Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2

Influenza A virus’s (IAV’s) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV’s genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem–loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term ‘programmable antivirals’, with implications for antiviral prophylaxis and post-exposure therapy

Formation of Complexes at Plasmodesmata for Potyvirus Intercellular Movement Is Mediated by the Viral Protein P3N-PIPO

Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants

Tubule-Guided Cell-to-Cell Movement of a Plant Virus Requires Class XI Myosin Motors

Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells

Arabidopsis Plasmodesmal Proteome

The multicellular nature of plants requires that cells should communicate in order to coordinate essential functions. This is achieved in part by molecular flux through pores in the cell wall, called plasmodesmata. We describe the proteomic analysis of plasmodesmata purified from the walls of Arabidopsis suspension cells. Isolated plasmodesmata were seen as membrane-rich structures largely devoid of immunoreactive markers for the plasma membrane, endoplasmic reticulum and cytoplasmic components. Using nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer, 1341 proteins were identified. We refer to this list as the plasmodesmata- or PD-proteome. Relative to other cell wall proteomes, the PD-proteome is depleted in wall proteins and enriched for membrane proteins, but still has a significant number (35%) of putative cytoplasmic contaminants, probably reflecting the sensitivity of the proteomic detection system. To validate the PD-proteome we searched for known plasmodesmal proteins and used molecular and cell biological techniques to identify novel putative plasmodesmal proteins from a small subset of candidates. The PD-proteome contained known plasmodesmal proteins and some inferred plasmodesmal proteins, based upon sequence or functional homology with examples identified in different plant systems. Many of these had a membrane association reflecting the membranous nature of isolated structures. Exploiting this connection we analysed a sample of the abundant receptor-like class of membrane proteins and a small random selection of other membrane proteins for their ability to target plasmodesmata as fluorescently-tagged fusion proteins. From 15 candidates we identified three receptor-like kinases, a tetraspanin and a protein of unknown function as novel potential plasmodesmal proteins. Together with published work, these data suggest that the membranous elements in plasmodesmata may be rich in receptor-like functions, and they validate the content of the PD-proteome as a valuable resource for the further uncovering of the structure and function of plasmodesmata as key components in cell-to-cell communication in plants

Advances in research on the use of biochar in soil for remediation: a review

Purpose: Soil contamination mainly from human activities remains a major environmental problem in the contemporary world. Significant work has been undertaken to position biochar as a readily-available material useful for the management of contaminants in various environmental media notably soil. Here, we review the increasing research on the use of biochar in soil for the remediation of some organic and inorganic contaminants.  Materials and methods: Bibliometric analysis was carried out within the past 10 years to determine the increasing trend in research related to biochar in soil for contaminant remediation. Five exemplar contaminants were reviewed in both laboratory and field-based studies. These included two inorganic (i.e., As and Pb) and three organic classes (i.e., sulfamethoxazole, atrazine, and PAHs). The contaminants were selected based on bibliometric data and as representatives of their various contaminant classes. For example, As and Pb are potentially toxic elements (anionic and cationic, respectively), while sulfamethoxazole, atrazine, and PAHs represent antibiotics, herbicides, and hydrocarbons, respectively.  Results and discussion: The interaction between biochar and contaminants in soil is largely driven by biochar precursor material and pyrolysis temperature as well as some characteristics of the contaminants such as octanol-water partition coefficient (KOW) and polarity. The structural and chemical characteristics of biochar in turn determine the major sorption mechanisms and define biochar’s suitability for contaminant sorption. Based on the reviewed literature, a soil treatment plan is suggested to guide the application of biochar in various soil types (paddy soils, brownfield, and mine soils) at different pH levels (4–5.5) and contaminant concentrations ( 50 mg kg−1).  Conclusions: Research on biochar has grown over the years with significant focus on its properties, and how these affect biochar’s ability to immobilize organic and inorganic contaminants in soil. Few of these studies have been field-based. More studies with greater focus on field-based soil remediation are therefore required to fully understand the behavior of biochar under natural circumstances. Other recommendations are made aimed at stimulating future research in areas where significant knowledge gaps exist

The ER-membrane transport system is critical for intercellular trafficking of the NSm movement protein and Tomato Spotted Wilt Tospovirus

Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV

Abstract P3-01-17: Evaluation of “Systematic sonographic axillary staging” on clinically node positive breast cancer patients becoming clinically node negative after neoadjuvant chemotherapy

Abstract Purpose Accumulating evidence supports usage of sentinel lymph node biopsy (SLNB) for clinically node positive (cN+) breast cancer (BC) patients, who become clinically node negative (ycN-) after neoadjuvant chemotherapy (NAC). How to reassess the axilla after NAC is a challenging issue. In our institution, Systematic Sonographic Axillary Staging (SSAS) is currently being tested in this group of patients. The aim of this study is to analyze the early results of this approach. Method During initial presentation, systematic sonographic axillary staging is done preferentially by the surgeon. Needle biopsy is then performed of suspicious node(s). At completion of NAC, the axilla is again systematically staged sonographically, taking into account the pre-treatment disease locations. Any residual morphologically suspicious node undergoes a repeat needle biopsy. According to the evidence of residual disease, the patient is either scheduled for axillary dissection (AD) or for SLNB. We analyzed early results of SSAS on patients, who converted ycN (-) after NAC. Strictly adherence to SSAS protocol was the only inclusion criteria. Results A total of 25 patients were included. On repeat axillary USG after NAC, 11 patients had normal appearing and 14 patients had suspicious appearing lymph nodes (LNs). 14 patients underwent repeat biopsy; 9 patients had benign and 5 patients had malign pathology results. 20 patients underwent SLNB. Among 20 patients, 12 patients had negative SLNB and 8 patients had positive SLNB (4 patients had benign appearing LNs on repeat USG and 4 patients had benign biopsy results). 13 patients underwent AD (8 patients after positive SLNB and 5 patients directly after positive US-guided biopsy result). Among 8 patients with positive SLNB, 4 patients had macrometastatic disease (>2mm) on SLNB. In 3 of these 8 patients, no additional disease was found on AD, while 1 patient had only isolated tumor cell and 1 patient had only micrometastasis (Table 1). Table 1. Size of the axillary involvement among patients with positive SLNB Sentinel Lymph Node BiopsyAxillary DissectionPatient #LN* (+) / Total LNs removedSize of involvement (mm)LN* (+) / Total LNs removedSize of involvement (mm)11/611/50.122/42.50/5-31/363/19141/210/3-51/11.50/21-62/21.53/32.573/357/10384/447/133* LN = lymph node Conclusion Systematic sonographic axillary staging is a reliable approach to cN (+) patients, who become ycN0 after NAC. This observation should be tested in a larger study. Citation Format: Ozmen T, Lazaro M, Vinyard A, Avisar E. Evaluation of “Systematic sonographic axillary staging” on clinically node positive breast cancer patients becoming clinically node negative after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-01-17

Abstract P5-22-05: Evaluation of simplified lymphatic microsurgical preventing healing approach (SLYMPHA) for the prevention of breast cancer-related clinical lymphedema after axillary lymph node dissection

Abstract Background: Lymphedema (LE) is a serious complication of axillary lymph node dissection (ALND) with an incidence rate of 16%. Lymphatic Microsurgical Preventing Healing Approach (LYMPHA) has been proposed as an effective adjunct to ALND for the prevention of LE. This procedure however requires microsurgical techniques. The aim of this study was to assess the efficiency of Simplified-LYMPHA (SLYMPHA) in preventing LE in a prospective cohort of patients. Methods: All patients, undergoing ALND with or without SLYMPHA between January 2014 and December 2016 were included in the study. SLYMPHA is a slightly modified and simplified version of LYMPHA. It is performed by the operating surgeon performing the ALND. One or more lymphatic channels identified by reverse arm mapping are inserted using a sleeve technique into the cut end of a neighboring vein. During follow-up visits, tape-measuring limb circumference method was used to detect clinical LE. Demographic, clinical, surgical and pathologic factors were recorded. The incidence of clinical LE was compared between ALND with and without SLYMPHA. Univariate and multivariate analysis were used to assess the role of other factors in the appearance of clinical LE. Results: 406 patients were included in the study. SLYMPHA procedure was attempted in 81 patients and was completed successfully in 90% of patients. Early complication rates were similar between patients who underwent SLYMPHA and who did not (4% vs. 4.13%; p = 0.948). Median follow-up time was 15±13.73 [1-32] months. Patients, who underwent SLYMPHA, had a significantly lower rate of clinical LE both in univariate and multivariate analysis (3% vs 19%; p = 0.001; OR 0.12 [0.03-0.5]). Excising > 22 lymph nodes and a co-diagnosis of diabetes were also correlated with higher clinical LE rates on univariate analysis, but only excising > 22 lymph nodes remained to be significant on multivariate analysis. Conclusions: SLYMPHA is a safe and relatively simple method, which decreases incidence of clinical LE dramatically. It should be considered as an adjunct procedure to ALND for all patients during initial surgery. Citation Format: Ozmen T, Lazaro M, Zhou Y, Vinyard A, Avisar E. Evaluation of simplified lymphatic microsurgical preventing healing approach (SLYMPHA) for the prevention of breast cancer-related clinical lymphedema after axillary lymph node dissection [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-22-05