high Ki67 index (≥ 5%) determined using any of the four methods were associated with significantly decreased disease-specific survival and distant metastasis-free survival. CONCLUSIONS: We herein validate a machine learning-based deep-learning platform and an image analysis software with internal thresholding to generate accurate automatic Ki67 proliferation indices in medullary thyroid carcinoma. Manual Ki67 count remains useful when facing a tumour with a borderline Ki67 proliferation index of 3-7%. In daily practice, validation of alternative evaluation methods for the Ki67 index in MTC is required prior to implementation.

Polymerase IV Plays a Crucial Role in Pollen Development in Capsella.

In Arabidopsis (Arabidopsis thaliana), DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA transcripts. These transcripts are processed by DICER-LIKE3 into 24-nucleotide small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nucleotide epigenetically activated siRNAs, which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernible pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV, in the Brassicaceae species Capsella (Capsella rubella), caused postmeiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nucleotide and 24-nucleotide siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella compared with Arabidopsis, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.

In Vitro Evaluation of the Antiamoebic Activity of Kaempferol against Trophozoites of Entamoeba histolytica and in the Interactions of Amoebae with Hamster Neutrophils.

Entamoeba histolytica (E. histolytica) is a parasite in humans that provokes amoebiasis. The most employed drug is metronidazole (MTZ); however, some studies have reported that this drug induces genotoxic effects. Therefore, it is necessary to explore new compounds without toxicity that can eliminate E. histolytica. Flavonoids are polyphenolic compounds that have demonstrated inhibition of growth and dysregulation of amoebic proteins. Despite the knowledge acquired to date, action mechanisms are not completely understood. The present work evaluates the effect of kaempferol against E. histolytica trophozoites and in the interactions with neutrophils from hamster, which is a susceptibility model. Our study demonstrated a significant reduction in the amoebic viability of trophozoites incubated with kaempferol at 150 µM for 90 min. The gene expression analysis showed a significant downregulation of Pr (peroxiredoxin), Rr (rubrerythrin), and TrxR (thioredoxin reductase). In interactions with amoebae and neutrophils for short times, we observed a reduction in ROS (reactive oxygen species), NO (nitric oxide), and MPO (myeloperoxidase) neutrophil activities. In conclusion, we confirmed that kaempferol is an effective drug against E. histolytica through the decrease in E. histolytica antioxidant enzyme expression and a regulator of several neutrophil mechanisms, such as MPO activity and the regulation of ROS and NO.

Evaluating the Effect of Policy Prohibiting Body Checking on Physical Contacts in U15 and U18 Youth Ice Hockey Leagues.

OBJECTIVE: To compare physical contacts (PCs) and head contacts (HCs) in nonelite U15 (ages 13-14) and U18 (ages 15-17) ice hockey players in body checking (BC) and non-BC leagues. DESIGN: Cohort video analysis study. SETTING: Ice hockey arenas in Calgary, Canada. PARTICIPANTS: Players from 13 BC and 13 non-BC games at the nonelite U15 and U18 levels (n = 52 total games). ASSESSMENT OF RISK FACTORS: Games were videotaped and analyzed to compare PC variables between leagues allowing and prohibiting BC. MAIN OUTCOME MEASURES: Validated methodology for PC type (trunk PC and other types of PC with limb/stick/head), intensity (low and high intensity), and HC. Incidence rate ratios (IRRs) were estimated using Poisson regression (controlling for cluster by team game and offset by player minutes) to compare the incidence of PCs in BC and non-BC games. RESULTS: The rate of trunk PCs was lower in the non-BC leagues for both U15 (IRR = 0.50, 99% confidence interval [CI]: 0.43-0.58) and U18 (IRR = 0.56, 99% CI: 0.46-0.67) players. This was most significant for BC contacts (U15: IRR = 0.18, 99% CI: 0.11-0.29; U18: IRR = 0.16, 99% CI: 0.08-0.34), although also significant for body contacts (U15: IRR = 0.54, 99% CI: 0.46-0.64; U18: IRR = 0.58, 99% CI: 0.48-0.70), other PCs, (U15: IRR = 0.60, 99% CI: 0.45-0.78; U18: IRR = 0.71, 99% CI: 0.58-0.88), and HCs (U15: IRR = 0.40, 99% CI: 0.22-0.71; U18: IRR = 0.37, 99% CI: 0.25-0.57). CONCLUSIONS: The incidence of BC was 82% lower in the U15 and 84% lower in U18 levels not allowing BC, with lower HC and other PC rates also in non-BC leagues. These findings demonstrate high adherence with BC policy change in youth ice hockey.

Radiation and Dust Sensor for Mars Environmental Dynamic Analyzer Onboard M2020 Rover.

The Radiation and Dust Sensor is one of six sensors of the Mars Environmental Dynamics Analyzer onboard the Perseverance rover from the Mars 2020 NASA mission. Its primary goal is to characterize the airbone dust in the Mars atmosphere, inferring its concentration, shape and optical properties. Thanks to its geometry, the sensor will be capable of studying dust-lifting processes with a high temporal resolution and high spatial coverage. Thanks to its multiwavelength design, it will characterize the solar spectrum from Mars' surface. The present work describes the sensor design from the scientific and technical requirements, the qualification processes to demonstrate its endurance on Mars' surface, the calibration activities to demonstrate its performance, and its validation campaign in a representative Mars analog. As a result of this process, we obtained a very compact sensor, fully digital, with a mass below 1 kg and exceptional power consumption and data budget features.

Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis.

Environmentally induced changes in the epigenome help individuals to quickly adapt to fluctuations in the conditions of their habitats. We explored those changes in Arabidopsis thaliana plants subjected to multiple biotic and abiotic stresses, and identified transposable element (TE) activation in plants infested with the green peach aphid, Myzus persicae. We performed a genome-wide analysis mRNA expression, small RNA accumulation and DNA methylation Our results demonstrate that aphid feeding induces loss of methylation of hundreds of loci, mainly TEs. This loss of methylation has the potential to regulate gene expression and we found evidence that it is involved in the control of plant immunity genes. Accordingly, mutant plants deficient in DNA and H3K9 methylation (kyp) showed increased resistance to M. persicae infestation. Collectively, our results show that changes in DNA methylation play a significant role in the regulation of the plant transcriptional response and induction of defense response against aphid feeding.

smartPARE: An R Package for Efficient Identification of True mRNA Cleavage Sites.

Degradome sequencing is commonly used to generate high-throughput information on mRNA cleavage sites mediated by small RNAs (sRNA). In our datasets of potato (Solanum tuberosum, St) and Phytophthora infestans (Pi), initial predictions generated high numbers of cleavage site predictions, which highlighted the need of improved analytic tools. Here, we present an R package based on a deep learning convolutional neural network (CNN) in a machine learning environment to optimize discrimination of false from true cleavage sites. When applying smartPARE to our datasets on potato during the infection process by the late blight pathogen, 7.3% of all cleavage windows represented true cleavages distributed on 214 sites in P. infestans and 444 sites in potato. The sRNA landscape of the two organisms is complex with uneven sRNA production and cleavage regions widespread in the two genomes. Multiple targets and several cases of complex regulatory cascades, particularly in potato, was revealed. We conclude that our new analytic approach is useful for anyone working on complex biological systems and with the interest of identifying cleavage sites particularly inferred by sRNA classes beyond miRNAs.

Metabolomic Analysis of The Chemical Diversity of South Africa Leaf Litter Fungal Species Using an Epigenetic Culture-Based Approach.

Microbial natural products are an invaluable resource for the biotechnological industry. Genome mining studies have highlighted the huge biosynthetic potential of fungi, which is underexploited by standard fermentation conditions. Epigenetic effectors and/or cultivation-based approaches have successfully been applied to activate cryptic biosynthetic pathways in order to produce the chemical diversity suggested in available fungal genomes. The addition of Suberoylanilide Hydroxamic Acid to fermentation processes was evaluated to assess its effect on the metabolomic diversity of a taxonomically diverse fungal population. Here, metabolomic methodologies were implemented to identify changes in secondary metabolite profiles to determine the best fermentation conditions. The results confirmed previously described effects of the epigenetic modifier on the metabolism of a population of 232 wide diverse South Africa fungal strains cultured in different fermentation media where the induction of differential metabolites was observed. Furthermore, one solid-state fermentation (BRFT medium), two classic successful liquid fermentation media (LSFM and YES) and two new liquid media formulations (MCKX and SMK-II) were compared to identify the most productive conditions for the different populations of taxonomic subgroups.

Seasonal variation of the estrous cycle length, corpus luteum area, and size of the pre-ovulatory follicle in Criollo Limonero heifers.

In order to determine whether seasonal variations may influence the estrous cycle length (ECL), corpus luteum size (CLS), maximum area of CL (MACL), day of cycle with maximum area of CL (DCMACL), and pre-ovulatory follicles size (PFS), ten Criollo Limonero heifers were subjected to daily ultrasound ovary scanning throughout their estrous cycles during three seasons: hot-dry (HD), hot-humid (HH), and wind-rain (WR). The effect of season on ECL, MACL, DCMACL, and PFS was analyzed with an ANOVA (PROC GLM, SAS), whereas, for the effect of season on CLS, an ANOVA with repeated measures (PROC MIXED, SAS) was used. Results showed no effect (P > 0.05) of season on ECL, MACL, and DCMACL. However, size of PFS was larger (P < 0.02) during the WR season and the CLS tended (P < 0.09) to be lower during the HH. In conclusion, the relative stability of ECL, MACL, DCMACL, PFS, and CLS measures suggests no major seasonal variations which could imply adaptation capability of Criollo Limonero cattle to the tropical environment.

Dynamic architecture and regulatory implications of the miRNA network underlying the response to stress in melon.

miRNAs are small RNAs that regulate mRNAs at both transcriptional and posttranscriptional level. In plants, miRNAs are involved in the regulation of different processes including development and stress-response. Elucidating how stress-responsive miRNAs are regulated is key to understand the global response to stress but also to develop efficient biotechnological tools that could help to cope with stress. Here, we describe a computational approach based on sRNA sequencing, transcript quantification and degradome data to analyse the accumulation, function and structural organization of melon miRNAs reactivated under seven biotic and abiotic stress conditions at two and four days post-treatment. Our pipeline allowed us to identify fourteen stress-responsive miRNAs (including evolutionary conserved such as miR156, miR166, miR172, miR319, miR398, miR399, miR894 and miR408) at both analysed times. According to our analysis miRNAs were categorized in three groups showing a broad-, intermediate- or narrow- response range. miRNAs reactive to a broad range of environmental cues appear as central components in the stress-response network. The strictly coordinated response of miR398 and miR408 (broad response-range) to the seven stress treatments during the period analysed here reinforces this notion. Although both, the amplitude and diversity of the miRNA-related response to stress changes during the exposition time, the architecture of the miRNA-network is conserved. This organization of miRNA response to stress is also conserved in rice and soybean supporting the conservation of miRNA-network organization in other crops. Overall, our work sheds light into how miRNA networks in plants organize and function during stress.

tRNA-derived small RNAs target transposable element transcripts.

tRNA-derived RNA fragments (tRFs) are 18-26 nucleotide small RNAs that are not random degradation products, but are rather specifically cleaved from mature tRNA transcripts. Abundant in stressed or viral-infected cells, the function and potential targets of tRFs are not known. We identified that in the unstressed wild-type male gamete containing pollen of flowering plants, and analogous reproductive structure in non-flowering plant species, tRFs accumulate to high levels. In the reference plant Arabidopsis thaliana, tRFs are processed by Dicer-like 1 and incorporated into Argonaute1 (AGO1), akin to a microRNA. We utilized the fact that many plant small RNAs direct cleavage of their target transcripts to demonstrate that the tRF-AGO1 complex acts to specifically target and cleave endogenous transposable element (TE) mRNAs produced from transcriptionally active TEs. The data presented here demonstrate that tRFs are bona-fide regulatory microRNA-like small RNAs involved in the regulation of genome stability through the targeting of TE transcripts.

Stress response regulation by epigenetic mechanisms: changing of the guards.

Plants are sessile organisms that lack a specialized immune system to cope with biotic and abiotic stress. Instead, plants have complex regulatory networks that determine the appropriate distribution of resources between the developmental and the defense programs. In the last years, epigenetic regulation of repeats and gene expression has evolved as an important player in the transcriptional regulation of stress-related genes. Here, we review the current knowledge about how different stresses interact with different levels of epigenetic control of the genome. Moreover, we analyze the different examples of transgenerational epigenetic inheritance and connect them with the known features of genome epigenetic regulation. Although yet to be explored, the interplay between epigenetics and stress resistance seems to be a relevant and dynamic player of the interaction of plants with their environments.

tRNA-derived small RNAs: New players in genome protection against retrotransposons.

Previously believed to be mere random degradation products, tRNA-derived small RNAs have been lately connected to a series of functions that include, surprisingly, genome protection against retrotransposons. tRNAs have been known for a long time to be involved in the replication cycle of retroviruses, pararetroviruses and retrotransposons as primers of their reverse transcription. tRNA-derived small RNAs, as functional small RNAs or as mere tRNA degradation products, have emerged as important players in the regulation of genic transcription. Nevertheless, the involvement of functional sRNAs derived from tRNA transcripts in transposon posttranscriptional control is a regulatory layer that remained elusive until now. Here I review the recent discoveries in the field that connect tRNA-derived small RNAs and retrotransposon control.