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The unique property of the pulmonary circulation to constrict in response to hypoxia, rather than dilate, brings advantages in both health and disease. Hypoxic pulmonary vasoconstriction (HPV) acts to optimise ventilation-perfusion matching - this is important clinically both in focal disease (such as pneumonia) and in one-lung ventilation during anaesthesia for thoracic surgery. However, during global hypoxia such as that encountered at high altitude, generalised pulmonary vasoconstriction can lead to pulmonary hypertension. There is now a growing body of evidence that links the hypoxia-inducible factor (HIF) pathway and pulmonary vascular tone - in both acute and chronic settings. Genetic and pharmacological alterations to all key components of this pathway (VHL - von Hippel-Lindau ubiquitin E3 ligase; PHD2 - prolyl hydroxylase domain protein 2; HIF1 and HIF2) have clear effects on the pulmonary circulation, particularly in hypoxia. Furthermore, knowledge of the molecular biology of the prolyl hydroxylase enzymes has led to an extensive and ongoing body of research into the importance of iron in both HPV and pulmonary hypertension. This review will explore these relationships in more detail and discuss future avenues of research., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.

Atoh1 overexpression is essential for hair cell (HC) regeneration in the sensory epithelium of mammalian auditory and vestibular organs. However, Atoh1 overexpression alone cannot induce fully mature and functional HCs in the mammalian inner ear. In the current study, we investigated the effect of Atoh1 constitutive overexpression in native HCs by manipulating Atoh1 expression at different developmental stages. We demonstrated that constitutive overexpression of Atoh1 in native vestibular HCs did not affect cell survival but did impair vestibular function by interfering with the subtype differentiation of HCs and hair bundle development. In contrast, Atoh1 overexpression in cochlear HCs impeded their maturation, eventually leading to gradual HC loss in the cochlea and hearing dysfunction. Our study suggests that time-restricted Atoh1 expression is essential for the differentiation and survival of HCs in the inner ear, and this is pivotal for both hearing and vestibular function re-establishment through Atoh1 overexpression-induced HC regeneration strategies., (© 2023. The Author(s).)

Cochlear hair cells (HCs) in the inner ear are responsible for sound detection. For HC fate specification, the master transcription factor Atoh1 is both necessary and sufficient. Atoh1 expression is dynamic and tightly regulated during development, but the cis -regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized Atoh1 enhancer, defined here as Eh1, failed to impair HC development. By using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we discovered two additional Atoh1 enhancers: Eh2 and Eh3. Notably, Eh2 deletion was sufficient for impairing HC development, and concurrent deletion of Eh1 and Eh2 or all three enhancers resulted in nearly complete absence of HCs. Lastly, we showed that Atoh1 binds to all three enhancers, consistent with its autoregulatory function. Our findings reveal that the cooperative action of three distinct enhancers underpins effective Atoh1 regulation during HC development, indicating potential therapeutic approaches for HC regeneration.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the pathogenic agent of Coronavirus-Induced Disease-2019 (COVID-19), a multi-organ syndrome which primarily targets the respiratory system. In this review, considering the large amount of data pointing out the role of the Aryl hydrocarbon Receptor (AhR) in the inflammatory response and in the modulation of innate and adaptive immunity, we describe some mechanisms that strongly suggest its involvement in the management of COVID-19's inflammatory framework. It regulates both the expression of Angiotensin Converting Enzyme-2 (ACE-2) and its stabilizing partner, the Broad neutral Amino acid Transporter 1 (B 0 AT1). It induces Indolamine 2,3 dioxygenase (IDO-1), the enzyme which, starting from Tryptophan (Trp), produces Kynurenine (Kyn, Beta-Anthraniloyl-L-Alanine). The accumulation of Kyn and the depletion of Trp arrest T cell growth and induce apoptosis, setting up an immune-tolerant condition, whereas AhR and interferon type I (IFN-I) build a mutual inhibitory loop that also involves NF-kB and limits the innate response. AhR/Kyn binding boosts the production of Interleukin-6 (IL-6), thus reinforcing the inflammatory state and counteracting the IDO-dependent immune tolerance in the later stage of COVID-19. Taken together, these data depict a framework where sufficient clues suggest the possible participation of AhR in the management of COVID-19 inflammation, thus indicating an additional therapeutic target for this disease.

bHLH family proteins play an important role in plant stress response. However, the molecular mechanism regulating the salt response of bHLH is largely unknown. This study aimed to investigate the function and regulating mechanism of the sweet sorghum SbbHLH85 during salt stress. The results showed that SbbHLH85 was different from its homologs in other species. Also, it was a new atypical bHLH transcription factor and a key gene for root development in sweet sorghum. The overexpression of SbbHLH85 resulted in significantly increased number and length of root hairs via ABA and auxin signaling pathways, increasing the absorption of Na + . Thus, SbbHLH85 plays a negative regulatory role in the salt tolerance of sorghum. We identified a potential interaction partner of SbbHLH85, which was phosphate transporter chaperone PHF1 and modulated the distribution of phosphate, through screening a yeast two-hybrid library. Both yeast two-hybrid and BiFC experiments confirmed the interaction between SbbHLH85 and PHF1. The overexpression of SbbHLH85 led to a decrease in the expression of PHF1 as well as the content of Pi. Based on these results, we suggested that the increase in the Na + content and the decrease in the Pi content resulted in the salt sensitivity of transgenic sorghum., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

NPAS2, a circadian rhythm gene encoding the neuronal PAS domain protein 2 (NPAS2), has received widespread attention because of its complex functions in cells and diverse roles in disease progression, especially tumorigenesis. NPAS2 binds with DNA at E-box sequences and forms heterodimers with another circadian protein, brain and muscle ARNT-like protein 1 (BMAL1). Nucleotide variations of the NPAS2 gene have been shown to influence the overall survival and risk of death of cancer patients, and differential expression of NPAS2 has been linked to patient outcomes in breast cancer, lung cancer, non-Hodgkin's lymphoma, and other diseases. Here, we review the latest advances in our understanding of NPAS2 with the aim of drawing attention to its potential clinical applications and prospects., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Iron (Fe) is an essential micronutrient whose availability is limiting in many soils. During Fe deficiency, plants alter the expression of many genes to increase Fe uptake, distribution, and utilization. In a genetic screen for suppressors of Fe sensitivity in the E3 ligase mutant bts-3 , we isolated an allele of the bHLH transcription factor (TF) ILR3 , ilr3-4 We identified a striking leaf bleaching phenotype in ilr3 mutants that was suppressed by limiting light intensity, indicating that ILR3 is required for phototolerance during Fe deficiency. Among its paralogs that are thought to be partially redundant, only ILR3 was required for phototolerance as well as repression of genes under Fe deficiency. A mutation in the gene-encoding PYE, a known transcriptional repressor under Fe deficiency, also caused leaf bleaching. We identified singlet oxygen as the accumulating reactive oxygen species (ROS) in ilr3-4 and pye , suggesting photosensitivity is due to a PSII defect resulting in ROS production. During Fe deficiency, ilr3-4 and pye chloroplasts retain normal ultrastructure and, unlike wild type (WT), contain stacked grana similar to Fe-sufficient plants. Additionally, we found that the D1 subunit of PSII is destabilized in WT during Fe deficiency but not in ilr3-4 and pye , suggesting that PSII repair is accelerated during Fe deficiency in an ILR3- and PYE-dependent manner. Collectively, our results indicate that ILR3 and PYE confer photoprotection during Fe deficiency to prevent the accumulation of singlet oxygen, potentially by promoting reduction of grana stacking to limit excitation and facilitate repair of the photosynthetic machinery., Competing Interests: The authors declare no competing interest.

Hypothalamic Kiss1 neurons control gonadotropin-releasing hormone release through the secretion of kisspeptin. Kiss1 neurons serve as a nodal center that conveys essential regulatory cues for the attainment and maintenance of reproductive function. Despite this critical role, the mechanisms that control kisspeptin synthesis and release remain largely unknown. Using Drop-Seq data from the arcuate nucleus of adult mice and in situ hybridization, we identified Nescient Helix-Loop-Helix 2 ( Nhlh2 ), a transcription factor of the basic helix-loop-helix family, to be enriched in Kiss1 neurons. JASPAR analysis revealed several binding sites for NHLH2 in the Kiss1 and Tac2 (neurokinin B) 5' regulatory regions. In vitro luciferase assays evidenced a robust stimulatory action of NHLH2 on human KISS1 and TAC3 promoters. The recruitment of NHLH2 to the KISS1 and TAC3 promoters was further confirmed through chromatin immunoprecipitation. In vivo conditional ablation of Nhlh2 from Kiss1 neurons using Kiss1 Cre : Nhlh2 fl/fl mice induced a male-specific delay in puberty onset, in line with a decrease in arcuate Kiss1 expression. Females retained normal reproductive function albeit with irregular estrous cycles. Further analysis of male Kiss1 Cre :Nhlh2 fl/fl mice revealed higher susceptibility to metabolic challenges in the release of luteinizing hormone and impaired response to leptin. Overall, in Kiss1 neurons, Nhlh2 contributes to the metabolic regulation of kisspeptin and NKB synthesis and release, with implications for the timing of puberty onset and regulation of fertility in male mice., Competing Interests: SL, RT, EM, KF, CF, LN, JC, RC, UK, CA, AL, VN No competing interests declared, (© 2021, Leon et al.)

Congenital heart defects (CHD) affect approximately 1% of all live births, and often require complex surgeries at birth. We have previously demonstrated abnormal placental vascularization in human placentas from fetuses diagnosed with CHD. Hand1 has roles in both heart and placental development and is implicated in CHD development. We utilized two conditionally activated Hand1 A126fs/+ murine mutant models to investigate the importance of cell-specific Hand1 on placental development in early ( Nkx2-5 Cre ) and late ( Cdh5 Cre ) pregnancy. Embryonic lethality occurred in Nkx2-5 Cre /Hand1 A126fs/+ embryos with marked fetal demise occurring after E10.5 due to a failure in placental labyrinth formation and therefore the inability to switch to hemotrophic nutrition or maintain sufficient oxygen transfer to the fetus. Labyrinthine vessels failed to develop appropriately and vessel density was significantly lower by day E12.5. In late pregnancy, the occurrence of Cdh5 Cre+ ;Hand1 A126fs/+ fetuses was reduced from 29% at E12.5 to 20% at E18.5 and remaining fetuses exhibited reduced fetal and placental weights, labyrinth vessel density and placenta angiogenic factor mRNA expression. Our results demonstrate for the first time the necessity of Hand1 in both establishment and remodeling of the exchange area beyond early pregnancy and in patterning vascularization of the placental labyrinth crucial for maintaining pregnancy and successful fetal growth.

[Figure: see text].

Lily (Lilium spp.), with its beautiful flower, is an important horticultural crop and a popular ornamental plant, but because the abundant pollen pollutes the flowers and surroundings, its use is restricted. To solve this problem, the mechanism of pollen development in lily needs to be analyzed. However, the complex and delicate process of anther development in lily remains largely unknown. In this study, LoUDT1, a bHLH transcription factor (TF), was isolated and identified in lily. LoUDT1 was closely related to OsUDT1 of Oryza sativa and AtDYT1 of Arabidopsis. It was localized in the cytoplasm and nucleus and showed no transcriptional activation in yeast cells. LoUDT1 interacted with another bHLH TF, LoAMS, and the interaction depended on their BIF domains. LoUDT1 and LoAMS were both expressed in the anthers but showed different expression patterns. LoUDT1 was continuously expressed during the entire development of anthers, whereas LoAMS was only highly expressed early in anther development. With overexpression of LoUDT1 in Arabidopsis, normal anther development was affected and defective pollens were produced, which caused partial male sterility of transgenic plants. These defects depended on the level of LoUDT1 accumulation. By contrast, with the appropriate expression of LoUDT1 in a dyt1-3 mutant, normal pollen grains were produced, showing partial fertility. Thus, LoUDT1 might be a key regulator of anther development in lily. By further increasing the understanding of anther development, the results of this study can provide a theoretical basis for the molecular breeding of pollen-free lilies., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Erythropoietin (Epo) is the critical hormone for erythropoiesis. In adults, Epo is mainly produced by a subset of interstitial fibroblasts in the kidney, with minor amounts being produced in the liver and the brain. In this study, we used the immortalized renal interstitial fibroblast cell line FAIK F3-5 to investigate the ability of the bioactive sphingolipid sphingosine 1-phosphate (S1P) to stimulate Epo production and to reveal the mechanism involved. Stimulation of cells with exogenous S1P under normoxic conditions (21% O 2 ) led to a dose-dependent increase in Epo mRNA and protein levels and subsequent release of Epo into the medium. S1P also enhanced the stabilization of HIF-2α, a key transcription factor for Epo expression. S1P-stimulated Epo mRNA and protein expression was abolished by HIF-2α mRNA knockdown or by the HIF-2 inhibitor compound 2. Furthermore, the approved S1P receptor modulator FTY720, and its active form FTY720-phosphate, both exerted a similar effect on Epo expression as S1P. The effect of S1P on Epo was antagonized by the selective S1P 1 and S1P 3 antagonists NIBR-0213 and TY-52156, but not by the S1P 2 antagonist JTE-013. Moreover, inhibitors of the classical MAPK/ERK, the p38-MAPK, and inhibitors of protein kinase (PK) C and D all blocked the effect of S1P on Epo expression. Finally, the S1P and FTY720 effects were recapitulated in the Epo-producing human neuroblastoma cell line Kelly, suggesting that S1P receptor-dependent Epo synthesis is of general relevance and not species-specific. In summary, these data suggest that, in renal interstitial fibroblasts, which are the primary source of plasma Epo, S1P 1 and 3 receptor activation upregulates Epo under normoxic conditions. This may have a therapeutic impact on disease situations such as chronic kidney disease, where Epo production is impaired, causing anemia, but it may also have therapeutic value as Epo can mediate additional tissue-protective effects in various organs.

The physiologically hypoxic intervertebral disc and cartilage rely on the hypoxia-inducible factor (HIF) family of transcription factors to mediate cellular responses to changes in oxygen tension. During homeostatic development, oxygen-dependent prolyl hydroxylases, circadian clock proteins and metabolic intermediates control the activities of HIF1 and HIF2 in these tissues. Mechanistically, HIF1 is the master regulator of glycolytic metabolism and cytosolic lactate levels. In addition, HIF1 regulates mitochondrial metabolism by promoting flux through the tricarboxylic acid cycle, inhibiting downsteam oxidative phosphorylation and controlling mitochondrial health through modulation of the mitophagic pathway. Accumulation of metabolic intermediates from HIF-dependent processes contribute to intracellular pH regulation in the disc and cartilage. Namely, to prevent changes in intracellular pH that could lead to cell death, HIF1 orchestrates a bicarbonate buffering system in the disc, controlled by carbonic anhydrase 9 (CA9) and CA12, sodium bicarbonate cotransporters and an intracellular H + /lactate efflux mechanism. In contrast to HIF1, the role of HIF2 remains elusive; in disorders of the disc and cartilage, its function has been linked to both anabolic and catabolic pathways. The current knowledge of hypoxic cell metabolism and regulation of HIF1 activity provides a strong basis for the development of future therapies designed to repair the degenerative disc.

Background: Identifying genes involved in salt tolerance in the recretohalophyte Limonium bicolor could facilitate the breeding of crops with enhanced salt tolerance. Here we cloned the previously uncharacterized gene LbHLH and explored its role in salt tolerance., Results: The 2,067-bp open reading frame of LbHLH encodes a 688-amino-acid protein with a typical helix-loop-helix (HLH) domain. In situ hybridization showed that LbHLH is expressed in salt glands of L. bicolor. LbHLH localizes to the nucleus, and LbHLH is highly expressed during salt gland development and in response to NaCl treatment. To further explore its function, we heterologously expressed LbHLH in Arabidopsis thaliana under the 35S promoter. The overexpression lines showed significantly increased trichome number and reduced root hair number. LbHLH might interact with GLABRA1 to influence trichome and root hair development, as revealed by yeast two-hybrid analysis. The transgenic lines showed higher germination percentages and longer roots than the wild type under NaCl treatment. Analysis of seedlings grown on medium containing sorbitol with the same osmotic pressure as 100 mM NaCl demonstrated that overexpressing LbHLH enhanced osmotic resistance., Conclusion: These results indicate that LbHLH enhances salt tolerance by reducing root hair development and enhancing osmotic resistance under NaCl stress.

The hypocotyls of germinating seedlings elongate in a search for light to enable autotrophic sugar production. Upon exposure to light, photoreceptors that are activated by blue and red light halt elongation by preventing the degradation of the hypocotyl-elongation inhibitor HY5 and by inhibiting the activity of the elongation-promoting transcription factors PIFs. The question of how sugar affects hypocotyl elongation and which cell types stimulate and stop that elongation remains unresolved. We found that overexpression of a sugar sensor, Arabidopsis hexokinase 1 (HXK1), in guard cells promotes hypocotyl elongation under white and blue light through PIF4. Furthermore, expression of PIF4 in guard cells is sufficient to promote hypocotyl elongation in the light, while expression of HY5 in guard cells is sufficient to inhibit the elongation of the hy5 mutant and the elongation stimulated by HXK1. HY5 exits the guard cells and inhibits hypocotyl elongation, but is degraded in the dark. We also show that the inhibition of hypocotyl elongation by guard cells' HY5 involves auto-activation of HY5 expression in other tissues. It appears that guard cells are capable of coordinating hypocotyl elongation and that sugar and HXK1 have the opposite effect of light on hypocotyl elongation, converging at PIF4.

The loss of auditory sensory hair cells (HCs) is the most common cause of sensorineural hearing loss (SNHL). As the main sound transmission structure in the cochlea, it is necessary to maintain the normal shape and survival of HCs. In this review, we described and summarized the signaling pathways that regulate the development and survival of auditory HCs in SNHL. The role of the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channels, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant. The molecular interactions of these signaling pathways play an important role in the survival of HCs, which may provide a theoretical basis and possible therapeutic interventions for the treatment of hearing loss., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Yao Liu et al.)

Plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. To this date, the regulation of thermomorphogenesis has been exclusively shown to intersect with light signalling pathways. To identify regulators of thermomorphogenesis that are conserved in flowering plants, we map changes in protein phosphorylation in both dicots and monocots exposed to warm temperature. We identify MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)/TARGET OF TEMPERATURE3 (TOT3) as a regulator of thermomorphogenesis that impinges on brassinosteroid signalling in Arabidopsis thaliana. In addition, we show that TOT3 plays a role in thermal response in wheat, a monocot crop. Altogether, the conserved thermal regulation by TOT3 expands our knowledge of thermomorphogenesis beyond the well-studied pathways and can contribute to ensuring food security under a changing climate.

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum ( DMS dSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum ( DLS dSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1 + neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinson's disease, the dSPN-Npas1 + projection was dramatically strengthened. As DLS dSPN-Npas1 + projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinson's disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states. SIGNIFICANCE STATEMENT In the classic basal ganglia model, the striatum is described as a divergent structure: it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal subpathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this subpathway undergoes changes in a Parkinson's disease model. In particular, our results suggest that the increase in strength of this subpathway contributes to the slowness or reduced movements observed in Parkinson's disease., (Copyright © 2021 the authors.)

The aryl hydrocarbon receptor (AHR) controls several inflammatory and metabolic pathways involved in various diseases, including the development of arthritis. Here, we investigated the role of AHR activation in IL-22-dependent acute arthritis using the K/BxN serum transfer model. We observed an overall reduction of cytokine expression in Ahr-deficient mice, along with decreased signs of joint inflammation. Conversely, we report worsened arthritis symptoms in Il-22 deficient mice. Pharmacological stimulation of AHR with the agonist VAG539, as well as injection of recombinant IL-22, given prior arthritogenic triggering, attenuated inflammation and reduced joint destruction. The protective effect of VAG539 was abrogated in Il-22 deficient mice. Finally, conditional Ahr depletion of Rorc-expressing cells was sufficient to attenuate arthritis, thereby uncovering a previously unsuspected role of AHR in type 3 innate lymphoid cells during acute arthritis., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

Root hair (RH) is essential for plant nutrient acquisition and the plant-environment communication. Here we report that transcription factors MYB30 and ETHYLENE INSENSITIVE3 (EIN3) modulate RH growth/elongation in Arabidopsis in an antagonistic way. The MYB30 loss-of-function mutant displays enhanced RH length, whereas the RH elongation in MYB30-overexpressing plants is highly repressed. MYB30 physically interacts with EIN3, a master transcription factor in ethylene signaling. MYB30 directly binds the promoter region of ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4) and represses its transcription. RSL4 loss-of-function suppresses the enhanced RH growth in myb30 mutant plants. Ethylene enhances MYB30-EIN3 complex formation, and reduces the association between MYB30 and RSL4 promotor via the action of EIN3. MYB30 and EIN3 antagonistically regulate the expression of RSL4 and a subset of core RH genes in a genome-wide way. Taken together, our work revealed a novel transcriptional network that modulates RH growth in plants., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Congenital cranial dysinnervation disorders (CCDDs) are a heterogeneous group of neurodevelopmental phenotypes caused by a primary disturbance of innervation due to deficient, absent, or misguided cranial nerves. Although some CCDDs genes are known, several clinical phenotypes and their aetiologies remain to be elucidated. We describe a 12-year-old boy with hypotonia, developmental delay, sensorineural hearing loss, and keratoconjunctivitis due to lack of corneal reflex. He had a long expressionless face, severe oromotor dysfunction, bilateral agenesis/severe hypoplasia of the VIII nerve with marked atresia of the internal auditory canals and cochlear labyrinth malformation. Trio-exome sequencing identified a homozygous loss of function variant in the NEUROG1 gene (NM_006161.2: c.202G > T, p.Glu68*). NEUROG1 is considered a causal candidate for CCDDs based on (i) the previous report of a patient with a homozygous gene deletion and developmental delay, deafness due to absent bilateral VIII nerves, and severe oromotor dysfunction; (ii) a second patient with a homozygous NEUROG1 missense variant and corneal opacity, absent corneal reflex and intellectual disability; and (iii) the knockout mouse model phenotype which highly resembles the disorder observed in humans. Our findings support the growing compelling evidence that loss of NEUROG1 leads to a very distinctive disorder of cranial nerves development., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Non-alcoholic fatty liver (NAFL) and related syndromes affect one-third of the adult population in industrialized and developing countries. Lifestyle and caloric oversupply are the main causes of such array of disorders, but the molecular mechanisms underlying their etiology remain elusive. Nuclear Protein 1 (NUPR1) expression increases upon cell injury in all organs including liver. Recently, we reported NUPR1 actively participates in the activation of the Unfolded Protein Response (UPR). The UPR typically maintains protein homeostasis, but downstream mediators of the pathway regulate metabolic functions including lipid metabolism. As increases in UPR and NUPR1 in obesity and liver disease have been well documented, the goal of this study was to investigate the roles of NUPR1 in this context. To establish whether NUPR1 is involved in these liver conditions we used patient-derived liver biopsies and in vitro and in vivo NUPR1 loss of functions models. First, we analyzed NUPR1 expression in a cohort of morbidly obese patients (MOPs), with simple fatty liver (NAFL) or more severe steatohepatitis (NASH). Next, we explored the metabolic roles of NUPR1 in wild-type (Nupr1 +/+ ) or Nupr1 knockout mice (Nupr1 -/- ) fed with a high-fat diet (HFD) for 15 weeks. Immunohistochemical and mRNA analysis revealed NUPR1 expression is inversely correlated to hepatic steatosis progression. Mechanistically, we found NUPR1 participates in the activation of PPAR-α signaling via UPR. As PPAR-α signaling is controlled by UPR, collectively, these findings suggest a novel function for NUPR1 in protecting liver from metabolic distress by controlling lipid homeostasis, possibly through the UPR., (© 2021 Federation of American Societies for Experimental Biology.)

Cigarette smoke is a prevalent respiratory toxicant that remains a leading cause of death worldwide. Cigarette smoke induces inflammation in the lungs and airways that contributes to the development of diseases such as lung cancer and chronic obstructive pulmonary disease (COPD). Due to the presence of aryl hydrocarbon receptor (AhR) ligands in cigarette smoke, activation of the AhR has been implicated in driving this inflammatory response. However, we have previously shown that the AhR suppresses cigarette smoke-induced pulmonary inflammation, but the mechanism by which the AhR achieves its anti-inflammatory function is unknown. In this study, we use the AhR antagonist CH-223191 to inhibit AhR activity in mice. After an acute (3-day) cigarette smoke exposure, AhR inhibition was associated with significantly enhanced neutrophilia in the airways in response to cigarette smoke, mimicking the phenotype of AhR-deficient mice. We then used genetically-modified mouse strains which express an AhR that can bind ligand but either cannot translocate to the nucleus or bind its cognate response element, to show that these features of the AhR pathway are not required for the AhR to suppress pulmonary neutrophilia. Finally, using the non-toxic endogenous AhR ligand FICZ, we provide proof-of-concept that activation of pulmonary AhR attenuates smoke-induced inflammation. Collectively, these results support the importance of AhR activity in mediating its anti-inflammatory function in response to cigarette smoke. Further investigation of the precise mechanisms by which the AhR exerts is protective functions may lead to the development of therapeutic agents to treat people with chronic lung diseases that have an inflammatory etiology, but for which few therapeutic options exist., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rico de Souza, Traboulsi, Wang, Fritz, Eidelman and Baglole.)

Background: Long non-coding RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) was found to be elevated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLSs). However, whether HAND2-AS1 functions as an exosomal lncRNA related to mesenchymal stem cells (MSCs) in RA progression is unknown., Methods: The expression of HAND2-AS1, microRNA (miR)-143-3p, and tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) was detected using quantitative real-time polymerase chain reaction and Western blot. Cell proliferation, apoptosis, migration, and invasion were detected using cell counting kit-8, flow cytometry, and wound healing and transwell assays. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL)-6 were analyzed using enzyme-linked immunosorbent assay. The level of phosphorylated-p65 was examined by Western blot. The binding interaction between miR-143-3p and HAND2-AS1 or TNFAIP3 was confirmed by the dual-luciferase reporter and RIP assays. Exosomes were isolated by ultracentrifugation and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot., Results: HAND2-AS1 was lowly expressed in RA synovial tissues, and HAND2-AS1 re-expression suppressed the proliferation, motility, and inflammation and triggered the apoptosis in RA-FLSs via the inactivation of NF-κB pathway. Mechanistically, HAND2-AS1 directly sponged miR-143-3p and positively regulated TNFAIP3 expression, the target of miR-143-3p. Moreover, the effects of HAND2-AS1 on RA-FLSs were partially attenuated by miR-143-3p upregulation or TNFAIP3 knockdown. HAND2-AS1 could be packaged into hMSC-derived exosomes and absorbed by RA-FLSs, and human MSC-derived exosomal HAND2-AS1 also repressed above malignant biological behavior of RA-FLSs., Conclusion: MSC-derived exosomes participated in the intercellular transfer of HAND2-AS1 and suppressed the activation of RA-FLSs via miR-143-3p/TNFAIP3/NF-κB pathway, which provided a novel insight into the pathogenesis and treatment of RA.

Bone metastatic prostate cancer is one of the most common malignancies in developed countries and the second leading cause of cancer-related death in men. There remains no effective treatment for metastatic prostate cancer. We investigate here the anticancer effects of botanical component p-hydroxycinnamic acid (HCA) on the PC-3 cells in vitro model of bone metastatic human prostate cancer. Culturing with HCA (10-1000 nM) suppressed colony formation and growth of PC-3 cells. Mechanistically, culturing with HCA decreased protein levels of Ras, PI3K, Akt, MAPK, NF-κB p65 and β-catenin related to processes of cell signaling and transcription, and it increased levels of p21, p53, retinoblastoma and regucalcin, which are suppressors in carcinogenesis. These alterations can lead to suppression of cell growth. Furthermore, culturing with HCA increased cell death and caspase-3 levels. The effects of HCA on the growth and death of PC-3 cells were blocked by culturing with CH223191, an antagonist of aryl hydrocarbon receptor (AHR), suggesting that HCA effects are partly involved in AHR signaling. Interestingly, HCA suppressed the stimulatory effects of Bay K 8644, an agonist of L-type calcium channel, on the growth of PC-3 cells. Coculturing of PC-3 cells and preosteoblastic MC-3T3 E1 cells increased osteoblastic mineralization. This increase was not attenuated by treatment of HCA that stimulated mineralization. Notably, osteoclastogenesis from preosteoclastic RAW264.7 cells was enhanced by coculturing with PC-3 cells, and this enhancement was suppressed by treatment with HCA (10-1000 nM). Thus, HCA has anticancer effects on bone metastatic human prostate cancer, potentially providing a novel therapeutic tool.

Anthocyanin accumulation is a striking symptom of plant environmental response and plays an important role in plant adaptation to adverse stimuli. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) is a member of the PIFs family that directly interacts with light-activated phytochromes, and it can not only regulate various light responses but also optimize growth as a key integrator of multiple signaling pathways. However, the mechanism by which PIF4 participates in the regulation of anthocyanin accumulation remains to be elucidated. In this study, we found that anthocyanin accumulation was effectively induced by white light in Arabidopsis Col-0, but such an effect was impaired in the overexpression line PIF4OX. Consistently, the transcript level of PAP1 that encodes a key transcript factor involved in regulating anthocyanin biosynthesis was significantly decreased in PIF4OX compared with Col-0. Moreover, the expression of PAP1 was markedly lower in pap1-D/PIF4OX than pap1-D, as a result, the phenotype that highly accumulates anthocyanins in leaves of pap1-D caused by PAP1 overexpressing was almost eliminated in pap1-D/PIF4OX. Analyses through chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) revealed that PIF4 could directly bind to the G-box motif present in the promoter of PAP1. Furthermore, transient transcriptional expression analysis showed that PIF4 could weaken the transcriptional activity of the PAP1 promoter, and the G-box motif is necessary for the effect of PIF4. Subsequently, when the seedlings shifted from darkness to light and grew under constant red light and short-day photoperiod, it was found that the PAP1 transcription level and anthocyanin content in pif4-2/pap1-D were significantly higher than pap1-D, implying that PIF4 mutation can strengthen PAP1's effect on anthocyanin biosynthesis under these conditions. Taken together, the results indicate that PIF4 negatively regulates anthocyanin accumulation in Arabidopsis through transcriptional suppression of PAP1 by directly binding to the G-box motif of the promoter., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development., Results: Here, we show that HIF-2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2α in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2α reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest., Conclusions: Taken together, these results suggest that expression levels of HIF-2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development., (© 2020 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association of Anatomists.)

Background: Osteoarthritis (OA) is a leading cause of disability. The incidence of OA is progressively rising due to the diminishing levels of physical activity and ever-expanding aging population. However, the mainstay for OA treatment only can improve symptoms without delay the progression of this severe disease. This study aimed to explore the biological role and clinical function of lncRNA HAND2-AS1 in OA., Methods: Blood samples and synovial fluid were collected from OA patients and normal subjects. HAND2-AS1 expression was detected by qRT-PCR and IL-6 expression was detected by ELISA. The plasma levels of HAND2-AS1 were also detected in different ages, stages, and gender of OA patients and controls. Furthermore, the ROC curve was used to analyze whether HAND2-AS1 can distinguish OA patients from normal subjects. Also, Pearson correlation coefficient analysis was used to analyze the correlation between lncRNA HAND2-AS1 and IL-6. In addition, Western blot was used to detect the IL-6 level upon HAND2-AS1 over-expression in chondrocytes and qRT-PCR was used to detect the HAND2-AS1 level after endogenous IL-6 treatment., Results: HAND2-AS1 and IL-6 were dysregulated in plasma and synovial fluid of OA patients. The expression of HAND2-AS1 in plasma of OA patients was decreased with aging and progression. Furthermore, HAND2-AS1 downregulation effectively distinguished OA patients from the healthy controls. Over-expression of HAND2-AS1 inhibited IL-6 expression in chondrocytes, while treatment with exogenous IL-6 did not affect HAND2-AS1 expression., Conclusion: HAND2-AS1 effectively distinguished OA patients from the healthy controls and regulates IL-6 expression in human chondrocytes., Trial Registration: ChiCTR, ChiCTR2000038635 . Registered 11 February 2019.

Ascorbic acid (AsA) is an antioxidant and enzyme co-factor that is vital to plant development and abiotic stress tolerance. However, the regulation mechanisms of AsA biosynthesis in plants remain poorly understood. Here, we report a basic helix-loop-helix 55 (ZmbHLH55) transcription factor that regulates AsA biosynthesis in maize. Analysis of publicly available transcriptomic data revealed that ZmbHLH55 is co-expressed with several genes of the GDP-mannose pathway. Experimental data showed that ZmbHLH55 forms homodimers localized to the cell nuclei, and it exhibits DNA binding and transactivation activity in yeast. Under salt stress conditions, knock down mutant (zmbhlh55) in maize accumulated lower levels of AsA compared with wild type, accompanied by lower antioxidant enzymes activity, shorter root length, and higher malondialdehyde (MDA) level. Gene expression data from the WT and zmbhlh55 mutant, showed that ZmbHLH55 positively regulates the expression of ZmPGI2, ZmGME1, and ZmGLDH, but negatively regulates ZmGMP1 and ZmGGP. Furthermore, ZmbHLH55-overexpressing Arabidopsis, under salt conditions, showed higher AsA levels, increased rates of germination, and elevated antioxidant enzyme activities. In conclusion, these results have identified previously unknown regulation mechanisms for AsA biosynthesis, indicating that ZmbHLH55 may be a potential candidate to enhance plant salt stress tolerance in the future., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anther development is a complex process regulated by a myriad of transcription factors belonging to distinct protein families. In this study, we focus on the functional characterization of OsbHLH35, a basic Helix-Loop-Helix (bHLH) TF that regulates anther development in rice. Plants overexpressing OsbHLH35 presented small and curved anthers, leading to a reduction of 72 % on seed production. Rice transgenic plants expressing GUS reporter gene under the control of OsbHLH35 promoter (pOsbHLH35::GUS) showed that this TF specifically accumulates in anthers at the meiosis stage and in other spikelet tissues. Yeast one-hybrid screening identified three members of the Growth-Regulating Factor (GRF) family, OsGRF3, OsGRF4, and OsGRF11, as transcriptional regulators of OsbHLH35. Transactivation assay showed that OsGRF11 negatively regulates OsbHLH35 expression in Arabidopsis protoplasts. This regulation was also observed in planta through the analysis of transgenic plants overexpressing OsGRF11 (OsGRF11OE), confirming that OsGRF11 is a negative regulator of OsbHLH35 in rice. Our data suggest that OsbHLH35 plays an essential role in anther development in rice and the fine control of its expression is crucial to ensure proper seed production., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The immune microenvironment of tumors can play a critical role in promoting or inhibiting tumor progression depending on the context. We present evidence that tumor-associated macrophages/microglia (TAMs) can promote tumor progression in the sonic hedgehog subgroup of medulloblastoma (SHH-MB). By combining longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) and immune profiling of a sporadic mouse model of SHH-MB, we found the density of TAMs is higher in the ~50% of tumors that progress to lethal disease. Furthermore, reducing regulatory T cells or eliminating B and T cells in Rag1 mutants does not alter SHH-MB tumor progression. As TAMs are a dominant immune component in tumors and are normally dependent on colony-stimulating factor 1 receptor (CSF1R), we treated mice with a CSF1R inhibitor, PLX5622. Significantly, PLX5622 reduces a subset of TAMs, prolongs mouse survival, and reduces the volume of most tumors within 4 weeks of treatment. Moreover, concomitant with a reduction in TAMs the percentage of infiltrating cytotoxic T cells is increased, indicating a change in the tumor environment. Our studies in an immunocompetent preclinical mouse model demonstrate TAMs can have a functional role in promoting SHH-MB progression. Thus, CSF1R inhibition could have therapeutic potential for a subset of SHH-MB patients.

Background: The basic helix-loop-helix (bHLH) proteins, a large transcription factors family, are involved in plant growth and development, and defensive response to various environmental stresses. The resurrection plant Myrothamnus flabellifolia is known for its extremely strong drought tolerance, but few bHLHs taking part in abiotic stress response have been unveiled in M. flabellifolia., Results: In the present research, we cloned and characterized a dehydration-inducible gene, MfbHLH38, from M. flabellifolia. The MfbHLH38 protein is localized in the nucleus, where it may act as a transcription factor. Heterologous expression of MfbHLH38 in Arabidopsis improved the tolerance to drought and salinity stresses, as determined by the studies on physiological indexes, such as contents of chlorophyll, malondialdehyde (MDA), proline (Pro), soluble protein, and soluble sugar, water loss rate of detached leaves, reactive oxygen species (ROS) accumulation, as well as antioxidant enzyme activities. Besides, MfbHLH38 overexpression increased the sensitivity of stomatal closure to mannitol and abscisic acid (ABA), improved ABA level under drought stress, and elevated the expression of genes associated with ABA biosynthesis and ABA responding, sucha as NCED3, P5CS, and RD29A., Conclusions: Our results presented evidence that MfbHLH38 enhanced tolerance to drought and salinity stresses in Arabidopsis through increasing water retention ability, regulating osmotic balance, decreasing stress-induced oxidation damage, and possibly participated in ABA-dependent stress-responding pathway.

Smoking is the largest preventable cause of mortality and the largest environmental driver of epigenetic aging. Contingency management-based strategies can be used to treat smoking but require objective methods of verifying quitting status. Prior studies have suggested that cg05575921 methylation reverts as a function of smoking cessation, but that it can be used to verify the success of smoking cessation has not been unequivocally demonstrated. To test whether methylation can be used to verify cessation, we determined monthly cg05575921 levels in a group of 67 self-reported smokers undergoing biochemically monitored contingency management-based smoking cessation therapy, as part of a lung imaging protocol. A total of 20 subjects in this protocol completed three months of cotinine verified smoking cessation. In these 20 quitters, the reversion of cg05575921 methylation was dependent on their initial smoking intensity, with methylation levels in the heaviest smokers reverting to an average of 0.12% per day over the 3-month treatment period. In addition, we found suggestive evidence that some individuals may have embellished their smoking history to gain entry to the study. Given the prominent effect of smoking on longevity, we conclude that DNA methylation may be a useful tool for guiding and incentivizing contingency management-based approaches for smoking cessation.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has emerged as an important player in asthma control. AhR is responsive to environmental molecules and endogenous or dietary metabolites and regulates innate and adaptive immune responses. Binding of this receptor by different ligands has led to seemingly opposite responses in different asthma models. In this review, we present two sides of the same coin, with the beneficial and deleterious roles of AhR evaluated using known endogenous or exogenous ligands, deficient mice or antagonists. On one hand, AhR has an anti-inflammatory role since its activation in dendritic cells blocks the generation of pro-inflammatory T cells or shifts macrophages toward an anti-inflammatory M2 phenotype. On the other hand, AhR activation by particle-associated polycyclic aromatic hydrocarbons from the environment is pro-inflammatory, inducing mucus hypersecretion, airway remodelling, dysregulation of antigen presenting cells and exacerbates asthma features. Data concerning the role of AhR in cells from asthmatic patients are also reviewed, since AhR could represent a potential target for therapeutic immunomodulation.

Cardiac inflammation and fibrosis triggered by left ventricular pressure overload are the major causes of heart dysfunction. Differentiated embryonic chondrocyte gene 1 (Dec1) is a basic helix-loop-helix transcription factor that is comprehensively involved in inflammation and tissue fibrosis, but its role in cardiac hypertrophy remains unclear. This study explored the effects of Dec1 on cardiac fibrosis, inflammation, and apoptosis in hypertrophic conditions. Transverse aortic constriction (TAC) was performed to induce cardiac hypertrophy in wild-type (WT) mice and in Dec1 knock out (KO) mice for 4 weeks. Using the TAC mouse model, prominent differences in cardiac hypertrophy at the morphological, functional, and molecular levels were delineated by Masson's Trichrome and TUNEL staining, immunohistochemistry, RT-PCR and Western Blot. DNA microarray and microRNA (miRNA) array analyses were carried out to identify gene and miRNA expression patterns. Dec1KO mice exhibited a more severe hypertrophic heart, whereas WT mice showed a more pronounced perivascular fibrosis after TAC at 4 weeks. The Dec1 deficiency promoted M2 phenotype macrophages. Dec1KO TAC mice showed fewer apoptotic cells than WT TAC mice. APEX1, WNT16, FGF10 and MMP-10 were differentially expressed according to DNA microarray analysis and expression levels of those genes and the corresponding miRNAs (miR-295, miR-200 b, miR-130a, miR-92a) showed the same trends. Furthermore, luciferase reporter assay confirmed that FGF10 is the direct target gene of miR-130. In conclusion, a Dec1 deficiency protects the heart from perivascular fibrosis, regulates M1/M2 macrophage polarization and reduces cell apoptosis, which may provide a novel insight for the treatment of cardiac hypertrophy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low-phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate-deprived conditions are TMO5- and cytokinin-dependent. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Direct reprogramming has been widely explored to generate various types of neurons for neurobiological research and translational medicine applications, but there is still no efficient reprogramming method to generate retinal ganglion cell (RGC)-like neurons, which are the sole projection neurons in the retina. Here, we show that three transcription factors, Ascl1, Brn3b, and Isl1, efficiently convert fibroblasts into RGC-like neurons (iRGCs). Furthermore, we show that the competence of cells to enter iRGC reprogramming route is determined by the cell-cycle status at a very early stage of the process. The iRGC reprogramming route involves intermediate states that are characterized by a transient inflammatory-like response followed by active epigenomic and transcriptional modifications. Our study provides an efficient method to generate iRGCs, which would be a valuable cell source for potential glaucoma cell replacement therapy and drug screening studies, and reveals the key cellular events that govern successful neuronal fate reprogramming., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hair cells (HCs) in the mammalian cochleae cannot spontaneously regenerate once damaged, resulting in permanent hearing loss. It has been shown that Atoh1 overexpression induces hair cell-like cells (HCLCs) in the cochlea of newborn rodents, but this is hard to achieve in adult mammals. In this study, we used a three-dimensional cochlear culture system and an adenoviral-mediated delivery vector to overexpress Atoh1 in adult mouse cochleae. HCLCs were successfully induced from 3 days after virus infection (3 DVI) in vitro, and the number increased with time. HCLCs were myosin7a positive and distinguishable from remnant HCs in a culture environment. Meanwhile, patch-clamp results showed that noninactive outward potassium currents (sustained outward potassium currents) could be recorded in HCLCs and that their magnitude increased with time, similar to normal HCs. Furthermore, transient HCN currents were recorded in some HCLCs, indicating that the HCLCs experienced a developmental stage similar to normal HCs. We also compared the electrophysiological features of HCLCs from adult mice with native HCs and found the HCLCs gradually matured, similar to the normal HCs. Meanwhile, HCLCs from adult mice possessed the same bundles as developmental HCs. However, these HCLCs did not express prestin, which is a special marker for outer hair cells (OHCs), even at 13 DVI. These results demonstrate that Atoh1 overexpression induces HCLC formation in the adult mammalian cochlea and that these HCLCs were functional and experienced a developmental process similar to that of normal HCs., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Lingyi Kong et al.)

There is strong evidence that exposure to fine particulate matter (PM 2.5 ) and a high-fat diet (HFD) increase the risk of mortality from atherosclerotic cardiovascular diseases. Recent studies indicate that PM 2.5 generated by combustion activates the Aryl Hydrocarbon Receptor (AHR) and inflammatory cytokines contributing to PM 2.5 -mediated atherogenesis. Here we investigate the effects of components of a HFD on PM-mediated activation of AHR in macrophages. Cells were treated with components of a HFD and AHR-activating PM and the expression of biomarkers of vascular inflammation was analyzed. The results show that glucose and triglyceride increase AHR-activity and PM 2.5 -mediated induction of cytochrome P450 (CYP)1A1 mRNA in macrophages. Cholesterol, fructose, and palmitic acid increased the PM- and AHR-mediated induction of proinflammatory cytokines in macrophages. Treatment with palmitic acid significantly increased the expression of inflammatory cytokines and markers of vascular injury in human aortic endothelial cells (HAEC) after treatment with PM 2.5 . The PM 2.5 -mediated activation of the atherogenic markers C-reactive protein (CRP) and S100A9, a damage-associated molecular pattern molecule, was found to be AHR-dependent and involved protein kinase A (PKA) and a CCAAT/enhancer-binding protein (C/EBP) binding element. This study identified nutritional factors interacting with AHR signaling and contributing to PM 2.5 -induced markers of atherogenesis and future cardiovascular risk.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Recent studies have shown that AhR is a novel master regulator of the mucosal immune system, including lungs and intestine. To elucidate the role of AhR in chronic severe asthma, AhR wild-type and knockout mice (AhR -/- ) were sensitized and challenged with ovalbumin for 4 weeks. To uncover the underlying mechanisms, inflammatory cells profile and cytokines production were analyzed in bronchial lavage fluid (BALF) and lung tissue. Compared to wild-type mice, AhR -/- mice had exacerbated asthma symptoms, including airway inflammation, mucus production, airway hyperresponsiveness, and airway remodeling. BALF monocytes, neutrophils, eosinophils, and lymphocytes were all enhanced in OVA-immunized AhR -/- mice. In OVA-immunized AhR -/- mice, T helper (Th) 17 cell-specific cytokine IL-17A, as well as airway remodeling factors, including epithelial-mesenchymal transition (EMT) markers and vascular endothelial growth factor (VEGF), were all enhanced in lung tissue. Moreover, human cohort studies showed that AhR gene expression in bronchial epithelial cells decreases in severe asthma patients. Loss of AhR leads to worsening of allergic asthma symptoms, indicating its importance in maintaining normal lung function and mediating disease severity., (© 2020 Federation of American Societies for Experimental Biology.)

Objective: Recent evidence indicates that inhibition of prolyl hydroxylase domain (PHD) proteins can exert beneficial effects to improve metabolic abnormalities in mice and humans. However, the underlying mechanisms are not clearly understood. This study was designed to address this question., Methods: A pan-PHD inhibitor compound was injected into WT and liver-specific hypoxia-inducible factor (HIF)-2α KO mice, after onset of obesity and glucose intolerance, and changes in glucose and glucagon tolerance were measured. Tissue-specific changes in basal glucose flux and insulin sensitivity were also measured by hyperinsulinemic euglycemic clamp studies. Molecular and cellular mechanisms were assessed in normal and type 2 diabetic human hepatocytes, as well as in mouse hepatocytes., Results: Administration of a PHD inhibitor compound (PHDi) after the onset of obesity and insulin resistance improved glycemic control by increasing insulin and decreasing glucagon sensitivity in mice, independent of body weight change. Hyperinsulinemic euglycemic clamp studies revealed that these effects of PHDi treatment were mainly due to decreased basal hepatic glucose output and increased liver insulin sensitivity. Hepatocyte-specific deletion of HIF-2α markedly attenuated these effects of PHDi treatment, showing PHDi effects are HIF-2α dependent. At the molecular level, HIF-2α induced increased Irs2 and cyclic AMP-specific phosphodiesterase gene expression, leading to increased and decreased insulin and glucagon signaling, respectively. These effects of PHDi treatment were conserved in human and mouse hepatocytes., Conclusions: Our results elucidate unknown mechanisms for how PHD inhibition improves glycemic control through HIF-2α-dependent regulation of hepatic insulin and glucagon sensitivity., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Hypoxia can be defined as a relative deficiency in the amount of oxygen reaching the tissues. Hypoxia-inducible factors (HIFs) are critical regulators of the mammalian response to hypoxia. In normal circumstances, HIF-1α protein turnover is rapid, and hyperglycemia further destabilizes the protein. In addition to their role in diabetes pathogenesis, HIFs are implicated in development of the microvascular and macrovascular complications of diabetes. Improving glucose control in people with diabetes increases HIF-1α protein and has wide-ranging benefits, some of which are at least partially mediated by HIF-1α. Nevertheless, most strategies to improve diabetes or its complications via regulation of HIF-1α have not currently proven to be clinically useful. The intersection of HIF biology with diabetes is a complex area in which many further questions remain, especially regarding the well-conducted studies clearly describing discrepant effects of different methods of increasing HIF-1α, even within the same tissues. This Review presents a brief overview of HIFs; discusses the range of evidence implicating HIFs in β cell dysfunction, diabetes pathogenesis, and diabetes complications; and examines the differing outcomes of HIF-targeting approaches in these conditions.

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA), a widespread disorder of breathing. This Review focuses on the role of hypoxia-inducible factors (HIFs) in hypertension, type 2 diabetes (T2D), and cognitive decline in experimental models of IH patterned after O2 profiles seen in OSA. IH increases HIF-1α and decreases HIF-2α protein levels. Dysregulated HIFs increase reactive oxygen species (ROS) through HIF-1-dependent activation of pro-oxidant enzyme genes in addition to reduced transcription of antioxidant genes by HIF-2. ROS in turn activate chemoreflex and suppress baroreflex, thereby stimulating the sympathetic nervous system and causing hypertension. We also discuss how increased ROS generation by HIF-1 contributes to IH-induced insulin resistance and T2D as well as disrupted NMDA receptor signaling in the hippocampus, resulting in cognitive decline.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which senses environmental, dietary or metabolic signals to mount a transcriptional response, vital in health and disease. As environmental stimuli and metabolic products have been shown to impact the central nervous system (CNS), a burgeoning area of research has been on the role of the AHR in ocular and non-ocular neurodegenerative diseases. Herein, we summarize our current knowledge, of AHR-controlled cellular processes and their impact on regulating pathobiology of select ocular and neurodegenerative diseases. We catalogue animal models generated to study the role of the AHR in tissue homeostasis and disease pathogenesis. Finally, we discuss the potential of targeting the AHR pathway as a therapeutic strategy, in the context of the maladies of the eye and brain.

Light responses mediated by the photoreceptors play crucial roles in regulating different aspects of plant growth and development. An E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)1/SUPPRESSOR OF PHYA (SPA), one of the central repressors of photomorphogenesis, is critical for maintaining skotomorphogenesis. It targets several positive regulators of photomorphogenesis for degradation in darkness. Recently, we revealed that basic helix-loop-helix factors, HECATEs (HECs), function as positive regulators of photomorphogenesis by directly interacting and antagonizing the activity of another group of repressors called PHYTOCHROME-INTERACTING FACTORs (PIFs). It was also shown that HECs are partially degraded in the dark through the ubiquitin/26S proteasome pathway. However, the underlying mechanism of HEC degradation in the dark is still unclear. Here, we show that HECs also interact with both COP1 and SPA proteins in darkness, and that HEC2 is directly targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway. Moreover, COP1-mediated polyubiquitylation and degradation of HEC2 are enhanced by PIF1. Therefore, the ubiquitylation and subsequent degradation of HECs are significantly reduced in both cop1 and pif mutants. Consistent with this, the hec mutants partially suppress photomorphogenic phenotypes of both cop1 and pifQ mutants. Collectively, our work reveals that the COP1/SPA-mediated ubiquitylation and degradation of HECs contributes to the coordination of skoto/photomorphogenic development in plants., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants have the ability to regenerate whole plant body parts, including shoots and roots, in vitro from callus derived from a variety of tissues. However, the underlying mechanisms for this de novo organogenesis, which is based on the totipotency of callus cells, are poorly understood. Here, we report that a microRNA (miRNA)-mediated posttranscriptional regulation plays an important role in de novo shoot regeneration. We found that mutations in HUA ENHANCER 1 (HEN1), a gene encoding a small RNA methyltransferase, cause cytokinin-related defects in de novo shoot regeneration. A hen1 mutation caused a large reduction in the miRNA319 (miR319) level and a subsequent increase in its known target (TCP3 and TCP4) transcript levels. TCP transcription factors redundantly inhibited shoot regeneration and directly activated the expression of a negative regulator of cytokinin response ARABIDOPSIS THALIANA RESPONSE REGULATOR 16 (ARR16). A tcp4 mutation at least partly rescued the shoot-regeneration defect and derepression of ARR16 in hen1. These findings demonstrate that the miR319-TCP3/4-ARR16 axis controls de novo shoot regeneration by modulating cytokinin responses., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Hypoxic environments occur normally at high altitude, or in underground burrows and in deep sea habitats. They also occur pathologically in human ischemia and in hypoxic solid tumors. Hypoxia in various cancer types and its related molecular mechanisms are associated with a poor clinical outcome. This review will discuss how hypoxia can influence two aspects of tumorigenesis, namely the direct, cell-intrinsic oncogenic effects, as well as the indirect effects on tumor progression mediated by an altered tumor microenvironment. We will also discuss recent progress in identifying the functional roles of hypoxia-related factors (HIFs), along with their regulators and downstream target genes, in cancer stem cells and therapy. Importantly, we propose, using convergent evolution schemes to identify novel biomarkers for both hypoxia adaptation and hypoxic solid tumors as an important strategy in the future., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)