Fabrication and Imaging of Monolayer Phosphorene with Preferred Edge Configurations via Graphene-Assisted Layer-by-Layer Thinning.

Phosphorene, a monolayer of black phosphorus (BP), is an elemental two-dimensional material with interesting physical properties, such as high charge carrier mobility and exotic anisotropic in-plane properties. To fundamentally understand these various physical properties, it is critically important to conduct an atomic-scale structural investigation of phosphorene, particularly regarding various defects and preferred edge configurations. However, it has been challenging to investigate mono- and few-layer phosphorene because of technical difficulties arising in the preparation of a high-quality sample and damages induced during the characterization process. Here, we successfully fabricate high-quality monolayer phosphorene using a controlled thinning process with transmission electron microscopy and subsequently perform atomic-resolution imaging. Graphene protection suppresses the e-beam-induced damage to multilayer BP and one-side graphene protection facilitates the layer-by-layer thinning of the samples, rendering high-quality monolayer and bilayer regions. We also observe the formation of atomic-scale crystalline edges predominantly aligned along the zigzag and (101) terminations, which is originated from edge kinetics under e-beam-induced sputtering process. Our study demonstrates a new method to image and precisely manipulate the thickness and edge configurations of air-sensitive two-dimensional materials.

Size-Dependent Phase Transformation of Noble Metal Nanomaterials.

As an important aspect of crystal phase engineering, controlled crystal phase transformation of noble metal nanomaterials has emerged as an effective strategy to explore novel crystal phases of nanomaterials. In particular, it is of significant importance to observe the transformation pathway and reveal the transformation mechanism in situ. Here, the phase transformation behavior of face-centered cubic (fcc) Au nanoparticles (fcc-AuNPs), adhering to the surface of 4H nanodomains in 4H/fcc Au nanorods, referred to as 4H-AuNDs, during in situ transmission electron microscopy imaging is systematically studied. It is found that the phase transformation is dependent on the ratio of the size of the monocrystalline nanoparticle (NP) to the diameter of 4H-AuND. Furthermore, molecular dynamics simulation and theoretical modeling are used to explain the experimental results, giving a size-dependent phase transformation diagram which provides a general guidance to predict the phase transformation pathway between fcc and 4H Au nanomaterials. Impressively, this method is general, which is used to study the phase transformation of other metal NPs, such as Pd, Ag, and PtPdAg, adhering to 4H-AuNDs. The work opens an avenue for selective phase engineering of nanomaterials which may possess unique physicochemical properties and promising applications.

Myhre syndrome: A first familial recurrence and broadening of the phenotypic spectrum.

Myhre syndrome is a rare multisystem connective tissue disorder, characterized by short stature, facial dysmorphology, variable intellectual disability, skeletal abnormalities, arthropathy, cardiopathy, laryngotracheal anomalies, and stiff skin. So far, all molecularly confirmed cases harbored a de novo heterozygous gain-of-function mutation in SMAD4, encoding the SMAD4 transducer protein required for both transforming growth factor-beta and bone morphogenic proteins signaling. We report on four novel patients (one female proband and her two affected children, and one male proband) with Myhre syndrome harboring the recurrent c.1486C>T (p.Arg496Cys) mutation in SMAD4. The female proband presented with a congenital heart defect, vertebral anomalies, and facial dysmorphic features. She developed severe tracheal stenosis requiring a total laryngectomy. With assisted reproductive treatment, she gave birth to two affected children. The second proband presented with visual impairment following lensectomy in childhood, short stature, brachydactyly, stiff skin, and decreased peripheral sensitivity. Transmission electron microscopy (TEM) of the dermis shows irregular elastin cores with globular deposits and almost absent surrounding microfibrils and suggests age-related increased collagen deposition. We report on the first familial case of Myhre syndrome and illustrate the variable clinical spectrum of the disorder. Despite the primarily fibrotic nature of the disease, TEM analysis mainly indicates elastic fiber anomalies.

Exosome-associated hepatitis C virus in cell cultures and patient plasma.

Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

Pheno- and Genotyping of Three Novel Bacteriophage Genera That Target a Wheat Phyllosphere Sphingomonas Genus.

Bacteriophages are viral agents that infect and replicate within bacterial cells. Despite the increasing importance of phage ecology, environmental phages-particularly those targeting phyllosphere-associated bacteria-remain underexplored, and current genomic databases lack high-quality phage genome sequences linked to specific environmentally important bacteria, such as the ubiquitous sphingomonads. Here, we isolated three novel phages from a Danish wastewater treatment facility. Notably, these phages are among the first discovered to target and regulate a Sphingomonas genus within the wheat phyllosphere microbiome. Two of the phages displayed a non-prolate Siphovirus morphotype and demonstrated a narrow host range when tested against additional Sphingomonas strains. Intergenomic studies revealed limited nucleotide sequence similarity within the isolated phage genomes and to publicly available metagenome data of their closest relatives. Particularly intriguing was the limited homology observed between the DNA polymerase encoding genes of the isolated phages and their closest relatives. Based on these findings, we propose three newly identified genera of viruses: Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, following the latest ICTV binomial nomenclature for virus species. These results contribute to our current understanding of phage genetic diversity in natural environments and hold promising implications for phage applications in phyllosphere microbiome manipulation strategies.

Atomically Sharp, Closed Bilayer Phosphorene Edges by Self-Passivation.

Two-dimensional crystals' edge structures not only influence their overall properties but also dictate their formation due to edge-mediated synthesis and etching processes. Edges must be carefully examined because they often display complex, unexpected features at the atomic scale, such as reconstruction, functionalization, and uncontrolled contamination. Here, we examine atomic-scale edge structures and uncover reconstruction behavior in bilayer phosphorene. We use in situ transmission electron microscopy (TEM) of phosphorene/graphene specimens at elevated temperatures to minimize surface contamination and reduce e-beam damage, allowing us to observe intrinsic edge configurations. The bilayer zigzag (ZZ) edge was found to be the most stable edge configuration under e-beam irradiation. Through first-principles calculations and TEM image analysis under various tilting and defocus conditions, we find that bilayer ZZ edges undergo edge reconstruction and so acquire closed, self-passivated edge configurations. The extremely low formation energy of the closed bilayer ZZ edge and its high stability against e-beam irradiation are confirmed by first-principles calculations. Moreover, we fabricate bilayer phosphorene nanoribbons with atomically sharp closed ZZ edges. The identified bilayer ZZ edges will aid in the fundamental understanding of the synthesis, degradation, reconstruction, and applications of phosphorene and related structures.

Improvement of leaf K+ retention is a shared mechanism behind CeO2 and Mn3O4 nanoparticles improved rapeseed salt tolerance.

Salinity is a global issue limiting efficient agricultural production. Nanobiotechnology has been emerged as an effective approach to improve plant salt tolerance. However, little known is about the shared mechanisms between different nanomaterials-enabled plant salt tolerance. In this study, we found that both PNC [polyacrylic acid coated nanoceria (CeO2 nanoparticles)] and PMO (polyacrylic acid coated Mn3O4 nanoparticles) nanozymes improved rapeseed salt tolerance. PNC and PMO treated rapeseed plants showed significantly fresh weight, dry weight, higher chlorophyll content, Fv/Fm, and carbon assimilation rate than control plants under salt stress. Results from confocal imaging with reactive oxygen species (ROS) fluorescent dye and histochemical staining experiments showed that the ROS over-accumulation level in PNC and PMO treated rapeseed was significantly lower than control plants under salt stress. Confocal imaging results with K+ fluorescent dye showed that significantly higher cytosolic and vacuolar K+ signals were observed in PNC and PMO treated rapeseed than control plants under salt stress. This is further confirmed by leaf K+ content data. Furthermore, we found that PNC and PMO treated rapeseed showed significantly lower cytosolic Na+ signals than control plants under salt stress. While, compared with significantly higher vacuolar Na+ signals in PNC treated plants, PMO treated rapeseed showed significantly lower vacuolar Na+ signals than control plants under salt stress. These results are further supported by qPCR results of genes of Na+ and K+ transport. Overall, our results suggest that besides maintaining ROS homeostasis, improvement of leaf K+ retention could be a shared mechanism in nano-improved plant salt tolerance.

The Development of the Innovative Synthesis Methodology of Albumin Nanoparticles Supported by Their Physicochemical, Cytotoxic and Hemolytic Evaluation.

Many studies are being performed to develop effective carriers for controlled cytostatic delivery wherein albumin is a promising material due to its tendency to accumulate near cancer cells. The novelty of this work involves the development of the synthesis methodology of albumin nanoparticles and their biological and physicochemical evaluation. Albumin particles were obtained via the salt-induced precipitation and K3PO4 was used as a salting-out agent. Various concentrations of protein and salting-out agent solutions were mixed using a burette or a syringe system. It was proved that the size of the particles depended on the concentrations of the reagents and the methodology applied. As a result of a process performed using a burette and 2 M K3PO4, albumin spheres having a size 5-25 nm were obtained. The size of nanospheres and their spherical shape was confirmed via TEM analysis. The use of a syringe system led to preparation of particles of large polydispersity. The highest albumin concentration allowing for synthesis of homogeneous particles was 2 g/L. The presence of albumin in spheres was confirmed via the FT-IR technique and UV-Vis spectroscopy. All samples showed no cytotoxicity towards normal human dermal fibroblasts and no hemolytic properties against human erythrocytes (the hemolysis did not exceed 2.5%).

Investigating the photo-thermo-radiosensitization effects of folate-conjugated gold nanorods on KB nasopharyngeal carcinoma cells.

In this article, we report a targeted cancer treatment strategy using nano-photo-thermal therapy (NPTT) and radiotherapy (RT) methods. Gold nanorods (AuNRs) without and with folic acid (FA) conjugation were firstly synthesized and then characterized. Cytotoxicity of various methods; NPTT (nanoparticles; 808 nm laser; 2 W/cm2) or RT (6 MV X-ray; 2 Gy) or combination of NPTT and RT; was separately investigated on KB nasopharyngeal carcinoma cells. The effects of different treatments were studies using MTT assay, inductively coupled plasma-mass spectrometry (ICP-MS) and transmission electron microscopy (TEM). Both TEM and ICP-MS confirmed the internalization of nanoparticles into the KB cells. ICP-MS analysis revealed that AuNR-FA cell uptake was higher than AuNRs. Viability of the cells received NPTT was lower than those cells received laser or nanoparticles alone. Furthermore, it was found that combination of NPTT and RT notably decreased the viability of KB cells. Following such a combinatorial treatment (NPTT + RT), intensive damages were identified in TEM images obtained from treated cells. It may be concluded that AuNR-FA nanoconjugate is a good candidate to be used as a targeted sensitizer agent for nano-photo-thermo-radiotherapy of head and neck cancer cells.

Navigating methane metabolism: Enzymes, compartments, and networks.

Microbial methane utilization-a key node in the global carbon cycle-controls and often eliminates emission of methane into the atmosphere. The diversity and distribution of microbes capable of methane oxidation is astounding. However, from a biochemical point of view, only a very narrow set of unique enzymes underlies their metabolic capabilities. Despite this restriction, the successful integration of the enzymes into nonmethanotrophs, if judged by the ability of the trait to grow on methane, remains to be achieved. Failures and small victories with heterologous expression have highlighted a set of challenges linked to structure, compartmentalization, and regulation of the methanotrophic metabolic network. A better understanding of how these challenges are handled by cells of native methane-consuming bacteria is required. In this chapter we focus on key experimental aspects of working with native methanotrophic bacteria, including routine cultivation strategies, lab-scale bioreactor setups, genetic alteration, imaging, and basic -omic-level approaches.

Investigation of non-linear imaging in high-resolution transmission electron microscopy.

Transmission cross-coefficient theory and pseudo-weak-phase object approximation theory were combined to investigate the non-linear imaging in high-resolution transmission electron microscopy (HRTEM). The analytical expressions of linear and non-linear imaging components in diffractogram were obtained and changes of linear and non-linear components over sample thickness were analyzed. Moreover, the linear and non-linear components are found to be an odd and even-function of the defocus and Cs, respectively. Based on this, a method for separating the linear and non-linear contrasts in Cs-corrected (non-zero Cs conditions included) HRTEM images was proposed, and its effectiveness was confirmed by image simulations with AlN as an example.

Efficient in vitro gene therapy with PEG siRNA lipid nanocapsules for passive targeting strategy in melanoma.

Small interfering RNA (siRNA)-mediated gene therapy is a promising strategy to temporarily inhibit the expression of proteins implicated in carcinogenesis or chemotherapy resistance. Although intra-tumoral administration can be envisaged, studies currently focus on formulating nanomedicines for intravenous injection to target tumor sites as well as metastases. The development of synthetic nanoparticles and liposomes has advanced greatly during the last decade. The objective of this work consists in formulating and optimizing the encapsulation of siRNA into lipid nanocapsules (LNCs) for efficient gene therapy to target melanoma cells. SiRNA LNCs were prepared from DOTAP/DOPE lipoplexes, and the siRNA amount and lipid/siRNA charge ratio were assayed to improve the stability and the encapsulation yield. Cryo-TEM imaging of the siRNA lipoplexes and LNC morphology revealed specific organization of the siRNA DOTAP/DOPE lipoplexes as well as specific lipid microstructures that can be eliminated by purification. No cytotoxicity of the siRNA LNCs against the melanoma SK-Mel28 cell line was observed at concentrations of up to 500 ng/mL siRNA. In vitro siRNA transfection experiments, compared to Oligofectamine™, demonstrated interesting targeted gene silencing effects. Finally, complement activation assays confirmed the feasibility of the PEGylation of siRNA LNCs as part of a passive targeting strategy for future in vivo melanoma- and metastasis-targeting experiments.