Building the brain mosaic: an expanded view.

The complexity of the brain is closely tied to its nature as a genetic mosaic, wherein each cell is distinguished by a unique constellation of somatic variants that contribute to functional and phenotypic diversity. Postzygotic variation arising during neurogenesis is recognized as a key contributor to brain mosaicism; however, recent advances have broadened our understanding to include sources of neural genomic diversity that develop throughout the entire lifespan, from embryogenesis through aging. Moving beyond the traditional confines of neurodevelopment, in this review, we delve into the complex mechanisms that enable various origins of brain mosaicism.

TCTP regulates genotoxic stress and tumorigenicity via intercellular vesicular signaling.

Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp-/f-), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53-/- mice are prone to tumor formation, we derived tumor cells from Trp53-/-;Tctp-/f- double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53-/-;Tctp-/f- mice show highly prolonged survival. Treatment of Trp53-/- mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity.

Restoration of virulence in the attenuated Candid#1 vaccine virus requires reversion at both positions 168 and 427 in the envelope glycoprotein GPC.

Live-attenuated virus vaccines provide long-lived protection against viral disease but carry inherent risks of residual pathogenicity and genetic reversion. The live-attenuated Candid#1 vaccine was developed to protect Argentines against lethal infection by the Argentine hemorrhagic fever arenavirus, Junín virus. Despite its safety and efficacy in Phase III clinical study, the vaccine is not licensed in the US, in part due to concerns regarding the genetic stability of attenuation. Previous studies had identified a single F427I mutation in the transmembrane domain of the Candid#1 envelope glycoprotein GPC as the key determinant of attenuation, as well as the propensity of this mutation to revert upon passage in cell culture and neonatal mice. To ascertain the consequences of this reversion event, we introduced the I427F mutation into recombinant Candid#1 (I427F rCan) and investigated the effects in two validated small-animal models: in mice expressing the essential virus receptor (human transferrin receptor 1; huTfR1) and in the conventional guinea pig model. We report that I427F rCan displays only modest virulence in huTfR1 mice and appears attenuated in guinea pigs. Reversion at another attenuating locus in Candid#1 GPC (T168A) was also examined, and a similar pattern was observed. By contrast, virus bearing both revertant mutations (A168T+I427F rCan) approached the lethal virulence of the pathogenic Romero strain in huTfR1 mice. Virulence was less extreme in guinea pigs. Our findings suggest that genetic stabilization at both positions is required to minimize the likelihood of reversion to virulence in a second-generation Candid#1 vaccine.IMPORTANCELive-attenuated virus vaccines, such as measles/mumps/rubella and oral poliovirus, provide robust protection against disease but carry with them the risk of genetic reversion to the virulent form. Here, we analyze the genetics of reversion in the live-attenuated Candid#1 vaccine that is used to protect against Argentine hemorrhagic fever, an often-lethal disease caused by the Junín arenavirus. In two validated small-animal models, we find that restoration of virulence in recombinant Candid#1 viruses requires back-mutation at two positions specific to the Candid#1 envelope glycoprotein GPC, at positions 168 and 427. Viruses bearing only a single change showed only modest virulence. We discuss strategies to genetically harden Candid#1 GPC against these two reversion events in order to develop a safer second-generation Candid#1 vaccine virus.

Conversion or reversion of interferon gamma release assays for Mycobacterium tuberculosis infection: a systematic review and meta-analysis.

BACKGROUND: Interferon-gamma release assays (IGRA) are widely used for diagnosis of latent tuberculosis infection. However, with repeat testing, IGRA transformation (conversion or reversion) may be detected and is challenging to interpret. We reviewed the frequency of and risk factors for IGRA transformation. METHODS: We screened public databases for studies of human participants that reported the frequency of IGRA transformation. We extracted study and subject characteristics, details of IGRA testing and results. We calculated the pooled frequency of IGRA transformation (and transient transformation) and examined associated risk factors. RESULTS: The pooled frequency of IGRA conversion or reversion from 244 studies was estimated at 7.3% (95% CI 6.1-8.5%) or 22.8% (20.1-25.7%), respectively. Transient conversion or reversion were estimated at 46.0% (35.7-56.4%) or 19.6% (9.2-31.7%) of conversion or reversion events respectively. Indeterminate results seldom reverted to positive (1.2% [0.1-3.5%]). IGRA results in the borderline positive or negative range were associated with increased risk of conversion or reversion (pooled OR: conversion, 4.15 [3.00-5.30]; reversion, 4.06 [3.07-5.06]). BCG vaccination was associated with decreased risk of conversion (0.70, 0.56-0.84), cigarette smoking with decreased risk of reversion (0.44, 0.06-0.82), and female sex with decreased risk of either conversion or reversion (conversion, 0.66 [0.58-0.75]; reversion, 0.46 [0.31-0.61]). CONCLUSIONS: IGRA conversion is less common than reversion, and frequently transient. Research is needed to determine whether individuals with reversion would benefit from tuberculosis preventive treatment. Re-testing of people with indeterminate results is probably not indicated, since indeterminate results seldom revert to positive.

Phytochrome C and Low Temperature Promote the Protein Accumulation and Red Light Signaling of Phytochrome D.

Light affects almost every aspect of plant development. It is perceived by photoreceptors, among which phytochromes (PHY) are responsible for monitoring the red and far-red spectrum. Arabidopsis thaliana possesses five phytochrome genes (phyA-E). Whereas functions of phyA and phyB are extensively studied, our knowledge on other phytochromes is still rudimentary. To analyze phyD function we expressed it at high levels in different phytochrome-deficient genetic backgrounds. Overexpressed phyD-YFP can govern effective light signaling but only at low temperature and in cooperation with functional phyC. Under these conditions, phyD-YFP accumulates to high levels and opposite to phyB, this pool is stable in light. By comparing the photoconvertible phyD-YFP and phyB levels and their signaling in continuous and pulsed irradiation, we showed that phyD-YFP is a less efficient photoreceptor than phyB. This conclusion is supported by the facts that only a part of the phyD-YFP pool is photoconvertible and thermal reversion of phyD-YFP is faster than that of phyB. Our data suggest that the temperature-dependent function of phyD is based on the amount of phyD protein and not on its Pfr stability, as described for phyB. We also found that phyD-YFP and phyB-GFP associate with strongly overlapping genomic locations and mediate similar changes in gene expression, however the efficiency of phyD-YFP is lower. Based on these data we propose that under certain conditions, synergistic interaction of phyD and phyC can substitute phyB function in seedlings and in adult plants, thus increases the ability of plants to respond more flexibly to environmental changes.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Phytochrome B phosphorylation expanded: site-specific kinases are identified.

The phytochrome B (phyB) photoreceptor is a key participant in red and far-red light sensing, playing a dominant role in many developmental and growth responses throughout the whole life of plants. Accordingly, phyB governs diverse signaling pathways, and although our knowledge about these pathways is constantly expanding, our view about their fine-tuning is still rudimentary. Phosphorylation of phyB is one of the relevant regulatory mechanisms, and - despite the expansion of the available methodology - it is still not easy to examine. Phosphorylated phytochromes have been detected using various techniques for decades, but the first phosphorylated phyB residues were only identified in 2013. Since then, concentrated attention has been turned toward the functional role of post-translational modifications in phyB signaling. Very recently in 2023, the first kinases that phosphorylate phyB were identified. These discoveries opened up new research avenues, especially by connecting diverse environmental impacts to light signaling and helping to explain some long-term unsolved problems such as the co-action of Ca2+ and phyB signaling. This review summarizes our recent views about the roles of the identified phosphorylated phyB residues, what we know about the enzymes that modulate the phospho-state of phyB, and how these recent discoveries impact future investigations.

Effect of erenumab on the reversion from chronic migraine to episodic migraine in an Asian population: A post hoc analysis of the DRAGON study.

BACKGROUND: Erenumab is a fully human monoclonal antibody that selectively targets the calcitonin gene-related peptide receptor. It has been proven to be safe and efficacious in patients with episodic migraine (EM) and chronic migraine (CM) as demonstrated in phase 2 and 3 clinical trials including patients from Europe, Japan, and the United States. Reversion from CM to EM, as indicated by a reduction in the frequency of headache days, is an important indicator for efficacy outcome, though it has not been analyzed widely in patients with CM to date. OBJECTIVE: Primary results of the DRAGON study demonstrated the efficacy and safety of erenumab in patients with CM from China and other Asian countries. This post hoc analysis evaluated the rate of reversion from CM to EM in the overall population and in subgroups of patients defined by baseline demographic and clinical characteristics (age, body mass index, gender, prior preventive treatment failure, medication overuse status, and disease duration). METHODS: Reversion from CM to EM was defined as a reduction in headache frequency to < 45 headache days over the 12 weeks of the double-blind treatment period. In addition, migraine-related disability and disease impact on functional impairment were assessed within each treatment group in reverters and non-reverters using the Headache Impact Test-6 (HIT-6), Migraine Physical Function Impact Diary (MPFID), and modified Migraine Disability Assessment (mMIDAS). RESULTS: Overall, 557 patients with CM were randomized to monthly erenumab 70 mg (n = 279) or placebo (n = 278), of whom 52.3% (146 of 279) treated with erenumab reverted from CM to EM compared to 41.0% (114 of 278) in the placebo group (odds ratio [OR] 1.59, 95% confidence interval: 1.1-2.2; p = 0.007). Treatment with erenumab resulted in a greater mean change (standard error) from baseline in the HIT-6 total score for reverters versus non-reverters compared to placebo (erenumab: -9.5 [0.6] vs. -5.1 [0.5]; placebo: -8.9 [0.7] vs. -4.9 [0.5]). A similar pattern was observed for mMIDAS score in erenumab treatment groups versus placebo (erenumab: -22.1 [1.2] vs. -6.3 [1.8]; placebo: -19.9 [1.3] vs. -7.9 [1.6]). Substantial improvements were reported in MPFID-Physical Impairment (PI) and Everyday Activities (EA) scores in reverters versus non-reverters in erenumab treatment groups (MPFID-PI: -5.9 [0.3] vs. -1.9 [0.6]; MPFID-EA: -7.9 [0.4] vs. -3.4 [0.6]) and in placebo (MPFID-PI: -5.4 [0.4] vs. -1.0 [0.5]; MPFID-EA: -7.1 [0.5] vs. -3.2 [0.5]). CONCLUSIONS: This analysis demonstrated that a greater proportion of patients treated with erenumab reverted from CM to EM compared to patients treated with placebo. The reversion from CM to EM was reflected by the greater improvements in patient-reported outcomes in the erenumab group.

Harnessing the Photoperformance of N-Methyl-Quinolinone for Gated Photo-Driven Cyclability and Reversible Photoligation.

[2π + 2π]-photocycloadditions and their ability to trigger controlled and reversible photoligation through disparate wavelengths provide an attractive platform to unlock advanced functionalities in soft materials. Yet, among the limited amount of functional motifs enabling reversible photoreactions, cyclability is often overlooked due to poor reaction yield and orthogonality. In this study, the advantageous photocharacteristics of the previously underexplored N-methyl-quinolinone photoresponsive motif are leveraged to create a covalent gated system, enabling controlled formation and cleavage of covalent bonds on demand. A systematic evaluation of individual cycloadditions and reversions on the molecular scale, including reaction rates, conversions, and photoproducts, allows identification of the required conditions for generating controlled photoreactions with a remarkable degree of cyclability; while, maintaining high reaction yields. Ultimately, these controlled and cyclable reactions are translated to a macromolecular scale, showcasing a comparable performance in initiating reversible photoligation, as observed at the molecular level. In addition, it is also shown that this progressive methodology can be leveraged to gain a comprehensive understanding of cyclability and clarify the factors contributing to its decreasing yield. Overall, unlocking the potential of quinolinone derivatives through this step-by-step approach lays the foundation for the development of highly controlled and responsive polymer materials with unprecedented potential.

Directed energy deposition of 18NiM300 steel: effect of process and post processing conditions on microstructure and properties.

This current study investigates the effect of Direct Energy Deposition (DED) process conditions on the properties and microstructure of M300 maraging steel samples. The investigation centers on two key factors: laser power and deposition environment. The microstructure of this tool steel is analyzed by computing the Primary Cellular Arm Spacing. The findings revealed a significant influence of both inert atmosphere and laser power on cooling conditions. These different cooling rates influence the phase content as demonstrated by X-Ray Diffraction and Electron Backscatter Diffraction measurements. It was demonstrated the presence of different content of residual austenite at cell boundaries. These distinct microstructural features caused variations in the hardness values of the printed samples. Furthermore, a direct aging heat treatment was implemented, that was chosen from Differential Scanning Calorimetry measurements results. This heat treatment proves effective in achieving consistent hardness increases and eliminated the differences among samples built in different process conditions. This outcome suggests the possibility of selecting the most economically viable DED parameters for optimal results.

This study innovatively explores how DED process conditions impact M300 maraging steel microstructure, revealing crucial insights for optimizing properties and achieving consistent results, ensuring economically viable applications.

Predictors and outcomes of fluctuations in the clinical dementia rating scale.

INTRODUCTION: Reversion, or change in cognitive status from impaired to normal, is common in aging and dementia studies, but it remains unclear what factors predict reversion. METHODS: We investigated whether reverters, defined as those who revert from a Clinical Dementia Rating® (CDR®) scale score of 0.5 to CDR 0) differed on cognition and biomarkers from unimpaired participants (always CDR 0) and impaired participants (converted to CDR > 0 and had no reversion events). Models evaluated relationships between biomarker status, apolipoprotein E (APOE) ε4 status, and cognition. Additional models described predictors of reversion and predictors of eventual progression to CDR > 0. RESULTS: CDR reversion was associated with younger age, better cognition, and negative amyloid biomarker status. Reverters that eventually progressed to CDR > 0 had more visits, were older, and were more likely to have an APOE ε4 allele. DISCUSSION: CDR reversion occupies a transitional phase in disease progression between cognitive normality and overt dementia. Reverters may be ideal candidates for secondary prevention Alzheimer's disease (AD) trials. HIGHLIGHTS: Reverters had more longitudinal cognitive decline than those who remained cognitively normal. Predictors of reversion: younger age, better cognition, and negative amyloid biomarker status. Reverting from CDR 0.5 to 0 is a risk factor for future conversion to CDR > 0. CDR reversion may be a transitional phase in Alzheimer's Disease progression. CDR reverters may be ideal for Alzheimer's disease secondary prevention trials.

Reversed Corneal Fibroblasts Therapy Restores Transparency of Mouse Cornea after Injury.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Darwin and the White Shipwrecked Sailor: Beyond Blending Inheritance and the Jenkin Myth.

This paper revisits Fleeming Jenkin's anonymous review of Charles Darwin's Origin of Species, published in the North British Review in June 1867. This review is usually revered for its impact on Darwin's theory of descent with modification. Its classical interpretation states that Jenkin, a Professor of Engineering at the University of Edinburgh, made a compelling case against natural selection based on the fact of "blending inheritance" and the "swamping" of advantageous variations. Those themes, however, are strikingly absent from Jenkin's text. They were later read into Jenkin's text by scholars trying to explain how Darwinian selection was reconciled with Mendelian genes and the birth of the Modern Synthesis. While many scholars have tried to measure Jenkin's effect on Darwin, the value of the 1867 review remains unclear. This paper re-examines its content and concludes that Jenkin's "able review" was in fact written by an engineer whose competencies in biology were very low. Focusing on the figure of the shipwrecked white sailor isolated on an island inhabited by Black people, this paper also underlines the racial assumptions behind Jenkin's review. "Blending inheritance" is thus a theme linked to theoretical reworkings on the question of race and skin colors, taking its root in Galton's typology of heredity. Darwin was probably mostly unimpressed by Jenkin's review. The problems raised by the review were not so much "blending inheritance" and "swamping" but a conundrum of problems related to the effects of intercrossing on variation and reversion.

Recent Updates on the Therapeutic Prospects of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs (RECK) in Liver Injuries.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored glycoprotein, negatively regulates various membrane proteins involved in the tissue governing extracellular matrix (ECM) remodeling such as metalloproteases (MMPs) and the sheddases ADAM10 and ADAM17. The significance of the present review is to summarize the current understanding of the pathophysiological role of RECK, a newly discovered signaling pathway associated with different liver injuries. Specifically, this review analyzes published data on the downregulation of RECK expression in hepatic ischemia/reperfusion (I/R) injury, liver-related cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as in the progression of nonalcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). In addition, this review discusses the regulation of RECK by inducers, such as FXR agonists. The RECK protein has also been suggested as a potential diagnostic and prognostic marker for liver injury or as a biomarker with predictive value for drug treatment efficacy.

Predicting the reversion from mild cognitive impairment to normal cognition based on magnetic resonance imaging, clinical, and neuropsychological examinations.

BACKGROUND: Reversion from mild cognitive impairment (MCI) to normal cognition (NC) is not uncommon and indicates a better cognitive trajectory. This study aims to identify predictors of MCI reversion and develop a predicting model. METHOD: A total of 391 MCI subjects (mean age = 74.3 years, female = 61 %) who had baseline data of magnetic resonance imaging, clinical, and neuropsychological measurements were followed for two years. Multivariate logistic analyses were used to identify the predictors of MCI reversion after adjusting for age and sex. A stepwise backward logistic regression model was used to construct a predictive nomogram for MCI reversion. The nomogram was validated by internal bootstrapping and in an independent cohort. RESULT: In the training cohort, the 2-year reversion rate was 19.95 %. Predictors associated with reversion to NC were higher education level (p = 0.004), absence of APOE4 allele (p = 0.001), larger brain volume (p < 0.005), better neuropsychological measurements performance (p < 0.001), higher glomerular filtration rate (p = 0.035), and lower mean arterial pressure (p = 0.060). The nomogram incorporating five predictors (education, hippocampus volume, the Alzheimer's Disease Assessment Scale-Cognitive score, the Rey Auditory Verbal Learning Test-immediate score, and mean arterial pressure) achieved good C-indexes of 0.892 (95 % confidence interval [CI], 0.859-0.926) and 0.806 (95 % CI, 0.709-0.902) for the training and validation cohort. LIMITATION: Observational duration is relatively short; The predicting model warrant further validation in larger samples. CONCLUSION: This prediction model could facilitate risk stratification and early management for the MCI population.

The phenotypic reversion of cancer: Experimental evidences on cancer reversibility through epigenetic mechanisms (Review).

Different experimental models reveal that malignant cancer cells can be induced to change their phenotype into a benign one. This phenotypic transformation, confirmed both in vitro and in vivo, currently is known as 'tumor reversion'. This evidence raises a radical question among current cancer models: Is cancer reversible? How do genetic and epigenetic alterations hierarchically relate? Understanding the mechanisms of 'tumor reversion' represents a key point in order to evolve the actual cancer models and develop new heuristic models that can possibly lead to drugs that target epigenetic mechanisms, for example epigenetic drugs. Even though evidence of tumor reversion dates back to the 1950s, this remains a completely new field of research recently re­discovered thanks to the interest in cell reprogramming research, developmental biology and the increasing understanding of epigenetic mechanisms. In the current review, a comprehensive review of all the main experimental models on tumor reversion was presented.

Interaction between Aß and tau on reversion and conversion in mild cognitive impairment patients: After 2-year follow-up.

Background: The role of amyloid-ß (Aß) and tau in reversion and conversion in patients with mild cognitive impairment (MCI) remains unclear. This study aimed to investigate the influence of cerebrospinal fluid (CSF) Aß and tau on reversion and conversion and the temporal sequence of their pathogenicity in MCI patients. Methods: 179 MCI patients were recruited from the Alzheimer's Disease Neuroimaging Initiative database and classified into two groups based on cognitive changes after follow-up: reversal group (MCI to cognitively normal) and conversion group (MCI to Alzheimer's disease). CSF biomarkers and cognitive function were measured at baseline and 2-year follow-up. Partial correlation was used to analyze the association between CSF biomarkers and cognitive function, and multivariable logistic regression to identify independent risk factors for cognitive changes at baseline and 2-year follow-up. Receiver operating characteristic (ROC) curves were utilized to evaluate the predictive ability of these risk factors for cognitive changes. Results: The differences in cognitive function and CSF biomarkers between the two groups remained consistent with baseline after 2-year follow-up. After controlling for confounding variables, there was still a correlation between CSF biomarkers and cognitive function at baseline and 2-year follow-up. Multivariable regression analysis found that at baseline, only Aß level was independently associated with cognitive changes, while Aß and tau were both predictive factors after 2-year follow-up. ROC curve analysis revealed that the combination of Aß and tau [area under the curve (AUC) 0.91, sensitivity 84%, specificity 86%] in predicting cognitive changes after 2-year follow-up had better efficacy than baseline Aß alone (AUC 0.81). Conclusion: Aß may precede Tau in causing cognitive changes, and the interaction between the two mediates cognitive changes in patients. This study provides new clinical evidence to support the view that Aß pathology precedes tau pathology, which together contribute to the changes in cognitive function.

Measurement of Phytochrome B Thermal Reversion Rates In Vivo.

Thermal reversion of phytochromes is the light-independent but strongly temperature-dependent relaxation of the light-activated Pfr form of phytochromes back into the inactive Pr ground state. The thermal reversion rates of different phytochromes vary considerably. For phytochrome B (phyB), thermal reversion represents a critical parameter affecting phyB activity as it reduces the active phyB Pfr pool, accelerated by increasing temperatures. Phytochromes are dimers existing in three different states: Pfr-Pfr homodimer, Pfr-Pr heterodimer, and Pr-Pr homodimer. Consequently, thermal reversion occurs in two steps, with Pfr-Pfr to Pfr-Pr reversion being much slower than reversion from Pfr-Pr to Pr-Pr. To measure thermal reversion in vivo, the relative proportion of Pfr in relation to the total amount of phytochrome (Ptot) must be determined in living samples. This is accomplished by in vivo spectroscopy utilizing dual wavelength ratiospectrophotometers, optimized for assaying phytochromes in highly scattering plant material. The method is depending on the photoreversibility of phytochromes displaying light-induced absorbance changes in response to actinic irradiation. In this chapter, we describe the experimental design and explain step-by-step the calculations necessary to determine the thermal reversion rates of phyB in vivo, taking into account phytochrome dimerization.

QuantiFERON-TB reversion in children and adolescents with tuberculosis.

We analyzed 136 children with tuberculosis disease or infection and a positive QuantiFERON-TB (QFT) assay, followed-up for a median of 21 months (0.4-11years). QFT reversed in 16.9% of cases, with significant decreases in TB1 (-1.72 vs. -0.03 IU/ml, p=0.001) and TB2 (-1.65 vs. -0.43 IU/ml, p=0.005) levels compared to non-reverters. We found a higher QFT reversion rate among children under 5 years (25.0% vs 11.9%, p=0.042), and those with TST induration <15mm (29% vs 13.3%, p=0.055). Our data reveal that, although QFT test remained positive in the majority of children, reversion occurred in 16% of cases in a progressive and stable pattern. Younger age and reduced TST induration were associated with QFT reversion.

Gradual ER calcium depletion induces a progressive and reversible UPR signaling.

The unfolded protein response (UPR) is a widespread signal transduction pathway triggered by endoplasmic reticulum (ER) stress. Because calcium (Ca2+) is a key factor in the maintenance of ER homeostasis, massive Ca2+ depletion of the ER is a potent inducer of ER stress. Although moderate changes in ER Ca2+ drive the ubiquitous Ca2+ signaling pathways, a possible incremental relationship between UPR activation and Ca2+ changes has yet to be described. Here, we determine the sensitivity and time-dependency of activation of the three ER stress sensors, inositol-requiring protein 1 alpha (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 alpha (ATF6α) in response to controlled changes in the concentration of ER Ca2+ in human cultured cells. Combining Ca2+ imaging, fluorescence recovery after photobleaching experiments, biochemical analyses, and mathematical modeling, we uncover a nonlinear rate of activation of the IRE1α branch of UPR, as compared to the PERK and ATF6α branches that become activated gradually with time and are sensitive to more important ER Ca2+ depletions. However, the three arms are all activated within a 1 h timescale. The model predicted the deactivation of PERK and IRE1α upon refilling the ER with Ca2+. Accordingly, we showed that ER Ca2+ replenishment leads to the complete reversion of IRE1α and PERK phosphorylation in less than 15 min, thus revealing the highly plastic character of the activation of the upstream UPR sensors. In conclusion, our results reveal a dynamic and dose-sensitive Ca2+-dependent activation/deactivation cycle of UPR induction, which could tightly control cell fate upon acute and/or chronic stress.