A Full Evaporation Static Headspace Gas Chromatography Method with Nitrogen Phosphorous Detection for Ultrasensitive Analysis of Semi-volatile Nitrosamines in Pharmaceutical Products.

The recent detection of potent carcinogenic nitrosamine impurities in several human medicines has triggered product recalls and interrupted the supply of critical medications for hundreds of millions of patients, illuminating the need for increased testing of nitrosamines in pharmaceutical products. However, the development of analytical methods for nitrosamine detection is challenging due to high sensitivity requirements, complex matrices, and the large number and variety of samples requiring testing. Herein, we report an analytical method for the analysis of a common nitrosamine, N-nitrosodimethylamine (NDMA), in pharmaceutical products using full evaporation static headspace gas chromatography with nitrogen phosphorous detection (FE-SHSGC-NPD). This method is sensitive, specific, accurate, and precise and has the potential to serve as a universal method for testing all semi-volatile nitrosamines across different drug products. Through elimination of the detrimental headspace-liquid partition, a quantitation limit of 0.25 ppb is achieved for NDMA, a significant improvement upon traditional LC-MS methods. The extraction of nitrosamines directly from solid sample not only simplifies the sample preparation procedure but also enables the method to be used for different products as is or with minor modifications, as demonstrated by the analysis of NDMA in 10+ pharmaceutical products. The in situ nitrosation that is commonly observed in GC methods for nitrosamine analysis was completely inhibited by the addition of a small volume solvent containing pyrogallol, phosphoric acid, and isopropanol. Employing simple procedures and low-cost instrumentation, this method can be implemented in any analytical laboratory for routine nitrosamine analysis, ensuring patient safety and uninterrupted supply of critical medications. Graphical Abstract.

SHORT-ROOT paralogs mediate feedforward regulation of D-type cyclin to promote nodule formation in soybean.

Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.

Ground tissue circuitry regulates organ complexity in maize and Setaria.

Most plant roots have multiple cortex layers that make up the bulk of the organ and play key roles in physiology, such as flood tolerance and symbiosis. However, little is known about the formation of cortical layers outside of the highly reduced anatomy of Arabidopsis. Here, we used single-cell RNA sequencing to rapidly generate a cell-resolution map of the maize root, revealing an alternative configuration of the tissue formative transcription factor SHORT-ROOT (SHR) adjacent to an expanded cortex. We show that maize SHR protein is hypermobile, moving at least eight cell layers into the cortex. Higher-order SHR mutants in both maize and Setaria have reduced numbers of cortical layers, showing that the SHR pathway controls expansion of cortical tissue to elaborate anatomical complexity.

Teleneurology Expansion in Response to the COVID-19 Outbreak at a Tertiary Health System in New York City.

OBJECTIVE: To assess the implementation of teleneurology (TN), including patient and clinician experiences, during the coronavirus respiratory disease 2019 (COVID-19) pandemic. METHODS: We studied synchronous (video visit) and asynchronous (store-and-forward, patient-portal evaluation, remote monitoring) TN utilization in the Mount Sinai Health System Neurology Department in New York, 2 months before and after the start of our department's response to the pandemic in mid-March 2020. Weekly division meetings enabled ongoing assessments and analysis of barriers and facilitators according to the Consolidated Framework for Implementation Research and the Expert Recommendations for Implementing Change models. We used postvisit surveys of clinicians (from April 13 to May 15, 2020) and patients (from May 11 to 15, 2020) to determine technology platforms used, and TN experience and acceptability, using Likert scales (1 = very poor/unlikely to 5 = very good/likely). RESULTS: Over the 4-month period, 117 TN clinicians (n = 14 subspecialties) conducted 4,225 TN visits with 3,717 patients (52 pre- vs 4,173 post-COVID-19). No asynchronous TN services were delivered. Post-COVID-19, the number of TN clinicians, subspecialties performing TN, and visits increased by 963%, 133%, and 7,925%, respectively. Mean acceptability among patients and clinicians was 4.7 (SD 0.6) and 3.4 (SD 1.6), respectively. Most video visits were completed using Epic MyChart (78.5%) and Zoom (8.1%). TN implementation facilitators included Medicare geographic restriction waivers, development of clinician educational materials, and MyChart outreach programs for patients experiencing technical difficulties. CONCLUSIONS: A significant expansion of TN utilization accompanied the COVID-19 response. Patients found TN more acceptable than did clinicians. Proactive application of an implementation framework facilitated rapid and effective TN expansion.

Gateway-compatible vectors for functional analysis of proteins in cell type specific manner.

BACKGROUND: Genetically encoded fluorescent proteins are often used to label proteins and study protein function and localization in vivo. Traditional cloning methods mediated by restriction digestion and ligation are time-consuming and sometimes difficult due to the lack of suitable restriction sites. Invitrogen developed the Gateway cloning system based on the site-specific DNA recombination, which allows for digestion-free cloning. Most gateway destination vectors available for use in plants employ either the 35S or ubiquitin promoters, which confer high-level, ubiquitous expression. There are far fewer options for moderate, cell-type specific expression. RESULTS: Here we report on the construction of a Gateway-compatible cloning system (SWU vectors) to rapidly tag various proteins and express them in a cell-type specific manner in plants. We tested the SWU vectors using the HISTONE (H2B) coding sequence in stable transgenic plants. CONCLUSIONS: The SWU vectors are a valuable tool for low cost, high efficiency functional analysis of proteins of interest in specific cell types in the Arabidopsis root.

Protein complex stoichiometry and expression dynamics of transcription factors modulate stem cell division.

Stem cells divide and differentiate to form all of the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the quiescent center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here we gained insight into the differences between the QC and the cortex endodermis initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an ordinary differential equation model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction, coupled with experimental validation, showed that high levels of the SHR-SCR complex are associated with more CEI division but less QC division. Furthermore, our model prediction allowed us to propose the putative upstream SHR regulators SEUSS and WUSCHEL-RELATED HOMEOBOX 5 and to experimentally validate their roles in QC and CEI division. In addition, our model established the timing of QC and CEI division and suggests that SHR repression of QC division depends on formation of the SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.

A role for the endoplasmic reticulum in the cell-to-cell movement of SHORT-ROOT.

Plasmodesmata enable the trafficking of various signaling molecules, as well as viruses that exploit these channels for their intercellular movement. Viral movement relies on the endoplasmic reticulum (ER), which serves as a stable platform for the assembly of viral replication complexes and their subsequent shuttling toward plasmodesmata. The role of the ER in the intercellular movement of endogenous proteins is less clear. In the root meristem, the mobile transcription factor SHORT-ROOT (SHR) traffics between cell layers to regulate root radial patterning and differentiation. Movement of SHR is a regulated process that requires several cellular factors including the endomembrane system, intact microtubules and an endosome-associated protein named SHR-interacting-embryonic-lethal (SIEL). Recently, we found that KINESIN G (KinG) interacts with both SIEL and microtubules to support the cell-to-cell movement of SHR. Here, we provide evidence that both SHR-associated endosomes and KinG localize to the endoplasmic reticulum (ER) and that movement of SHR-associated endosomes occurs on the ER. Moreover, we show that compromised ER structure leads to a reduction in the cell-to-cell movement of SHR. Collectively, these results support the hypothesis that the ER plays a role in SHR movement.

Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose.

Both plants and animals must contend with changes in their environment. The ability to respond appropriately to these changes often underlies the ability of the individual to survive. In plants, an early response to environmental stress is an alteration in plasmodesmatal permeability with accompanying changes in cell to cell signaling. However, the ways in which plasmodesmata are modified, the molecular players involved in this regulation, and the biological significance of these responses are not well understood. Here, we examine the effects of nutrient scarcity and excess on plasmodesmata-mediated transport in the Arabidopsis thaliana root and identify two CALLOSE SYNTHASES and two ß-1,3-GLUCANASES as key regulators of these processes. Our results suggest that modification of plasmodesmata-mediated signaling underlies the ability of the plant to maintain root growth and properly partition nutrients when grown under conditions of excess nutrients.

KinG Is a Plant-Specific Kinesin That Regulates Both Intra- and Intercellular Movement of SHORT-ROOT.

Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR.

Symplastic signaling instructs cell division, cell expansion, and cell polarity in the ground tissue of Arabidopsis thaliana roots.

Cell-to-cell communication is essential for the development and patterning of multicellular organisms. In plants, plasmodesmata (PD) provide direct routes for intercellular signaling. However, the role that PD-mediated signaling plays in plant development has not been fully investigated. To gain a comprehensive view of the role that symplastic signaling plays in Arabidopsis thaliana, we have taken advantage of a synthetic allele of CALLOSE SYNTHASE3 (icals3m) that inducibly disrupts cell-to-cell communication specifically at PD. Our results show that loss of symplastic signaling to and from the endodermis has very significant effects on the root, including an increase in the number of cell layers in the root and a misspecification of stele cells, as well as ground tissue. Surprisingly, loss of endodermal signaling also results in a loss of anisotropic elongation in all cells within the root, similar to what is seen in radially swollen mutants. Our results suggest that symplastic signals to and from the endodermis are critical in the coordinated growth and development of the root.

Delaying in vivo exposure to a tarantula with very brief exposure to phobic stimuli.

BACKGROUND AND OBJECTIVES: Research has documented the very brief exposure (VBE) effect: the reduction of phobic fear by continuous presentation of masked phobic pictures. In prior studies, phobic participants approached a live tarantula immediately after the masked stimuli were presented. This study tested the hypothesis that VBE would reduce phobic avoidance of the tarantula 24 h later. METHOD: 86 spider-phobic participants were identified with a fear questionnaire and Behavioral Avoidance Test (BAT) with a live tarantula indicative of a DSM-IV diagnosis of Specific Phobia. One week later, they were randomly assigned in double-blind fashion to presentation of a continuous series of 25 trials of masked images of either spiders or flowers (33-ms each), i.e., to VBE or control exposure. The participants gave subjective distress ratings just before and after these exposures. Then they engaged in the BAT again either immediately thereafter or 24 h later to measure changes in avoidance of the tarantula. RESULTS: Masked images of spiders reduced avoidance of the tarantula both immediately after exposure and 24 h later without causing subjective distress. The effect sizes at these two time points did not significantly differ from each other. LIMITATIONS: We did not manipulate awareness of the spider images by presenting them unmasked to a third group. Conclusions about the effect of awareness of the stimuli cannot be drawn. CONCLUSIONS: VBE induces a process of fear reduction before phobic individuals engage in in vivo exposure, which is more distressing. Thus, VBE may help phobic-resistant individuals start treatment more gradually.

Techniques for assessing the effects of pharmacological inhibitors on intercellular protein movement.

Intercellular protein movement is an important mechanism in plant development. Here we present an integrated protocol that utilizes an inducible system to block plasmodesmata-dependent movement and assessment of fluorescent recovery after photobleaching (FRAP) to identify compounds that influence intercellular protein movement.

A plausible mechanism, based upon Short-Root movement, for regulating the number of cortex cell layers in roots.

Formation of specialized cells and tissues at defined times and in specific positions is essential for the development of multicellular organisms. Often this developmental precision is achieved through intercellular signaling networks, which establish patterns of differential gene expression and ultimately the specification of distinct cell fates. Here we address the question of how the Short-root (SHR) proteins from Arabidopsis thaliana (AtSHR), Brachypodium distachyon (BdSHR), and Oryza sativa (OsSHR1 and OsSHR2) function in patterning the root ground tissue. We find that all of the SHR proteins function as mobile signals in A. thaliana and all of the SHR homologs physically interact with the AtSHR binding protein, Scarecow (SCR). Unlike AtSHR, movement of the SHR homologs was not limited to the endodermis. Instead, the SHR proteins moved multiple cell layers and determined the number of cortex, not endodermal, cell layers formed in the root. Our results in A. thaliana are consistent with a mechanism by which the regulated movement of the SHR transcription factor determines the number of cortex cell layers produced in the roots of B. distachyon and O. sativa. These data also provide a new model for ground tissue patterning in A. thaliana in which the ability to form a functional endodermis is spatially limited independently of SHR.

Intercellular protein movement: deciphering the language of development.

Development in multicellular organisms requires the coordinated production of a large number of specialized cell types through sophisticated signaling mechanisms. Non-cell-autonomous signals are one of the key mechanisms by which organisms coordinate development. In plants, intercellular movement of transcription factors and other mobile signals, such as hormones and peptides, is essential for normal development. Through a combination of different approaches, a large number of non-cell-autonomous signals that control plant development have been identified. We review some of the transcriptional regulators that traffic between cells, as well as how changes in symplasmic continuity affect and are affected by development. We also review current models for how mobile signals move via plasmodesmata and how movement is inhibited. Finally, we consider challenges in and new tools for studying protein movement.

The movement of the non-cell-autonomous transcription factor, SHORT-ROOT relies on the endomembrane system.

Plant cells are able to convey positional and developmental information between cells through the direct transfer of transcription factors. One well studied example of this is the SHORT-ROOT (SHR) protein, which moves from the stele into the neighboring ground tissue layer to specify endodermis. While it has been shown that SHR trafficking relies on plasmodesmata (PD), and interaction with the SHR INTERACTING EMBRYONIC LETHAL (SIEL) protein, little information is known about how SHR trafficking is controlled or how SIEL promotes the movement of SHR. Here we show that SHR can move from multiple different cell types in the root. Analysis of subcellular localization indicates that in the cytoplasm of root or leaf cells, SHR localizes to endosomes in a SIEL-dependent manner. Interference of early and late endosomes disrupts intercellular movement of SHR. Our findings reveal an essential role for the plant endomembrane, independent of secretion, in the intercellular trafficking of SHR.

Identification of SHRUBBY, a SHORT-ROOT and SCARECROW interacting protein that controls root growth and radial patterning.

The timing and extent of cell division is particularly important for the growth and development of multicellular organisms. Roots of the model organism Arabidopsis thaliana have been widely studied as a paradigm for organ development in plants. In the Arabidopsis root, the plant-specific GRAS family transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR) are key regulators of root growth and of the asymmetric cell divisions that separate the ground tissue into two separate layers: the endodermis and cortex. To elucidate the role of SHR in root development, we identified 17 SHR-interacting proteins. Among those isolated was At5g24740, which we named SHRUBBY (SHBY). SHBY is a vacuolar sorting protein with similarity to the gene responsible for Cohen syndrome in humans. Hypomorphic alleles of shby caused poor root growth, decreased meristematic activity and defects in radial patterning that are characterized by an increase in the number of cell divisions in the ground tissue that lead to extra cells in the cortex and endodermis, as well as additional cell layers. Analysis of genetic and molecular markers indicates that SHBY acts in a pathway that partially overlaps with SHR, SCR, PLETHORA1 and PLETHORA2 (PLT1 and PLT2). The shby-1 root phenotype was partially phenocopied by treatment of wild-type roots with the proteosome inhibitor MG132 or the gibberellic acid (GA) synthesis inhibitor paclobutrazol (PAC). Our results indicate that SHBY controls root growth downstream of GA in part through the regulation of SHR and SCR.

Intact microtubules are required for the intercellular movement of the SHORT-ROOT transcription factor.

In both plants and animals, cell-to-cell signaling controls key aspects of development. In plants, cells communicate through direct transfer of transcription factors between cells. It is thought that most, if not all, mobile transcription factors move via plasmodesmata, membrane-lined channels that connect nearly all cells in the plant. However, the mechanisms by which these proteins access the plasmodesmata are not known. Using four independent assays, we examined the movement of the SHORT-ROOT (SHR) transcription factor under conditions that affect microtubule stability, organization or dynamics. We found that intact microtubules are required for cell-to-cell trafficking of SHR. Either chemical or genetic disruption of microtubules results in a significant reduction in SHR transport. Interestingly, inhibition of microtubules also results in mis-localization of the SHR-INTERACTING EMBRYONIC LETHAL (SIEL) protein, which has been shown to bind directly to SHR and is required for SHR movement. These results show that microtubules facilitate cell-to-cell transport of an endogenous plant protein.

SCARECROW reinforces SHORT-ROOT signaling and inhibits periclinal cell divisions in the ground tissue by maintaining SHR at high levels in the endodermis.

In contrast to development in animals, much of the patterning of plants occurs post-embryonically in specialized structures called meristems. The root apical meristem of Arabidopsis thaliana is a readily accessible structure that has been extensively studied to uncover the factors that control root growth and cellular patterning. Recently we showed that one of the key factors in patterning the root, the mobile transcription factor SHORT-ROOT (SHR), acts in a concentration-dependent manner to initiate or suppress asymmetric divisions in the endodermis. The amount of SHR varies dynamically in the endodermis with the age of the root. Here we show that this variation is controlled in part through the activity of the transcription factor, SCARECROW (SCR), which regulates SHR movement and therefore its effective concentration and function in the endodermis.