Capturing the Onset of PRC2-Mediated Repressive Domain Formation.

Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.

Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation.

Classical dendritic cells (cDCs) are essential for immune responses and differentiate from hematopoietic stem cells via intermediate progenitors, such as monocyte-DC progenitors (MDPs) and common DC progenitors (CDPs). Upon infection, cDCs are activated and rapidly express host defense-related genes, such as those encoding cytokines and chemokines. Chromatin structures, including nuclear compartments and topologically associating domains (TADs), have been implicated in gene regulation. However, the extent and dynamics of their reorganization during cDC development and activation remain unknown. In this study, we comprehensively determined higher-order chromatin structures by Hi-C in DC progenitors and cDC subpopulations. During cDC differentiation, chromatin activation was initially induced at the MDP stage. Subsequently, a shift from inactive to active nuclear compartments occurred at the cDC gene loci in CDPs, which was followed by increased intra-TAD interactions and loop formation. Mechanistically, the transcription factor IRF8, indispensable for cDC differentiation, mediated chromatin activation and changes into the active compartments in DC progenitors, thereby possibly leading to cDC-specific gene induction. Using an infection model, we found that the chromatin structures of host defense-related gene loci were preestablished in unstimulated cDCs, indicating that the formation of higher-order chromatin structures prior to infection may contribute to the rapid responses to pathogens. Overall, these results suggest that chromatin structure reorganization is closely related to the establishment of cDC-specific gene expression and immune functions. This study advances the fundamental understanding of chromatin reorganization in cDC differentiation and activation.

Alu insertion variants alter gene transcript levels.

Alu are high copy number interspersed repeats that have accumulated near genes during primate and human evolution. They are a pervasive source of structural variation in modern humans. Impacts that Alu insertions may have on gene expression are not well understood, although some have been associated with expression quantitative trait loci (eQTLs). Here, we directly test regulatory effects of polymorphic Alu insertions in isolation of other variants on the same haplotype. To screen insertion variants for those with such effects, we used ectopic luciferase reporter assays and evaluated 110 Alu insertion variants, including more than 40 with a potential role in disease risk. We observed a continuum of effects with significant outliers that up- or down-regulate luciferase activity. Using a series of reporter constructs, which included genomic context surrounding the Alu, we can distinguish between instances in which the Alu disrupts another regulator and those in which the Alu introduces new regulatory sequence. We next focused on three polymorphic Alu loci associated with breast cancer that display significant effects in the reporter assay. We used CRISPR to modify the endogenous sequences, establishing cell lines varying in the Alu genotype. Our findings indicate that Alu genotype can alter expression of genes implicated in cancer risk, including PTHLH, RANBP9, and MYC These data show that commonly occurring polymorphic Alu elements can alter transcript levels and potentially contribute to disease risk.

Patterns of kidney function and risk assessment in a nationwide laboratory database: the Brazilian CHECK-CKD study.

BACKGROUND: Chronic kidney disease (CKD) is a global health problem with rising prevalence, morbidity, mortality, and associated costs. Early identification and risk stratification are key to preventing progression to kidney failure. However, there is a paucity of data on practice patterns of kidney function assessment to guide the development of improvement strategies, particularly in lower-income countries. METHODS: A retrospective observational analysis was conducted in a nationwide laboratory database in Brazil. We included all adult patients with at least one serum creatinine assessment between June 2018 and May 2021. Our primary objective was to determine the proportion of patients with estimated glomerular filtration rate (eGFR) evaluations accompanied by predicted levels of urinary albumin-to-creatinine ratio (pACR) assessments within 12 months. RESULTS: Out of 4,5323,332 serum creatinine measurements, 42% lacked pACR measurements within 12 months. Approximately 10.8% of tests suggested CKD, mostly at stage 3a. The proportion of serum creatinine exams paired with pACR assessment varied according to the CKD stage. Internal Medicine, Cardiology, and Obstetrics/Gynecology were the specialties requesting most of the creatinine tests. Nephrology contributed with only 1.1% of serum creatinine requests for testing. CONCLUSION: Our findings reveal that a significant proportion of individuals with a creatinine test lack an accompanying urinary albuminuria measurement in Brazil, contrary to the recommendations of the international guidelines. Non-Nephrologists perform most kidney function evaluations, even among patients with presumable advanced CKD. This highlights the urge to incorporate in clinical practice the early detection of CKD and to encourage more collaborative multidisciplinary care to improve CKD management.

Repetitive Elements: Different Subtypes Hint at Distinct Functions.

Lu et al. report that the association of different repeat types with distinct gene classes goes far beyond what has previously been shown and suggest that such relationship might be essential for gene function and regulation. As an example, they describe how long interspersed nuclear repeat (LINE1) transcripts are recruited together with associated genes to silent nuclear regions.

Analysis of Immune Intratumor Heterogeneity Highlights Immunoregulatory and Coinhibitory Lymphocytes as Hallmarks of Recurrence in Stage I Non-Small Cell Lung Cancer.

Our understanding of the molecular mechanisms underlying postsurgical recurrence of non-small cell lung cancer (NSCLC) is rudimentary. Molecular and T cell repertoire intratumor heterogeneity (ITH) have been reported to be associated with postsurgical relapse; however, how ITH at the cellular level impacts survival is largely unknown. Here we report the analysis of 2880 multispectral images representing 14.2% to 27% of tumor areas from 33 patients with stage I NSCLC, including 17 cases (relapsed within 3 years after surgery) and 16 controls (without recurrence ≥5 years after surgery) using multiplex immunofluorescence. Spatial analysis was conducted to quantify the minimum distance between different cell types and immune cell infiltration around malignant cells. Immune ITH was defined as the variance of immune cells from 3 intratumor regions. We found that tumors from patients having relapsed display different immune biology compared with nonrecurrent tumors, with a higher percentage of tumor cells and macrophages expressing PD-L1 (P =.031 and P =.024, respectively), along with an increase in regulatory T cells (Treg) (P =.018), antigen-experienced T cells (P =.025), and effector-memory T cells (P =.041). Spatial analysis revealed that a higher level of infiltration of PD-L1+ macrophages (CD68+PD-L1+) or antigen-experienced cytotoxic T cells (CD3+CD8+PD-1+) in the tumor was associated with poor overall survival (P =.021 and P =.006, respectively). A higher degree of Treg ITH was associated with inferior recurrence-free survival regardless of tumor mutational burden (P =.022), neoantigen burden (P =.021), genomic ITH (P =.012) and T cell repertoire ITH (P =.001). Using multiregion multiplex immunofluorescence, we characterized ITH at the immune cell level along with whole exome and T cell repertoire sequencing from the same tumor regions. This approach highlights the role of immunoregulatory and coinhibitory signals as well as their spatial distribution and ITH that define the hallmarks of tumor relapse of stage I NSCLC.

Code Integrity and Confidentiality: An Active Data Approach for Active and Healthy Ageing.

Internet of Things cybersecurity is gaining attention as the number of devices installed in IoT environments is exponentially increasing while the number of attacks successfully addressed to these devices are also proliferating. Security concerns have, however, been mainly addressed to service availability and information integrity and confidentiality. Code integrity, on the other hand, is not receiving proper attention, mainly because of the limited resources of these devices, thus preventing the implementation of advanced protection mechanisms. This situation calls for further research on how traditional mechanisms for code integrity can be adapted to IoT devices. This work presents a mechanism for code integrity in IoT devices based on a virtual-machine approach. A proof-of-concept virtual machine is presented, specially designed for providing code integrity during firmware updates. The proposed approach has been experimentally validated in terms of resource consumption among the most-widespread micro-controller units. The obtained results demonstrate the feasibility of this robust mechanism for code integrity.

Developing a Compost Quality Index (CQI) Based on the Electrochemical Quantification of Cd (HA) Reactivity.

The present work demonstrates the use of Cd2+ as a reactivity probe of the fulvic acids (FAs), humic acids (HAs) and dissolved organic matter (DOM) compost extracts. Significant differences were observed between the extracts, with the HA extract showing the highest reactivity. Comparing the different composts, the largest reactivity variation was again observed for HA then FA and finally DOM extracts. The Cd2+ binding extent was used to calculate the quality of composts and compared with a reference of uncomposted organic fertiliser (FLW), leading to the definition of an operational scale of compost quality. The parameter equivalent mass of fertiliser (mEF) was used for this scale sorted the seven composts from 0.353 to 1.09 kg FLW, for compost of sewage sludge (CSS) and vermicompost of domestic waste (CVDW), respectively. The significance of this parameter was verified through a correlation analysis between binding extent and the effect of compost application on lettuce crop growth in a field trial. The results demonstrate the potentiality of FA and HA extracts as markers of compost bioactivity and the use of Cd2+ as a reactivity probe.

Dynamic expression of Schlafen 11 (SLFN11) in circulating tumour cells as a liquid biomarker in small cell lung cancer.

INTRODUCTION: Small cell lung cancer (SCLC) is an aggressive malignancy with no established biomarkers. Schlafen 11(SLFN11), a DNA/RNA helicase that sensitises cancer cells to DNA-damaging agents, has emerged as a promising predictive biomarker for several drug classes including platinum and PARP inhibitors. Detection of SLFN11 in circulating tumour cells (CTCs) may provide a valuable alternative to tissue sampling. METHODS: SLFN11 expression was evaluated in tumour samples and characterised in circulating tumour cells (CTC) longitudinally to determine its potential role as a biomarker of response. RESULTS: Among 196 SCLC tumours, 51% expressed SLFN11 by IHC. In addition, 20/29 extra-thoracic high-grade neuroendocrine tumours expressed SLFN11 expression. In 64 blood samples from 42 SCLC patients, 83% (53/64) of samples had detectable CTCs, and SLFN11-positive CTCs were detected in 55% (29/53). Patients actively receiving platinum treatment had the lowest number of CTCs and a lower percentage of SLFN11-positive CTCs (p = 0.014). Analysis from patients with longitudinal samples suggest a decrease in CTC number and in SLFN11 expression that correlates with clinical response. CONCLUSIONS: SLFN11 levels can be monitored in CTCs from SCLC patients using non-invasive liquid biopsies. The ability to detect SLFN11 in CTCs from SCLC patients adds a valuable tool for the detection and longitudinal monitoring of this promising biomarker.

Adverse Radiation Therapy Effects in the Treatment of Head and Neck Tumors.

Whether used as a single modality or as part of a combined approach, radiation therapy (RT) plays an essential role in the treatment of several head and neck malignancies. Despite the improvement in radiation delivery techniques, normal structures in the vicinity of the target area remain susceptible to a wide range of adverse effects. Given their high incidence, some of these effects are referred to as expected postradiation changes (eg, mucositis, sialadenitis, and edema), while others are considered true complications, meaning they should not be expected and can even represent life-threatening conditions (eg, radionecrosis, fistulas, and radiation-induced neoplasms). Also, according to their timing of onset, these deleterious effects can be divided into four groups: acute (during RT), subacute (within weeks to months), delayed onset (within months to years), and very delayed onset (after several years).The authors provide a comprehensive review of the most important radiation-induced changes related to distinct head and neck sites, focusing on their typical cross-sectional imaging features and correlating them with the time elapsed after treatment. Radiologists should not only be familiar with these imaging findings but also actively seek essential clinical data at the time of interpretation (including knowledge of the RT dose and time, target site, and manifesting symptoms) to better recognize imaging findings, avoid pitfalls and help guide appropriate management. © RSNA, 2022.

CD73 expression defines immune, molecular, and clinicopathological subgroups of lung adenocarcinoma.

INTRODUCTION: CD73 is a membrane-bound enzyme crucial in adenosine generation. The adenosinergic pathway plays a critical role in immunosuppression and in anti-tumor effects of immune checkpoint inhibitors (ICI). Here, we interrogated CD73 expression in a richly annotated cohort of human lung adenocarcinoma (LUAD) and its association with clinicopathological, immune, and molecular features to better understand the role of this immune marker in LUAD pathobiology. MATERIALS AND METHODS: Protein expression of CD73 was evaluated by immunohistochemistry in 106 archived LUADs from patients that underwent surgical treatment without neoadjuvant therapy. Total CD73 (T +) was calculated as the average of luminal (L +) and basolateral (BL +) percentage membrane expression scores for each LUAD and was used to classify tumors into three groups based on the extent of T CD73 expression (high, low, and negative). RESULTS: CD73 expression was significantly and progressively increased across normal-appearing lung tissue, adenomatous atypical hyperplasia, adenocarcinoma in situ, minimally invasive adenocarcinoma, and LUAD. In LUAD, BL CD73 expression was associated with an increase in PD-L1 expression in tumor cells and increase of tumor-associated immune cells. Stratification of LUADs based on T CD73 extent also revealed that tumors with high expression of this enzyme overall exhibited significantly elevated immune infiltration and PD-L1 protein expression. Immune profiling demonstrated that T-cell inflammation and adenosine signatures were significantly higher in CD73-expressing lung adenocarcinomas relative to those lacking CD73. CONCLUSION: Our study suggests that higher CD73 expression is associated with an overall augmented host immune response, suggesting potential implications in the immune pathobiology of early stage lung adenocarcinoma. Our findings warrant further studies to explore the role of CD73 in immunotherapeutic response of LUAD.

Shaped by the Sun: the effect of exposure to sunlight on the evolution of spider bodies.

Body temperature can strongly influence fitness. Some Sun-exposed ectotherms thermoregulate by adjusting body posture according to the Sun's position. In these species, body elongation should reduce the risk of heat stress by allowing the exposure of a smaller body area to sunlight. Therefore, selection should favour more elongated bodies in Sun-exposed than in Sun-protected species. Diurnal orb-web spider species that sit on their webs are more likely to be Sun-exposed, on average, than nocturnal or diurnal shelter-building species. We measured the body elongation of orb-web spiders (Araneae, Araneidae) across 1024 species and classified them as Sun-protected or exposed based on the literature. We found that Sun-exposed species evolved more elongate bodies than Sun-protected ones. Further, we built a model combining traditional heat transfer models with models of thermoregulatory postures in orb-web spiders and meteorological data. The model indicates that body elongation in large orb-web spiders decreases the risk of high body temperatures. Overall, our results suggest that Sun exposure influenced the evolution of body shapes of orb-web spiders.

Values of plasma total proteins of free-living marmosets (Callithrix jacchus) in an urban forest.

BACKGROUND: Callithrix jacchus is a small primate widely used in experimentation, but data on plasma total protein (PTP) values of free-living animals inhabiting its own endemism region are non-existent. METHODS: Marmosets belonging to two free-living groups were captured for marking and obtaining biological material. The collected blood was centrifuged to obtain the plasma and thus to determine the concentration of PTP. RESULTS AND CONCLUSIONS: Females and adults had the highest mean PTP. Mean found were higher than other values described for other neotropical primates, and the high PTP values presented by the animals indicate dehydration. Due to the importance of total plasma proteins for health and management, obtaining these values as a reference is relevant for the free-living Callithrix genus and other neotropical primates.

Forest and connectivity loss simplify tropical pollination networks.

Mutualistic interactions between plants and pollinators play an essential role in the organization and persistence of biodiversity. The structure of interaction networks mediates the resilience of local communities and ecosystem functioning to environmental changes. Hence, network structure conservation may be more critical for maintaining biodiversity and ecological services than the preservation of isolated species in changing landscapes. Here, we intensively surveyed seven 36 km2 landscapes to empirically investigate the effects of forest loss and landscape configuration on the structure of plant-pollinator networks in understory vegetation of Brazilian Atlantic Forest. Our results indicate that forest loss and isolation affect the structure of the plant-pollinator networks, which were smaller in deforested landscapes, and less specialized as patch isolation increased. Lower nestedness and degree of specialization (H'2) indicated that the remaining plant and bee species tend to be generalists, and many of the expected specialized interactions in the network were already lost. Because generalist species generate a cohesive interaction core in these networks, these simplified networks might be resistant to loss of peripheral species, but may be susceptible to the extinction of the most generalist species. We suggest that such a network pattern is an outcome of landscapes with a few remaining isolated patches of natural habitat. Our results add a new perspective to studies of plant-pollinator networks in fragmented landscapes, showing that those interaction networks might also be used to indicate how changes in natural habitat affect biodiversity and biotic interactions.

Close proximity to Igh is a contributing factor to AID-mediated translocations.

Class switch recombination (CSR) has the potential to generate genomic instability in B cells as activation-induced cytidine deaminase (AID), which mediates this process, is known to target many sites outside Igh. Nonetheless we do not fully understand what factors influence AID targeting genome-wide. Given that errors in CSR can lead to dangerous, oncogenic chromosomal translocations it is important to identify the elements that determine which genes are at risk of being "hit" and could be involved in aberrant rearrangements. Here we have investigated the influence of nuclear organization in determining "off-target" activity and the choice of fusion partners. Our studies indicate that the vast majority of known AID-mediated Igh translocation partners are found in chromosomal domains that contact this locus during class switching. Further, these interaction domains can be used to identify other genes that are hit by AID.

Cold-Water Immersion Has No Effect on Muscle Stiffness After Exercise-Induced Muscle Damage.

OBJECTIVE: To analyze the effect of cryotherapy on muscle stiffness after exercise-induced muscle damage. DESIGN: A leg-to-leg comparison model. SETTING: University research laboratory. PARTICIPANTS: Thirty (30) untrained men (21.1 ± 1.6 years, 177.6 ± 6.4 cm, 75.9 ± 10.0 kg, and 15.9 ± 2.9% fat mass) with no history of lower-limb injury and no experience in resistance training. INTERVENTION: All participants underwent a plyometric exercise program to induce muscle damage; however, randomly, one leg was assigned to a treatment condition and subjected twice to cold-water immersion of the lower limb at 10°C (±1°C) for 10 minutes, while the other leg was assigned to control. MAIN OUTCOMES MEASURES: Longitudinal stiffness and passive transverse stiffness were evaluated on the soleus and gastrocnemius muscles at 4 moments: pre-exercise, immediately after exercise, 24 hours, and 72 hours after the damage protocol. Furthermore, pressure pain threshold (PPT) and maximal voluntary isometric contraction (MVIC) were also assessed in the same periods. RESULTS: No significant differences between control and cryotherapy were observed in regard to MVIC (P = 0.529), passive longitudinal stiffness (P = 0.315), and passive transverse stiffness (P = 0.218). Only a significant decrease was observed in PPT on the soleus muscle in the cryotherapy compared with the control leg immediately after exercise (P = 0.040). CONCLUSIONS: The results show that cryotherapy had no influence on muscle stiffness. However, cryotherapy had a positive effect on PPT immediately after exercise.

Chemical Composition and Antibacterial Activity of Lippia multiflora Moldenke Essential Oil from Different Regions of Angola.

The purpose of the study was to determine the chemical composition and antibacterial activity of Lippia multiflora Moldenke essential oils (EOs) collected in different regions of Angola. Antibacterial activity was evaluated using the agar wells technique and vapour phase test. Analysis of the oils by GC/MS identified thirty-five components representing 67.5 to 100% of the total oils. Monoterpene hydrocarbons were the most prevalent compounds, followed by oxygenated monoterpenes. The content of the compounds varied according to the samples. The main components were Limonene, Piperitenone, Neral, Citral, Elemol, p-cymene, Transtagetone, and Artemisia ketone. Only one of the eleven samples contained Verbenone as the majority compound. In the vapour phase test, a single oil was the most effective against all the pathogens studied. The principal component analysis (PCA) and hierarchical cluster analysis (HCA) of components of the selected EOs and inhibition zone diameter values of agar wells technique allowed us to identify a variability between the plants from the two provinces, but also intraspecific variability between sub-groups within a population. Each group of essential oils constituted a chemotype responsible for their bacterial inhibition capacity. The results presented here suggest that Angolan Lippia multiflora Moldenke has antibacterial properties and could be a potential source of antimicrobial agents for the pharmaceutical and food industry.

Determination of antipsychotic drugs in oral fluid using dried saliva spots by gas chromatography-tandem mass spectrometry.

The present work describes the optimization and validation of an analytical method for the determination of six antipsychotic drugs (chlorpromazine, levomepromazine, cyamemazine, clozapine, haloperidol, and quetiapine) in oral fluid samples after solvent extraction from dried saliva spots, by gas chromatography coupled to tandem mass spectrometry. The method was fully validated, and the included parameters were selectivity, linearity, limits of quantification, precision and accuracy, stability, and recovery. The method was linear for all compounds from 10 to 400 ng/mL, except for haloperidol (5-100 ng/mL), presenting coefficients of determination higher than 0.99. Inter- and intra-day precision and accuracy were in conformity with the criteria usually seen in bioanalytical method validation; i.e., coefficients of variation were lower than 15% and an accuracy of 15% or better for all studied drugs. The recoveries obtained with this miniaturized technique ranged from 63 to 97%. The herein described method is the first to be reported using the dried saliva spots approach for the analysis of these antypshychotic drugs, proving great sensitivity apart from its simple and fast procedure. The method was considered a good alternative to the conventional techniques to be applied in clinical and toxicological analyses, even more taking into account the extremely low sample volume used (50 µL). Graphical abstract.

The phenotypic relationship between residual intake and gain and other feed efficiency traits in Nellore cattle.

This study aimed to compare feed efficiency measures of Nellore beef cattle on different residual intake and gain (RIG) classes. We used data from 610 animals weighing on average 236.33 kg and average of 283 days of age from feedlot performance tests carried out between 2005 and 2012. Animals were grouped based on RIG into three different classes: high RIG (> mean + 0.5 standard deviation (SD), most efficient; n = 193), medium RIG (mean ± 0.5 SD; n = 235), and low RIG (< mean - 0.5 SD, least efficient; n = 182). Residual feed intake (RFI), residual gain (RG), feed conversion ratio (FCR), feed efficiency (FE), relative growth rate (RGR), and Kleiber ratio (KR) of animals in each RIG class were compared by Tukey test at 1% of probability. Phenotypic correlations between variables were evaluated as well. Animals on high RIG class showed lower dry matter intake (P < 0.01) and higher average daily gain (P < 0.01) than low RIG animals. Consequently, high RIG animals had lower FCR (P < 0.01) and higher FE (P < 0.01) than those animals in low RIG class. The most efficient animals based on RIG were also the most efficient animals based on RG and RFI. RIG was negatively correlated to dry matter intake (P < 0.01) and FCR (P < 0.01), and a positive correlation was found between RIG and FE (P < 0.01). Therefore, RIG appears to be a good parameter to select animals with reduced dry matter intake and high productive performance.

Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions.

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3'Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.