Synthetic developmental biology: molecular tools to re-design plant shoots and roots.

Plant morphology and anatomy strongly influence agricultural yield. Crop domestication has strived for desirable growth and developmental traits, such as larger and more fruits and semi-dwarf architecture. Genetic engineering has accelerated rational, purpose-driven engineering of plant development, but it can be unpredictable. Developmental pathways are complex and riddled with environmental and hormonal inputs, as well as feedback and feedforward interactions, which occur at specific times and places in a growing multicellular organism. Rational modification of plant development would probably benefit from precision engineering based on synthetic biology approaches. This review outlines recently developed synthetic biology technologies for plant systems and highlights their potential for engineering plant growth and development. Streamlined and high-capacity genetic construction methods (Golden Gate DNA Assembly frameworks and toolkits) allow fast and variation-series cloning of multigene transgene constructs. This, together with a suite of gene regulation tools (e.g. cell type-specific promoters, logic gates, and multiplex regulation systems), is starting to enable developmental pathway engineering with predictable outcomes in model plant and crop species.

Intra-arterial chemotherapy for retinoblastoma: Experience from the pediatric ophthalmology referral center in Malaysia with literature review.

PURPOSE: We aimed to analyze our 4-year experience of intra-arterial chemotherapy (IAC) for retinoblastoma (RB) and to examine the tumor response, globe salvage, mortality, and safety profile of IAC in the Malaysian profile. MATERIALS AND METHODS: This was a retrospective, interventional case series. A total of 22 eyes of 20 patients with RB who underwent IAC using melphalan and topotecan from January 2018 to December 2021 in Hospital Kuala Lumpur were retrospectively reviewed. Tumor response, globe salvage, mortality, and safety profile of IAC were compared based on the International Classification of Retinoblastoma. RESULTS: The mean patient age at IAC was 21.3 months. An overall globe salvage rate of 63.6% was observed: more specifically, 100% for Group A, 75% for Groups B and C, 66.7% for Group D, and 42.9% for Group E. Poor tumor response after IAC was significantly associated with a lesser chance of globe salvage (P = 0.045). The overall rate of good tumor response following IAC was 77.3%. Specifically, rates of good tumor response in each group were 100%, 75%, 75%, 83.3% and 71.4% in group A, B, C, D and E, respectively. The mortality rate was 5%. Complications (per-catheterization) included cerebral infarct (2.2%), oxygen desaturation (2.2%), vomiting (26.1%), periorbital edema (8.8%), ptosis (6.5%), fever, femoral hematoma, and hyperpigmentation over lid (4.4% each). CONCLUSION: Four-year experience showed that IAC is a safe and effective method for RB management. Patients with a poor response after IAC may have a lower chance of globe salvage. Careful patient selection is of utmost importance to achieve the best outcome in a setting of limited health-care resources.

Mechanical conflict caused by a cell-wall-loosening enzyme activates de novo shoot regeneration.

Cellular heterogeneity is a hallmark of multicellular organisms. During shoot regeneration from undifferentiated callus, only a select few cells, called progenitors, develop into shoot. How these cells are selected and what governs their subsequent progression to a patterned organ system is unknown. Using Arabidopsis thaliana, we show that it is not just the abundance of stem cell regulators but rather the localization pattern of polarity proteins that predicts the progenitor's fate. A shoot-promoting factor, CUC2, activated the expression of the cell-wall-loosening enzyme, XTH9, solely in a shell of cells surrounding the progenitor, causing different mechanical stresses in these cells. This mechanical conflict then activates cell polarity in progenitors to promote meristem formation. Interestingly, genetic or physical perturbations to cells surrounding the progenitor impaired the progenitor and vice versa. These suggest a feedback loop between progenitors and their neighbors for shoot regeneration in the absence of tissue-patterning cues.

Quantitative Hole Mobility Simulation and Validation in Substituted Acenes.

Knowledge of the full phonon spectrum is essential to accurately calculate the dynamic disorder (σ) and hole mobility (µh) in organic semiconductors (OSCs). However, most vibrational spectroscopy techniques under-measure the phonons, thus limiting the phonon validation. Here, we measure and model the full phonon spectrum using multiple spectroscopic techniques and predict µh using σ from only the Γ-point and the full Brillouin zone (FBZ). We find that only inelastic neutron scattering (INS) provides validation of all phonon modes, and that σ in a set of small molecule semiconductors can be miscalculated by up to 28% when comparing Γ-point against FBZ calculations. A subsequent mode analysis shows that many modes contribute to σ and that no single mode dominates. Our results demonstrate the importance of a thoroughly validated phonon calculation, and a need to develop design rules considering the full spectrum of phonon modes.

Age, Wound Size, and Position of Injury - Dependent Vascular Regeneration Assay in Growing Leaves.

Recurring damage to the aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assays for aerial plant parts such as the stem and inflorescence stalk are well established, those for leaf vasculature remain unexplored. Recently, we established a new vascular regeneration assay in growing leaves and discovered the underlying molecular mechanism. Here, we describe the detailed stepwise method for the incision and regeneration assay used to study leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy, and other experimental approaches, we further show that the age of the leaf as well as the position and size of the injury determine the overall success rate of regeneration. This easy-to-master vascular regeneration assay is an efficient and rapid method to study the mechanism of vascular regeneration in growing leaves. The assay can be readily combined with cellular and molecular biology techniques.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

Regrowing the damaged or lost body parts.

Plants display extraordinary ability to revive tissues and organs lost or damaged in injury. This is evident from the root tip restoration and classical experiments in stem demonstrating re-establishment of vascular continuity. While recent studies have begun to unravel the mechanistic understanding of tissue restoration in response to injury in underground plant organs, the molecular mechanisms of the same in aerial organs remain to be ventured deeper. Here, we discuss the possibility of unearthing the regulatory mechanism that can confer universal regeneration potential to plant body and further provide a comprehensive understanding of how tissue and organ regeneration gets triggered in response to mechanical injury and later gets terminated after re-patterning and regaining the appropriate size.

A Study on the Impact of the Novel Biochemical Parameter-Calcium Score in Preventing the Progression of the Cardiovascular Diseases to Invasive Interventions.

Cardiovascular diseases is increasing its pace day by day. Though the traditional biomarkers are made available the novel biomarkers are being incorporated to predict the risk of cardiovascular diseases for earlier detection. The present study aimed to investigate the impact of calcium score level the novel biochemical parameter in preventing the progression of the cardiovascular patients to PTCA (percutaneous transluminal coronary angioplasty) and CABG (coronary artery bypass grafting). Four hundred cardiovascular patients irrespective of sex were randomly selected from Visakhapatnam district Andhrapradesh. Information of subjects was collected using an interview schedule. Data collected were consolidated and tabulated. From this group a sub sample of 50 patients was selected and grouped as primordial, secondary and post PTCA. The subjects were then analyzed for their biochemical parameters before and after intervention. Statistical analysis was done and interpreted. An extensive evidence of calcium score was shown among 61.2% of the cardiovascular patients, a minimum evidence of 22.2% and moderate evidence of about 16.5%. The paired sample t-test is employed to observe any statistical significant difference between the before and after treatment effects. The analysis for the calcium score level was found to be significantly lower (mean difference=424.0134; t=13.297; df=49; p=0.01) in post intervention (mean=88.3766±88.40) than pre-intervention (512.39±260.79812). The present study identified calcium score the novel biochemical parameter as a key preventive measure among the usual biochemical management conducted by the clinicians to diagnosis and confirm the progression of the disease.

Correlation between Color and Elasticity in Anomia ephippium Shells: Biological Design to Enhance the Mechanical Properties.

Anomia ephippium (Oyster) shell has an inorganic "pseudo nacre" microstructure that is colored (white, yellow, orange, and red) with varying concentrations of organic polyene pigments. Using Brillouin laser light scattering, we measured the elastic constants of A. ephippium and Abalone prismatic and nacre microstructures. We find a direct correlation between the polyene concentration and the measured elastic stiffnesses of A. ephippium shells, suggesting that polyene pigment enhances the shell's primary function of mechanical protection. Further, Young's, bulk, and shear moduli of A. ephippium shells are found to surpass that of ultrastrong Abalone nacre. By studying both microstructures, we demonstrate that pseudo nacre is stiffer than nacre. This work sheds light on the structure-property relation of pseudo nacre and organic constituents that may potentially be a paradigm shift for the future mimicry of super strong biomaterials.

Intermediate Developmental Phases During Regeneration.

The initial view that regeneration can be a continuum in terms of regulatory mechanisms is gradually changing, and recent evidence points towards the presence of discrete regulatory steps and intermediate phases. Furthermore, regeneration presents an excellent example of a process generating order and pattern, i.e. a self-organization process. It is likely that the process traverses a set of intermediate phases before reaching an endpoint. Although some progress has been made in deciphering the identity of these intermediate phases, a lot more work is needed to derive a comprehensive and complete picture. Here, we discuss the intermediate developmental phases in plant regeneration and compare them with the possible intermediate developmental phases in animal regeneration.

Fungal Production and Manipulation of Plant Hormones.

Living organisms are part of a highly interconnected web of interactions, characterised by species nurturing, competing, parasitizing and preying on one another. Plants have evolved cooperative as well as defensive strategies to interact with neighbour organisms. Among these, the plant-fungus associations are very diverse, ranging from pathogenic to mutualistic. Our current knowledge of plant-fungus interactions suggests a sophisticated coevolution to ensure dynamic plant responses to evolving fungal mutualistic/pathogenic strategies. The plant-fungus communication relies on a rich chemical language. To manipulate the plant defence mechanisms, fungi produce and secrete several classes of biomolecules, whose modeof- action is largely unknown. Upon perception of the fungi, plants produce phytohormones and a battery of secondary metabolites that serve as defence mechanism against invaders or to promote mutualistic associations. These mutualistic chemical signals can be co-opted by pathogenic fungi for their own benefit. Among the plant molecules regulating plant-fungus interaction, phytohormones play a critical role since they modulate various aspects of plant development, defences and stress responses. Intriguingly, fungi can also produce phytohormones, although the actual role of fungalproduced phytohormones in plant-fungus interactions is poorly understood. Here, we discuss the recent advances in fungal production of phytohormone, their putative role as endogenous fungal signals and how fungi manipulate plant hormone balance to their benefits.

Shoot regeneration: a journey from acquisition of competence to completion.

Plants display an extraordinary ability to regenerate complete shoot systems from a tissue fragment or even from a single cell. Upregulation of the determinants of pluripotency during a precise window of time in response to external inductive cues is a key decisive factor for shoot regeneration. A burst of recent studies has begun to provide an understanding of signaling molecules that are instrumental in the making of the regenerative mass, as well as the developmental regulators that are seminal in shaping the pluripotent state. Here, we discuss how signaling molecules, waves of mutually exclusive stem cell regulators and epigenetic modifiers could contribute to cellular heterogeneity in an island of regenerative mass, thus leading to de novo shoot regeneration.

De novo assembly of plant body plan: a step ahead of Deadpool.

While in the movie Deadpool it is possible for a human to recreate an arm from scratch, in reality plants can even surpass that. Not only can they regenerate lost parts, but also the whole plant body can be reborn from a few existing cells. Despite the decades old realization that plant cells possess the ability to regenerate a complete shoot and root system, it is only now that the underlying mechanisms are being unraveled. De novo plant regeneration involves the initiation of regenerative mass, acquisition of the pluripotent state, reconstitution of stem cells and assembly of regulatory interactions. Recent studies have furthered our understanding on the making of a complete plant system in the absence of embryonic positional cues. We review the recent studies probing the molecular mechanisms of de novo plant regeneration in response to external inductive cues and our current knowledge of direct reprogramming of root to shoot and vice versa. We further discuss how de novo regeneration can be exploited to meet the demands of green culture industries and to serve as a general model to address the fundamental questions of regeneration across the plant kingdom.

Protocol: a method to study the direct reprogramming of lateral root primordia to fertile shoots.

BACKGROUND: Plants have the remarkable property to elaborate entire body plan from any tissue part. The conversion of lateral root primordium (LRP) to shoot is an ideal method for plant propagation and for plant researchers to understand the mechanism underlying trans-differentiation. Until now, however, a robust method that allows the efficient conversion of LRP to shoot is lacking. This has limited our ability to study the dynamic phases of reprogramming at cellular and molecular levels. RESULTS: Here we present an efficient protocol for the direct conversion of LRP to a complete fertile shoot system. This protocol can be readily applied to the various ecotypes of Arabidopsis. We show that, the conversion process is highly responsive to developmental stages of LRP and changes in external environmental stimuli such as temperature. The entire conversion process can be adequately analyzed by histological and imaging techniques. As a demonstration, using a battery of cell fate specific markers, we show that confocal time-lapse imaging can be employed to uncover the early molecular events, intermediate developmental phases and relative abundance of stem cell regulators during the conversion of LRP to shoot. CONCLUSION: Our method is highly efficient, independent of genotypes tested and suitable to study the reprogramming of LRP to shoot in intact plants as well as in excised roots.

Guest dependent Brillouin and Raman scattering studies of zeolitic imidazolate framework-8 (ZIF-8) under external pressure.

We have investigated the pressure dependence of the acoustic modes of zeolitic imidazolate framework (ZIF-8) in different pressure transmitting mediums and also under non-hydrostatic conditions using high pressure Brillouin spectroscopy. Our study shows the pressure induced flexibility and dynamics of ZIF-8 framework as well as a huge increase in the acoustic velocities on applying external pressure, illustrating the role of guest in enhancing the elastic properties of the framework. In fact, the elastic constant C11 of the guest incorporated ZIF-8 increases by ∼183% on applying a pressure of only 1.47 GPa. The pressure transmitting medium also plays an important role in controlling the gate opening behaviour of ZIF-8. Pressure dependent Raman study shows significant changes in the modes of ZIF-8 as well as that of that of the pressure transmitting medium which is entrapped within the framework, indicating that the interaction between the framework and guest is responsible for the medium dependent changes observed in the Brillouin spectra.

Effect of pore occupancy on the acoustic properties of zeolitic imidazolate framework (ZIF)-8: A Brillouin spectroscopic study at ambient and low temperatures.

Brillouin spectroscopy is used to study the effect of pore occupancy on the elastic constants by incorporating various guest molecules into zeolitic imidazolate framework (ZIF)-8. A systematic study on the effect of mass and polarizability of the guest has been carried out by incorporating alcohols of varying chain lengths at room temperature. The interaction between the guest and host affects the elastic properties, lifetimes and guest dynamics inside the pores. The elastic anisotropy was seen to reduce upon incorporation of the guests. We have also studied the temperature dependence of the acoustic modes on gas adsorption to understand the framework flexibility. The Brillouin shift of the acoustic modes increases upon temperature dependent gas adsorption with transverse acoustic modes exhibiting a larger shift. This suggests a hardening of otherwise low shear modulus of ZIF-8. Our findings give insight into the role of guest molecules and temperature in tuning the elastic properties of ZIF-8 which is important for practical applications.