Understanding the Genetic Basis of C4 Kranz Anatomy with a View to Engineering C3 Crops.

One of the most remarkable examples of convergent evolution is the transition from C3 to C4 photosynthesis, an event that occurred on over 60 independent occasions. The evolution of C4 is particularly noteworthy because of the complexity of the developmental and metabolic changes that took place. In most cases, compartmentalized metabolic reactions were facilitated by the development of a distinct leaf anatomy known as Kranz. C4 Kranz anatomy differs from ancestral C3 anatomy with respect to vein spacing patterns across the leaf, cell-type specification around veins, and cell-specific organelle function. Here we review our current understanding of how Kranz anatomy evolved and how it develops, with a focus on studies that are dissecting the underlying genetic mechanisms. This research field has gained prominence in recent years because understanding the genetic regulation of Kranz may enable the C3-to-C4 transition to be engineered, an endeavor that would significantly enhance crop productivity.

Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW during leaf patterning in C4 grasses.

Leaves comprise a number of different cell-types that are patterned in the context of either the epidermal or inner cell layers. In grass leaves, two distinct anatomies develop in the inner leaf tissues depending on whether the leaf carries out C3 or C4 photosynthesis. In both cases a series of parallel veins develops that extends from the leaf base to the tip but in ancestral C3 species veins are separated by a greater number of intervening mesophyll cells than in derived C4 species. We have previously demonstrated that the GRAS transcription factor SCARECROW (SCR) regulates the number of photosynthetic mesophyll cells that form between veins in the leaves of the C4 species maize, whereas it regulates the formation of stomata in the epidermal leaf layer in the C3 species rice. Here we show that SCR is required for inner leaf patterning in the C4 species Setaria viridis but in this species the presumed ancestral stomatal patterning role is also retained. Through a comparative mutant analysis between maize, setaria and rice we further demonstrate that loss of NAKED-ENDOSPERM (NKD) INDETERMINATE DOMAIN (IDD) protein function exacerbates loss of function scr phenotypes in the inner leaf tissues of maize and setaria but not rice. Specifically, in both setaria and maize, scr;nkd mutants exhibit an increased proportion of fused veins with no intervening mesophyll cells. Thus, combined action of SCR and NKD may control how many mesophyll cells are specified between veins in the leaves of C4 but not C3 grasses. Together our results provide insight into the evolution of cell patterning in grass leaves and demonstrate a novel patterning role for IDD genes in C4 leaves.

SCARECROW is deployed in distinct contexts during rice and maize leaf development.

The flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is downregulated in Osscr1;Osscr2 mutants, indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants, and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

Redundant SCARECROW genes pattern distinct cell layers in roots and leaves of maize.

The highly efficient C4 photosynthetic pathway is facilitated by 'Kranz' leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here, we demonstrate that, in the C4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeologue: ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both the leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate - mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature.

A genetically encoded near-infrared fluorescent calcium ion indicator.

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators.

MetAP2 inhibitor treatment of high-fat and -fructose-fed dogs: impact on the response to oral glucose ingestion and a hyperinsulinemic hyperglycemic clamp.

We examined the methionine aminopeptidase 2 inhibitor fumagillin in dogs consuming a high-fat and -fructose diet (HFFD). In pilot studies (3 dogs that had consumed HFFD for 3 yr), 8 wk of daily treatment with fumagillin reduced food intake 29%, weight 6%, and the glycemic excursion during an oral glucose tolerance test (OGTT) 44%. A second group of dogs consumed the HFFD for 17 wk: pretreatment (weeks 0-4), treatment with fumagillin (FUM; n = 6), or no drug (Control, n = 8) (weeks 4-12), washout period (weeks 12-16), and fumagillin or no drug for 1 wk (week 17). OGTTs were performed at 0, 4, 11, and 16 wk. A hyperinsulinemic hyperglycemic clamp was performed in week 12; 4 chow-fed dogs underwent identical clamps. Kilocalories per day intake during the treatment period was 2,067 ± 50 (Control) versus 1,824 ± 202 (FUM). Body weights (kg) increased 1.9 ± 0.3 vs. 2.7 ± 0.8 (0-4 wk) and 1.2 ± 0.2 vs. -0.02 ± 0.9 (4-12 wk) in Control versus fumagillin. The OGTT glycemic response was 30% greater in Control versus fumagillin at 11 wk. Net hepatic glucose uptake (NHGU; mg·kg-1·min-1) in the Chow, Control, and fumagillin dogs was ~1.5 ± 0.6, -0.1 ± 0.1, and 0.3 ± 0.4 (with no portal glucose infusion) and 3.1 ± 0.6, 0.5 ± 0.3, and 1.5 ± 0.5 (portal glucose infusion at 4 mg·kg-1·min-1), respectively. Fumagillin improved glucose tolerance and NHGU in HFFD dogs, suggesting methionine aminopeptidase 2 (MetAP2) inhibitors have the potential for improving glycemic control in prediabetes and diabetes.

Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases.

Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (n = 2) or with unknown underlying genetic defect (n = 3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients.

Depth and durability of response to ibrutinib in CLL: 5-year follow-up of a phase 2 study.

The safety and efficacy of ibrutinib (420 mg) in chronic lymphocytic leukemia (CLL) were evaluated in a phase 2 study; 51 patients had TP53 aberration (TP53 cohort) and 35 were enrolled because of age 65 years or older (elderly cohort). Both cohorts included patients with treatment-naive (TN) and relapsed/refractory (RR) CLL. With the median follow-up of 4.8 years, 49 (57.0%) of 86 patients remain on study. Treatment was discontinued for progressive disease in 20 (23.3%) patients and for adverse events in 5 (5.8%). Atrial fibrillation occurred in 18 (20.9%) patients for a rate of 6.4 per 100 patient-years. No serious bleeding occurred. The overall response rate at 6 months, the primary study endpoint, was 95.8% for the TP53 cohort (95% confidence interval, 85.7%-99.5%) and 93.9% for the elderly cohort (95% confidence interval, 79.8%-99.3%). Depth of response improved with time: at best response, 14 (29.2%) of 48 patients in the TP53 cohort and 9 (27.3%) of 33 in the elderly cohort achieved a complete response. Median minimal residual disease (MRD) in peripheral blood was 3.8 × 10-2 at 4 years, with MRD-negative (<10-4) remissions in 5 (10.2%) patients. In the TP53 cohort, the estimated 5-year progression-free survival (PFS) was 74.4% in TN-CLL compared with 19.4% in RR-CLL (P = .0002), and overall survival (OS) was 85.3% vs 53.7%, respectively (P = .023). In the elderly cohort, the estimated 5-year PFS and OS in RR-CLL were 64.8% and 71.6%, respectively, and no event occurred in TN-CLL. Long-term administration of ibrutinib was well tolerated and provided durable disease control for most patients. This trial was registered at www.clinicaltrials.gov as #NCT01500733.

Correction to: A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

Understanding the Fluorescence Change in Red Genetically Encoded Calcium Ion Indicators.

For over 20 years, genetically encoded Ca2+ indicators have illuminated dynamic Ca2+ signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand how they work. Various fluorescence colors of these indicators have been developed, including red. Red ones are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a red fluorescent protein that is circularly permuted and fused to a Ca2+-sensing domain. When Ca2+ binds, a conformational change in the sensing domain causes a change in fluorescence. Three factors can contribute to this fluorescence change: 1) a shift in the protonation equilibrium of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient or the two-photon cross section, depending on if it is excited with one or two photons. Here, we conduct a systematic study of the photophysical properties of a range of red Ca2+ indicators to determine which factors are the most important. In total, we analyzed nine indicators, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We find that these could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases, the magnitude of the change in fluorescence was larger with two-photon excitation compared to one-photon because of a change in the two-photon cross section, by up to a factor of two.

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics.

Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca2+ entry through a Gd3+-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca2+ entry.

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

Preclinical Efficacy and Safety of the Novel Antidiabetic, Antiobesity MetAP2 Inhibitor ZGN-1061.

Methionine aminopeptidase 2 (MetAP2) inhibition is a promising approach to treating diabetes, obesity, and associated metabolic disorders. Beloranib, a MetAP2 inhibitor previously investigated for treatment of Prader-Willi syndrome, was associated with venous thrombotic adverse events likely resulting from drug effects on vascular endothelial cells (ECs). Here, we report the pharmacological characterization of ZGN-1061, a novel MetAP2 inhibitor being investigated for treatment of diabetes and obesity. Four weeks of subcutaneous administration of ZGN-1061 to diet-induced obese (DIO) insulin-resistant mice produced a 25% reduction in body weight, primarily due to reduced fat mass, that was comparable to beloranib. ZGN-1061 also produced improvements in metabolic parameters, including plasma glucose and insulin, and, in HepG2 cells, initiated gene changes similar to beloranib that support observed in vivo pharmacodynamics. In vitro studies in ECs demonstrated that ZGN-1061 effects on EC proliferation and coagulation proteins were greatly attenuated, or absent, relative to beloranib, due to lower intracellular drug concentrations, shorter half-life of inhibitor-bound MetAP2 complex, and reduced cellular enzyme inhibition. In dogs, ZGN-1061 was more rapidly absorbed and cleared, with a shorter half-life than beloranib. Unlike beloranib, ZGN-1061 did not increase coagulation markers in dogs, and ZGN-1061 had a greatly improved safety profile in rats relative to beloranib. In conclusion, ZGN-1061 and beloranib demonstrated similar efficacy in a mouse model of obesity, while ZGN-1061 had a markedly improved safety profile in multiple in vitro and in vivo models. The lower duration of exposure characteristic of ZGN-1061 is expected to provide a meaningfully enhanced clinical safety profile.

Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange.

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca(2+) concentration dynamics and other second-messenger signaling activities.

Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes in Neuroimaging.

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in the mammalian brain and can be readily applied to neuroimaging.

Haploidentical Related Donor Hematopoietic Stem Cell Transplantation for Dedicator-of-Cytokinesis 8 Deficiency Using Post-Transplantation Cyclophosphamide.

Dedicator-of-cytokinesis 8 (DOCK8) deficiency, a primary immunodeficiency disease, can be reversed by allogeneic hematopoietic stem cell transplantation (HSCT); however, there are few reports describing the use of alternative donor sources for HSCT in DOCK8 deficiency. We describe HSCT for patients with DOCK8 deficiency who lack a matched related or unrelated donor using bone marrow from haploidentical related donors and post-transplantation cyclophosphamide (PT/Cy) for graft-versus-host disease (GVHD) prophylaxis. Seven patients with DOCK8 deficiency (median age, 20 years; range, 7 to 25 years) received a haploidentical related donor HSCT. The conditioning regimen included 2 days of low-dose cyclophosphamide, 5 days of fludarabine, 3 days of busulfan, and 200 cGy total body irradiation. GVHD prophylaxis consisted of PT/Cy 50 mg/kg/day on days +3 and +4 and tacrolimus and mycophenolate mofetil starting at day +5. The median times to neutrophil and platelet engraftment were 15 and 19 days, respectively. All patients attained >90% donor engraftment by day +30. Four subjects developed acute GVHD (1 with maximum grade 3). No patient developed chronic GVHD. With a median follow-up time of 20.6 months (range, 9.5 to 31.7 months), 6 of 7 patients are alive and disease free. Haploidentical related donor HSCT with PT/Cy represents an effective therapeutic approach for patients with DOCK8 deficiency who lack a matched related or unrelated donor.

The impact of widespread regulatory neofunctionalization on homeolog gene evolution following whole-genome duplication in maize.

Whole-genome duplications are a widespread feature of plant genome evolution, having been detected in all flowering plant lineages. Despite the prevalence of these events, the extent to which duplicated genes (homeolog gene pairs) functionally diverge (neofunctionalization) is unclear. We present a genome-wide analysis of molecular evolution and regulatory neofunctionalization in maize (Zea mays L.). We demonstrate that 13% of all homeolog gene pairs in maize are regulatory neofunctionalized in leaves, and that regulatory neofunctionalized genes experience enhanced purifying selection. We show that significantly more genes have been regulatory neofunctionalized in foliar leaves than in husk leaves and that both leaf types have experienced selection for distinct functional roles. Furthermore, we demonstrate that biased subgenome expression dominance occurs only in the presence of regulatory neofunctionalization and that in nonregulatory neofunctionalized genes subgenome dominance is progressively acquired during development. Taken together, our study reveals several novel insights into the evolution of maize, genes, and gene expression, and provides a general model for gene evolution following whole-genome duplication in plants.

C4 Photosynthesis in the Rice Paddy: Insights from the Noxious Weed Echinochloa glabrescens.

The C4 pathway is a highly complex trait that increases photosynthetic efficiency in more than 60 plant lineages. Although the majority of C4 plants occupy disturbed, arid, and nutrient-poor habitats, some grow in high-nutrient, waterlogged conditions. One such example is Echinochloa glabrescens, which is an aggressive weed of rice paddies. We generated comprehensive transcriptome datasets for C4 E. glabrescens and C3 rice to identify genes associated with adaption to waterlogged, nutrient-replete conditions, but also used the data to better understand how C4 photosynthesis operates in these conditions. Leaves of E. glabrescens exhibited classical Kranz anatomy with lightly lobed mesophyll cells having low chloroplast coverage. As with rice and other hygrophytic C3 species, leaves of E. glabrescens accumulated a chloroplastic phosphoenolpyruvate carboxylase protein, albeit at reduced amounts relative to rice. The arid-grown species Setaria italica (C4) and Brachypodium distachyon (C3) were also found to accumulate chloroplastic phosphoenolpyruvate carboxylase. We identified a molecular signature associated with C4 photosynthesis in nutrient-replete, waterlogged conditions that is highly similar to those previously reported from C4 plants that grow in more arid conditions. We also identified a cohort of genes that have been subjected to a selective sweep associated with growth in paddy conditions. Overall, this approach highlights the value of using wild species such as weeds to identify adaptions to specific conditions associated with high-yielding crops in agriculture.