Unique among the known plant and animal viral suppressors of RNA silencing, the 2b protein interacts directly with both small interfering RNA (siRNA) and ARGONAUTE1 (AGO1) and AGO4 proteins and is targeted to the nucleolus. However, it is largely unknown which regions of the 111-residue 2b protein determine these biochemical properties and how they contribute to its diverse silencing suppressor activities. Here, we identified a functional nucleolar localization signal encoded within the 61-amino acid N-terminal double-stranded RNA (dsRNA) binding domain (dsRBD) that exhibited high affinity for short and long dsRNA. However, physical interaction of 2b with AGOs required an essential 33-residue region C-terminal to the dsRBD and was sufficient to inhibit the in vitro AGO1 Slicer activity independently of its dsRNA binding activities. Furthermore, the direct 2b-AGO interaction was not essential for the 2b suppression of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) in vivo. Lastly, we found that the 2b-AGO interactions in vivo also required the nucleolar targeting of 2b and had the potential to redistribute both the 2b and AGO proteins in nucleus. These findings together suggest that 2b may suppress PTGS and RdDM in vivo by binding and sequestering siRNA and the long dsRNA precursor in a process that is facilitated by its interactions with AGOs in the nucleolus.

Satellite RNAs (satRNAs) depend on cognate helper viruses for replication, encapsidation, movement and transmission. Many satRNAs with different symptom modulation effects have been reported. The pathogenicity of satRNAs is thought to be the result of a direct interaction among the satRNA, helper viruses and host factors by unknown mechanisms. To understand the effect of satRNA of Cucumber mosaic virus (a severe field ShanDong strain, SD-CMV) on pathogenicity, and the possible involvement of host RNA silencing pathways in pathogenicity, we constructed biologically active CMV cDNA clones and a CMV-Δ2b mutant lacking the open reading frame of 2b, a silencing suppressor protein, in order to infect Nicotiana benthamiana and Arabidopsis with or without SD-satRNA. We found that SD-satRNA reduced the accumulation of the 2b protein and its coding RNA4A and attenuated the yellowing caused by SD-CMV infection. Small RNA analysis indicated that the 2b protein interfered with RNA silencing, specifically in the synthesis of CMV RNA3-derived small interfering RNAs (R3-siRNAs). The accumulation of R3-siRNAs in CMV-Δ2b infection was reduced in the presence of satRNA, for which greater accumulation of satRNA-derived siRNAs (satsiRNAs) was detected. Our results suggest that abundant SD-satRNA serving as target for RNA silencing may play a role in protecting helper CMV RNA, especially, subgenomic RNA4, from being targeted by RNA silencing. This compensates for the increase in RNA silencing resulting from the reduction in expression of the 2b suppressor in the presence of satRNA. Our data provide evidence that a plant silencing mechanism is involved in the pathogenicity of satRNA.

Hypericum perforatum (St John's wort) is a reservoir of diverse classes of biologically active and high value secondary metabolites, which captured the interest of both researchers and the pharmaceutical industry alike. Several studies and clinical trials have shown that H. perforatum extracts possess an astounding array of pharmacological properties. These properties include antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial activities; and are largely attributed to the naphtodianthrones and xanthones found in the genus. Hence, improving their production via genetic manipulation is an important strategy. In spite of the presence of contemporary genome editing tools, genetic improvement of this genus remains challenging without robust transformation methods in place. In the recent past, we found that H. perforatum remains recalcitrant to Agrobacterium tumefaciens mediated transformation partly due to the induction of plant defense responses coming into play. However, H. perforatum transformation is possible via a non-biological method, biolistic bombardment. Some research groups have observed the induction of hairy roots in H. perforatum after Agrobacterium rhizogenes co-cultivation. In this review, we aim at updating the available methods for regeneration and transformation of H. perforatum. In addition, we also propose a brief perspective on certain novel strategies to improve transformation efficiency in order to meet the demands of the pharmaceutical industry via metabolic engineering.

ObjectiveAcute organ embolism in children with Mycoplasma pneumoniae pneumonia (MPP) has been reported, but changes in coagulation are unclear. This study aimed to investigate changes in coagulation in children with MPP.MethodsA total of 185 children with MMP (cases) and 117 healthy children (controls) were recruited. We measured prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and plasma fibrinogen (FIB) and D-dimer levels.ResultsPlasma FIB (3.39 ± 0.96 g/L vs 2.93 ± 0.6 6g/L, t = 4.50) and D-dimer (326.45 ± 95.62mg/L vs 263.93 ± 103.32mg/L, t=5.36) in MPP children were higher than controls and PT (9.54 ± 4.97S vs 11.48 ± 5.96S, t=3.05) and APTT (31.41 ± 12.01S vs 38.38 ± 11.72S, t=4.95) were shorter than controls. FIB, D-dimer, PT, and APTT were not different between the high IgM-titre and low-titre groups. The areas under the receiver operating characteristic curves in cases and controls for plasma FIB and D-dimer levels were 0.654 (95% confidence interval [CI], 0.593–0.716, P = 0.031) and 0.682 (95% CI, 0.619–0.744, P = 0.032), respectively.ConclusionsChildren with MPP have a higher risk of blood coagulation and thrombosis. Controlling these problems should be considered as soon as possible.

Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved into H. perforatum L. recalcitrance to Agrobacterium tumefaciens-mediated transformation.

In order to provide an effective way to prevent or substantially delay the recurrence of invasive meningioma, and improve the curative effect of surgical treatment, we collected and analyzed the clinical manifestations, pathological features, preoperative imaging characteristics as well the data obtained during the surgical treatment of invasive meningioma. From February 2014 to February 2016, 59 patients with invasive meningioma were enrolled in this study. Invasive meningioma was confirmed in all patients by operation. Information about clinical manifestations, pathological features, preoperative imaging and surgical treatment were collected and analyzed. After surgery, pathological specimens were collected, and cases were confirmed as invasive meningioma by pathological examination. The course of disease ranged from 15 days to 7 years (average, 13.2 months). We used World Health Organization (WHO) criteria for classification of meningioma in the nervous system tumors as our reference. Symptoms were as follows: Intracranial hypertension (29 cases), cranial nerve dysfunction (10 cases), epilepsy (11 cases) and other symptoms (9 cases). We had 56 cases of WHO grade I; 6 cases of WHO grade II and 7 cases of WHO grade III. Surgical removal was: Simpson grade I (56 cases), Simpson grade II (2 cases), Simpson grade III and above (56 cases). We used before surgery imaging data to formulate our surgical plan. In general, during surgeries we did not proceed to complete resection, because in the majority of cases, some key structures were invaded and meningioma was very deep and any attempt for total resection could easily lead to significant damage to these structures.

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