Epithelial-derived interleukin-23 promotes oral mucosal immunopathology.

At mucosal surfaces, epithelial cells provide a structural barrier and an immune defense system. However, dysregulated epithelial responses can contribute to disease states. Here, we demonstrated that epithelial cell-intrinsic production of interleukin-23 (IL-23) triggers an inflammatory loop in the prevalent oral disease periodontitis. Epithelial IL-23 expression localized to areas proximal to the disease-associated microbiome and was evident in experimental models and patients with common and genetic forms of disease. Mechanistically, flagellated microbial species of the periodontitis microbiome triggered epithelial IL-23 induction in a TLR5 receptor-dependent manner. Therefore, unlike other Th17-driven diseases, non-hematopoietic-cell-derived IL-23 served as an initiator of pathogenic inflammation in periodontitis. Beyond periodontitis, analysis of publicly available datasets revealed the expression of epithelial IL-23 in settings of infection, malignancy, and autoimmunity, suggesting a broader role for epithelial-intrinsic IL-23 in human disease. Collectively, this work highlights an important role for the barrier epithelium in the induction of IL-23-mediated inflammation.

A Hematogenous Route for Medulloblastoma Leptomeningeal Metastases.

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma.

The molecular basis of phenotypic evolution: beyond the usual suspects.

It has been well documented that mutations in coding DNA or cis-regulatory elements underlie natural phenotypic variation in many organisms. However, the development of sophisticated functional tools in recent years in a wide range of traditionally non-model systems have revealed many 'unusual suspects' in the molecular bases of phenotypic evolution, including upstream open reading frames (uORFs), cryptic splice sites, and small RNAs. Furthermore, large-scale genome sequencing, especially long-read sequencing, has identified a cornucopia of structural variation underlying phenotypic divergence and elucidated the composition of supergenes that control complex multi-trait polymorphisms. In this review article we highlight recent studies that demonstrate this great diversity of molecular mechanisms producing adaptive genetic variation and the panoply of evolutionary paths leading to the 'grandeur of life'.

Rechargeable Na/Cl2 and Li/Cl2 batteries.

Lithium-ion batteries (LIBs) are widely used in applications ranging from electric vehicles to wearable devices. Before the invention of secondary LIBs, the primary lithium-thionyl chloride (Li-SOCl2) battery was developed in the 1970s using SOCl2 as the catholyte, lithium metal as the anode and amorphous carbon as the cathode1-7. This battery discharges by lithium oxidation and catholyte reduction to sulfur, sulfur dioxide and lithium chloride, is well known for its high energy density and is widely used in real-world applications; however, it has not been made rechargeable since its invention8-13. Here we show that with a highly microporous carbon positive electrode, a starting electrolyte composed of aluminium chloride in SOCl2 with fluoride-based additives, and either sodium or lithium as the negative electrode, we can produce a rechargeable Na/Cl2 or Li/Cl2 battery operating via redox between mainly Cl2/Cl- in the micropores of carbon and Na/Na+ or Li/Li+ redox on the sodium or lithium metal. The reversible Cl2/NaCl or Cl2/LiCl redox in the microporous carbon affords rechargeability at the positive electrode side and the thin alkali-fluoride-doped alkali-chloride solid electrolyte interface stabilizes the negative electrode, both are critical to secondary alkali-metal/Cl2 batteries.

Shedding light on rechargeable Na/Cl2 battery.

Advancing new ideas of rechargeable batteries represents an important path to meeting the ever-increasing energy storage needs. Recently, we showed rechargeable sodium/chlorine (Na/Cl2) (or lithium/chlorine Li/Cl2) batteries that used a Na (or Li) metal negative electrode, a microporous amorphous carbon nanosphere (aCNS) positive electrode, and an electrolyte containing dissolved aluminum chloride and fluoride additives in thionyl chloride [G. Zhu et al., Nature 596, 525-530 (2021) and G. Zhu et al., J. Am. Chem. Soc. 144, 22505-22513 (2022)]. The main battery redox reaction involved conversion between NaCl and Cl2 trapped in the carbon positive electrode, delivering a cyclable capacity of up to 1,200 mAh g-1 (based on positive electrode mass) at a ~3.5 V discharge voltage [G. Zhu et al., Nature 596, 525-530 (2021) and G. Zhu et al., J. Am. Chem. Soc. 144, 22505-22513 (2022)]. Here, we identified by X-ray photoelectron spectroscopy (XPS) that upon charging a Na/Cl2 battery, chlorination of carbon in the positive electrode occurred to form carbon-chlorine (C-Cl) accompanied by molecular Cl2 infiltrating the porous aCNS, consistent with Cl2 probed by mass spectrometry. Synchrotron X-ray diffraction observed the development of graphitic ordering in the initially amorphous aCNS under battery charging when the carbon matrix was oxidized/chlorinated and infiltrated with Cl2. The C-Cl, Cl2 species and graphitic ordering were reversible upon discharge, accompanied by NaCl formation. The results revealed redox conversion between NaCl and Cl2, reversible graphitic ordering/amorphourization of carbon through battery charge/discharge, and probed trapped Cl2 in porous carbon by XPS.

Genetic basis of nectar guide trichome variation between bumblebee- and self-pollinated monkeyflowers (Mimulus): role of the MIXTA-like gene GUIDELESS.

Nectar guide trichomes play crucial ecological roles in bee-pollinated flowers, as they serve as footholds and guides for foraging bees to access the floral rewards. However, the genetic basis of natural variation in nectar guide trichomes among species remains poorly understood. In this study, we performed genetic analysis of nectar guide trichome variation between two closely related monkeyflower (Mimulus) species, the bumblebee-pollinated Mimulus lewisii and self-pollinated M. parishii. We demonstrate that a MIXTA-like R2R3-MYB gene, GUIDELESS, is a major contributor to the nectar guide trichome length variation between the two species. The short-haired M. parishii carries a recessive allele due to non-synonymous substitutions in a highly conserved motif among MIXTA-like MYB proteins. Furthermore, our results suggest that besides GUIDELESS, additional loci encoding repressors of trichome elongation also contribute to the transition from bumblebee-pollination to selfing. Taken together, these results suggest that during a pollination syndrome switch, changes in seemingly complex traits such as nectar guide trichomes could have a relatively simple genetic basis, involving just a few genes of large effects.

miR-124-3p improves mitochondrial function of renal tubular epithelial cells in db/db mice.

Diabetic kidney disease (DKD) is one of the most serious complications of diabetes mellitus (DM) and the main cause of end-stage renal failure. However, the pathogenesis of DKD is complicated. In this study, we found that miR-124-3p plays a key role in regulating renal mitochondrial function and explored its possible mechanism in DKD progression by performing a series of in vitro and in vivo experiments. Decreased expression of miR-124-3p was found in db/db mice compared to db/m mice. Moreover, miR-124-3p down-regulated FOXQ1 by targeting FOXQ1 mRNA 3'-UTR in NRK-52E cells. Also, an increase in FOXQ1 and down-regulation of Sirt4 were found in db/db mouse kidney and renal tubular epithelial cells cultured with high glucose and high lipid. Overexpression of FOXQ1 could further down-regulate the expression of Sirt4 and aggravate the damage of mitochondria. Conversely, the knockdown of the FOXQ1 gene induced Sirt4 expression and partially restored mitochondrial function. To verify the effects of miR-124-3p on Sirt4 and mitochondria, we found that miR-124-3p mimics could up-regulate Sirt4 and inhibit ROS production and MitoSOX, thus restoring the number and morphology of mitochondria. These results showed that under high-glucose and high-lipid conditions, the down-regulation of miR-124-3p induces FOXQ1 in renal tubular epithelial cells, which in turn suppresses Sirt4 and leads to mitochondrial dysfunction, promoting the development of DKD.

Identification of M2 Macrophage-Related Key Genes in Advanced Atherosclerotic Plaques by Network-Based Analysis.

ABSTRACT: Atherosclerotic plaque accounts for major adverse cardiovascular events because of its vulnerability. The classically activated macrophage (M1) and alternatively activated macrophage (M2) are implicated in the progression and regression of plaque, respectively. However, the therapeutic targets related to M2 macrophages still remain largely elusive. In this study, cell-type identification by estimating relative subsets of RNA transcripts and weighted gene coexpression network analysis algorithms were used to establish a weighted gene coexpression network for identifying M2 macrophage-related hub genes using GSE43292 data set. The results showed that genes were classified into 7 modules, with the blue module (Cor = 0.67, P = 3e-05) being the one that was most related to M2 macrophage infiltration in advanced plaques, and then 99 hub genes were identified from blue module. Meanwhile, 1289 differentially expressed genes were produced in GSE43292 data set. Subsequently, the intersection genes of hub genes and differentially expressed genes, including AKTIP , ASPN , FAM26E , RAB23 , PLS3 , and PLSCR4 , were obtained by Venn diagrams and named as key genes. Further validation using data sets GSE100927 and GSE41571 showed that 6 key genes all downregulated in advanced and vulnerable plaques compared with early and stable plaque samples (|Log2 (fold change)| > 0.5, P < 0.05 or 0.001), respectively. Receiver operator characteristic curve analysis indicated that the 6 key genes might have potential diagnostic value. The validation of key genes in the model in vitro and in vivo also demonstrated decreased mRNA expressions of AKTIP , ASPN , FAM26E , RAB23 , PLS3 , and PLSCR4 ( P < 0.05 or 0.001). Collectively, we identified AKTIP, ASPN, FAM26E, RAB23, PLS3, and PLSCR4 as M2 macrophage-related key genes during atherosclerotic progression, proposing potential intervention targets for advanced atherosclerotic plaques.

Brigatinib, a newly discovered AXL inhibitor, suppresses AXL-mediated acquired resistance to osimertinib in EGFR-mutated non-small cell lung cancer.

In addition to the classical resistance mechanisms, receptor tyrosine-protein kinase AXL is a main mechanism of resistance to third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) osimertinib in EGFR-mutated non-small cell lung cancer (NSCLC). Developing an effective AXL inhibitor is important to sensitize osimertinib in clinical application. In this study we assessed the efficacy of brigatinib, a second-generation of anaplastic lymphoma kinase (ALK)-TKI, as a novel AXL inhibitor, in overcoming acquired resistance to osimertinib induced by AXL activation. We established an AXL-overexpression NSCLC cell line and conducted high-throughput screening of a small molecule chemical library containing 510 anti-tumor drugs. We found that brigatinib potently inhibited AXL expression, and that brigatinib (0.5 µM) significantly enhanced the anti-tumor efficacy of osimertinib (1 µM) in AXL-mediated osimertinib-resistant NSCLC cell lines in vitro. We demonstrated that brigatinib had a potential ability to bind AXL kinase protein and further inhibit its downstream pathways in NSCLC cell lines. Furthermore, we revealed that brigatinib might decrease AXL expression through increasing K48-linked ubiquitination of AXL and promoting AXL degradation in HCC827OR cells and PC-9OR cells. In AXL-high expression osimertinib-resistant PC-9OR and HCC827OR cells derived xenograft mouse models, administration of osimertinib (10 mg·kg-1·d-1) alone for 3 weeks had no effect, and administration of brigatinib (25 mg·kg-1·d-1) alone caused a minor inhibition on the tumor growth; whereas combination of osimertinib and brigatinib caused marked tumor shrinkages. We concluded that brigatinib may be a promising clinical strategy for enhancing osimertinib efficacy in AXL-mediated osimertinib-resistant NSCLC patients.

Robotic versus laparoscopic pelvic lateral lymph node dissection in locally advanced rectal cancer: a systemic review and meta-analysis.

BACKGROUND: There are few available studies that compare the feasibility, efficacy, and safety of robotic pelvic lateral lymph node dissection compared to laparoscopic pelvic lateral lymph node dissection (LPLND) in advanced rectal cancer. This meta-analysis aims to compare perioperative outcomes between robotic and LPLND. METHODS: We performed a systemic literature review of PubMed, Embase, and Web of Science databases. Perioperative parameters were extracted and pooled for analysis. This meta-analysis provided an analysis of heterogeneity and prediction intervals. RESULTS: Five studies were included: 567 patients divided between 266 robotic and 301 LPLND. Overall operation time was longer in the robotic group than laparoscopic group (difference in means = 67.11, 95% CI [30.80, 103.42], p < 0.001) but the difference in the pelvic lateral lymph dissection time was not statistically significant (difference in means = - 1.212, 95% CI [ - 11.594, 9.171], p = 0.819). There were fewer overall complications in the robotic than in the laparoscopic group (OR = 1.589, 95% CI [1.009, 2.503], p = 0.046), especially with respect to urinary retention (OR = 2.23, 95% CI [1.277, 3.894], p = 0.005). More pelvic lateral lymph nodes were harvested by robotic surgery than by laparoscopy (differences in means = - 1.992, 95% CI [ - 2.421, 1.563], p < 0.001). CONCLUSIONS: In this meta-analysis, robotic pelvic lateral lymph node dissection was associated with more pelvic lateral lymph nodes harvested and lower overall complications, especially urinary retention when compared to LPLND. Further studies are needed to reinforce these findings.

Nomogram to predict recurrence risk factors in patients with non-valvular paroxysmal atrial fibrillation after catheter radiofrequency ablation.

BACKGROUND: Radiofrequency catheter ablation (RFCA) is an effective method for controlling the heart rate of paroxysmal atrial fibrillation (PAF). However, recurrence is trouble under the RFCA. To gain a deeper understanding of the risk factors for recurrence in patients, we created a nomogram model to provide clinicians with treatment recommendations. METHODS: A total of two hundred thirty-three patients with PAF treated with RFCA at Guizhou Medical University Hospital between January 2021 and December 2022 were consecutively included in this study, and after 1 year of follow-up coverage, 166 patients met the nadir inclusion criteria. Patients with AF were divided into an AF recurrence group and a non-recurrence group. The nomogram was constructed using univariate and multivariate logistic regression analyses. By calculating the area under the curve, we analyzed the predictive ability of the risk scores (AUC). In addition, the performance of the nomogram in terms of calibration, discrimination, and clinical utility was evaluated. RESULTS: At the 12-month follow-up, 48 patients (28.92%) experienced a recurrence of AF after RFCA, while 118 patients (71.08%) maintained a sinus rhythm. In addition to age, sex, and TRV, LAD, and TTPG were independent predictors of recurrence of RFCA. The c-index of the nomogram predicted AF recurrence with an accuracy of .723, showing good decision curves and a calibrated nomogram, as determined by internal validation using a bootstrap sample size of 1000. CONCLUSION: We created a nomogram based on multifactorial logistic regression analysis to estimate the probability of recurrence in patients with atrial fibrillation 1 year after catheter ablation. This plot can be utilized by clinicians to predict the likelihood of recurrence.

Ancient and recent introgression shape the evolutionary history of pollinator adaptation and speciation in a model monkeyflower radiation (Mimulus section Erythranthe).

Inferences about past processes of adaptation and speciation require a gene-scale and genome-wide understanding of the evolutionary history of diverging taxa. In this study, we use genome-wide capture of nuclear gene sequences, plus skimming of organellar sequences, to investigate the phylogenomics of monkeyflowers in Mimulus section Erythranthe (27 accessions from seven species). Taxa within Erythranthe, particularly the parapatric and putatively sister species M. lewisii (bee-pollinated) and M. cardinalis (hummingbird-pollinated), have been a model system for investigating the ecological genetics of speciation and adaptation for over five decades. Across >8000 nuclear loci, multiple methods resolve a predominant species tree in which M. cardinalis groups with other hummingbird-pollinated taxa (37% of gene trees), rather than being sister to M. lewisii (32% of gene trees). We independently corroborate a single evolution of hummingbird pollination syndrome in Erythranthe by demonstrating functional redundancy in genetic complementation tests of floral traits in hybrids; together, these analyses overturn a textbook case of pollination-syndrome convergence. Strong asymmetries in allele sharing (Patterson's D-statistic and related tests) indicate that gene tree discordance reflects ancient and recent introgression rather than incomplete lineage sorting. Consistent with abundant introgression blurring the history of divergence, low-recombination and adaptation-associated regions support the new species tree, while high-recombination regions generate phylogenetic evidence for sister status for M. lewisii and M. cardinalis. Population-level sampling of core taxa also revealed two instances of chloroplast capture, with Sierran M. lewisii and Southern Californian M. parishii each carrying organelle genomes nested within respective sympatric M. cardinalis clades. A recent organellar transfer from M. cardinalis, an outcrosser where selfish cytonuclear dynamics are more likely, may account for the unexpected cytoplasmic male sterility effects of selfer M. parishii organelles in hybrids with M. lewisii. Overall, our phylogenomic results reveal extensive reticulation throughout the evolutionary history of a classic monkeyflower radiation, suggesting that natural selection (re-)assembles and maintains species-diagnostic traits and barriers in the face of gene flow. Our findings further underline the challenges, even in reproductively isolated species, in distinguishing re-use of adaptive alleles from true convergence and emphasize the value of a phylogenomic framework for reconstructing the evolutionary genetics of adaptation and speciation.

Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation.

BACKGROUND: Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. RESULTS: In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. CONCLUSIONS: The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

Exacerbated gastrointestinal symptoms and long COVID in IBD patients with SARS-CoV-2 infection: A multi-center study from taiwan.

BACKGROUND/PURPOSE: Limited studies have addressed the exacerbation of symptoms and long COVID in inflammatory bowel disease (IBD) patients following non-severe COVID-19 infection, particularly with post-COVID-19 vaccination. We aim to investigate factors associated with exacerbated gastrointestinal symptoms (EGS) and long COVID in IBD patients with non-severe COVID-19, which is most common situation in daily practice. METHODS: This is an observational study by multiple centers in Taiwan from May 2020 to March 2023. We collected clinical manifestation, data, and medication information from IBD patients with non-severe COVID-19. EGS was defined as increased frequency of diarrhea, bloody stool, and abdomen pain within 14 days after SARS-COV-2 infection. Long COVID was defined following the guidelines of the World Health Organization. RESULTS: Out of 90 patients, most of them (88.9%) received at least standard two doses of COVID-19 vaccination and the majority (87.8%) were mild diseases of COVID-19.30% of patients experienced EGS during COVID-19 with higher ESR levels serving as a predictive factor (Odds ratio: 3.6, 95% confidence interval: 1.2-10.5, P = 0.02). 38.1% of those patients developed long COVID. The patients who experienced EGS during COVID-19 and with a history of longer IBD duration showed a significant association with long COVID (p = 0.03 and p = 0.02). CONCLUSIONS: Our study revealed that EGS and long COVID occurred in one third of IBD patients with non-severe COVID-19, even though most of them had received the standard plus booster vaccination. We identified associated factors for EGS and long COVID, emphasizing the importance of post-COVID-19 follow-up in IBD patients.

Recent Progress in Two-Dimensional Nanomaterials for Flame Retardance and Fire-Warning Applications.

Graphene-like 2D nanomaterials, such as graphene, MXene, molybdenum disulfide, and boron nitride, present a promising avenue for eco-friendly flame retardants. Their inherent characteristics, including metal-like conductivity, high specific surface area, electron transport capacity, and solution processability, make them highly suitable for applications in both structural fire protection and fire alarm systems. This review offers an up-to-date exploration of advancements in flame retardant composites, utilizing pristine graphene-like nanosheets, versatile graphene-like nanosheets with multiple functions, and collaborative systems based on these nanomaterials. Moreover, graphene-like 2D nanomaterials exhibit considerable potential in the development of early fire alarm systems, enabling timely warnings. This review provides an overview of flame-retarding and fire-warning mechanisms, diverse multifunctional nanocomposites, and the evolving trends in the development of fire alarm systems anchored in graphene-like 2D nanomaterials and their derivatives. Ultimately, the existing challenges and prospective directions for the utilization of graphene-like 2D nanomaterials in flame retardant and fire-warning applications are put forward.

A New Family of High Oxidation State Antiperovskite Nitrides: La3MN5 (M=Cr, Mn and Mo).

Three new nitrides La3MN5 (M=Cr, Mn, and Mo) have been synthesized using a high pressure azide route. These are the first examples of ternary Cs3CoCl5-type nitrides, and show that this (MN4)NLa3 antiperovskite structure type may be used to stabilise high oxidation-state transition metals in tetrahedral molecular [MN4]n- nitridometallate anions. Magnetic measurements confirm that Cr and Mo are in the M6+ state, but the M=Mn phase has an anomalously small paramagnetic moment and large cell volume. Neutron powder diffraction data are fitted using an anion-excess La3MnN5.30 model (space group I4/mcm, a=6.81587(9) Šand c=11.22664(18) Šat 200 K) in which Mn is close to the +7 state. Excess-anion incorporation into Cs3CoCl5-type materials has not been previously reported, and this or other substitution mechanisms may enable many other high oxidation state transition metal nitrides to be prepared.

IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages.

Interleukin-36α (IL-36α) is essential for various inflammatory conditions, such as psoriasis and rheumatoid arthritis, whereas its role in tumor immunity is unclear. In this study, it was demonstrated that IL-36α could activate the NF-κB and MAPK signaling pathways in macrophages, leading to the expression of IL-1ß, IL-6, TNF-α, CXCL1, CXCL2, CXCL3, CXCL5 and iNOS. Importantly, IL-36α has significant antitumor effects, altering the tumor microenvironment and promoting the infiltration of MHC IIhigh macrophages and CD8+ T cells while decreasing the levels of monocyte myeloid-derived suppressor cells, CD4+ T cells and regulatory T cells. This ultimately results in the inhibition of tumor growth and migration. Furthermore, IL-36α synergized with the PD-L1 antibody increased the immune cells infiltration and enhanced the anti-tumor effect of the PD-L1 antibody on melanoma. Collectively, this study reveals a new role for IL-36α in promoting anti-tumor immune responses in macrophages and suggests its potential for cancer immunotherapy.

To stripe or not to stripe: the origin of a novel foliar pigmentation pattern in monkeyflowers (Mimulus).

The origin of phenotypic novelty is one of the most challenging problems in evolutionary biology. Although genetic regulatory network rewiring or co-option has been widely recognised as a major contributor, in most cases how such genetic rewiring/co-option happens is completely unknown. We have studied a novel foliar pigmentation pattern that evolved recently in the monkeyflower species Mimulus verbenaceus. Through genome-wide association tests using wild-collected samples, experimental crosses of laboratory inbred lines, gene expression analyses, and functional assays, we identified an anthocyanin-activating R2R3-MYB gene, STRIPY, as the causal gene triggering the emergence of the discrete, mediolateral anthocyanin stripe in the M. verbenaceus leaf. Chemical mutagenesis revealed the existence of upstream activators and repressors that form a 'hidden' prepattern along the leaf proximodistal axis, potentiating the unique expression pattern of STRIPY. Population genomics analyses did not reveal signatures of positive selection, indicating that nonadaptive processes may be responsible for the establishment of this novel trait in the wild. This study demonstrates that the origin of phenotypic novelty requires at least two separate phases, potentiation and actualisation. The foliar stripe pattern of M. verbenaceus provides an excellent platform to probe the molecular details of both processes in future studies.

A Tetratricopeptide Repeat Protein Regulates Carotenoid Biosynthesis and Chromoplast Development in Monkeyflowers (Mimulus).

Little is known about the factors regulating carotenoid biosynthesis in flowers. Here, we characterized the REDUCED CAROTENOID PIGMENTATION2 (RCP2) locus from two monkeyflower (Mimulus) species, the bumblebee-pollinated species Mimulus lewisii and the hummingbird-pollinated species Mimulus verbenaceus We show that loss-of-function mutations of RCP2 cause drastic down-regulation of the entire carotenoid biosynthetic pathway. The causal gene underlying RCP2 encodes a tetratricopeptide repeat protein that is closely related to the Arabidopsis (Arabidopsis thaliana) REDUCED CHLOROPLAST COVERAGE proteins. RCP2 appears to regulate carotenoid biosynthesis independently of RCP1, a previously identified R2R3-MYB master regulator of carotenoid biosynthesis. We show that RCP2 is necessary and sufficient for chromoplast development and carotenoid accumulation in floral tissues. Simultaneous down-regulation of RCP2 and two closely related paralogs, RCP2-L1 and RCP2-L2, yielded plants with pale leaves deficient in chlorophyll and carotenoids and with reduced chloroplast compartment size. Finally, we demonstrate that M. verbenaceus is just as amenable to chemical mutagenesis and in planta transformation as the more extensively studied M. lewisii, making these two species an excellent platform for comparative developmental genetics studies of closely related species with dramatic phenotypic divergence.