Immunity against selected piscine flagellates

This discussion is on immune response to Amyloodinium ocellatum, Cryptobia salmositica, Trypanoplasma borreli and Trypanosoma carassii. Piscidin and histone-like proteins enhance innate resistance to Amyloodinium. Fish that are naturally resistant to Cryptobia and Trypanoplasma can be bred. Cryptobia resistance in charr is controlled by a dominant Mendelian locus and protection is via the Alternative Pathway of Complement Activation. Studies on Cryptobia-tolerant charr may lead to production of transgenic Cryptobia-tolerant salmon. Innate response to T. borreli is associated with NO in macrophages. Transferrin regulates resistance and carp have been bred for transferrin genotypes. Recovered fish are protected from homologous challenge, and complement fixing antibodies are crucial in protection. Studies on antigens in T. carassii may lead to a vaccine. There are two vaccines against cryptobiosis; a single dose of the attenuated vaccine protects salmonids. On challenge fish inoculated with the metalloprotease-DNA vaccine do not have the disease and they recover faster.     

Creating perfused functional vascular channels using 3D bio-printing technology

We developed a methodology using 3D bio-printing technology to create a functional in vitro vascular channel with perfused open lumen using only cells and biological matrices. The fabricated vasculature has a tight, confluent endothelium lining, presenting barrier function for both plasma protein and high-molecular weight dextran molecule. The fluidic vascular channel is capable of supporting the viability of tissue up to 5 mm in distance at 5 million cells/mL density under the physiological flow condition. In static-cultured vascular channels, active angiogenic sprouting from the vessel surface was observed whereas physiological flow strongly suppressed this process. Gene expression analysis was reported in this study to show the potential of this vessel model in vascular biology research. The methods have great potential in vascularized tissue fabrication using 3D bio-printing technology as the vascular channel is simultaneously created while cells and matrix are printed around the channel in desired 3D patterns. It can also serve as a unique experimental tool for investigating fundamental mechanisms of vascular remodeling with extracellular matrix and maturation process under 3D flow condition.     

Mouse mammary tumor virus production by thymic epithelial cells in vivo

Exogenous mouse mammary tumor viruses (MMTV) replicate in the mammary glands of infected females, and so infect the suckling pups. We have previously shown that the virus is rapidly disseminated to all the lymphoid organs, including the thymus. The present electron microscope immunohistochemical study describes the viral production site in the thymus. Viral buds and viral proteins were restricted to the thymus medullary epithelial cells. MMTV-encoded proteins were identified on the free viral particles and on the budding ones, the ribosomes, the membrane of the endoplasmic reticulum, and on the membrane of the medullary type II epithelial cell vacuolar network. The thymus medullary epithelial cells can thus integrate the virus and allow viral replication. The results support earlier results indicating that in some experimental conditions, epithelial cells may be involved in MMTV-induced negative selection by showing that thymic epithelial cells do express MMTV-encoded proteins.     

Invasive Breast Carcinoma with Neuroendocrine Differentiation: A Single-Center Analysis of Clinical Features and Prognosis

Introduction Invasive breast cancer with neuroendocrine differentiation is a rare subtype of breast malignancy. Due to frequent changes in the definition of these lesions, the correct diagnosis, estimation of exact prevalence, and clinical behaviour of this entity may be challenging. The aim of this study was to evaluate the prevalence, clinical features, and outcomes in a large cohort of patients with breast cancer with neuroendocrine differentiation. Patients Twenty-seven cases of breast cancer with neuroendocrine differentiation have been included in this analysis. Twenty-one cases were identified by systematic immunohistochemical re-evaluation of 465 breast cancer specimens using the neuroendocrine markers chromogranin A and synaptophysin, resulting in a prevalence of 4.5%. A further six cases were identified by a review of clinical records. Results Median age at the time of diagnosis was 61 years. 70% of patients had T2 – 4 tumors and 37% were node-positive. The most common immunohistochemical subtype was HR-positive/HER2-negative (85%). 93% were positive for synaptophysin and 48% for chromogranin A. Somatostatin receptor type 2A status was positive in 12 of 24 analyzed tumors (50%). Neuroendocrine-specific treatment with somatostatin analogues was administered in two patients. The 5-year survival rate was 70%. Conclusions Breast cancer with neuroendocrine differentiation is mostly HR-positive/HER2-negative and the diagnosis is made at a higher TNM stage than in patients with conventional invasive breast carcinoma. Moreover, breast cancer with neuroendocrine differentiation was found to be associated with impaired prognosis in several retrospective trials. Due to somatostatin receptor 2A expression, somatostatin receptor-based imaging can be used and somatostatin receptor-targeted therapy can be offered in selected cases. Key words: neuroendocrine neoplasia of the breast, invasive breast cancer with neuroendocrine differentiation, neuroendocrine breast cancer, neuroendocrine markers, somatostatin receptor 2A     

High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na⁺ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter Glt_Ph. Since CitS and Glt_Ph are structurally unrelated, we conclude that the three-state elevators have evolved independently.

The cytoplasmic membrane forms a semipermeable barrier between the cellular interior and the periplasm of bacteria. The trafficking of molecules across the membrane is necessary for the cell’s metabolism and is mediated by membrane transporters. These proteins are embedded in the lipid bilayer and undergo conformational changes leading to alternating exposure of the substrate-binding site to either side of the membrane. In secondary active transporters, the transitions between these conformational changes are strictly coupled to the binding and unbinding of not only the primary transported substrate but also a secondary substrate, which leads to their combined transport. Secondary substrates are usually protons or sodium ions of which membrane gradients are maintained in cells, which thereby provides the free energy gain necessary for substrate transport (1).The secondary active transporter CitS mediates the accumulation of citrate in gram-negative bacteria by mechanistic coupling of substrate translocation to the transport of two Na⁺ ions across the cytoplasmic membrane. In the pathogen Klebsiella pneumoniae, CitS is responsible for citrate uptake in the anaerobic citrate degradation pathway (2). CitS belongs to the 2-HydrocyCarboxylate Transporter family. Other members of this family are capable of transporting mono-, di-, and tricarboxylates containing a 2-hydroxy group. These proteins are involved in several energy conservation pathways such as citrate fermentation, malolactic fermentation, citrolactic fermentation, and oxidative malate decarboxylation (3–5). Crystal structures have been solved of CitS from K. pneumoniae and Salmonella enterica (6). The quaternary structure revealed that CitS is a homodimer, with each protomer consisting of two domains: a dimerization domain located centrally and a transport domain located peripherally (SI Appendix, Fig. S1). This dimeric structural arrangement of CitS leads to the presence of two identical transport routes per complex.Before the first structure of CitS was revealed, the alternating access was explained as a “rocker switch” mechanism similarly to LacY and GlpT, two transporters for which crystal structures were available at the time (7, 8). The publication of the structure of CitS from S. enterica suggested that the translocation of the substrates occurs by an elevator mechanism, similarly to the aspartate transporters Glt_Ph and Glt_Tk (9, 10). The structure of CitS suggests that the dimerization domains serve as central membrane anchor, and the peripheral transport domains can move up and down through the bilayer during turnover as a rigid body. This movement leads to a displacement of the binding site by 17 Å perpendicular to the membrane plane, as well as a rotation by ∼35°, and shuttles the protein between the outward-facing and inward-facing conformations (11). This elevator-type mechanistic interpretation, however, was inferred solely from the static crystal structures, and dynamic experiments have not been reported to test this assumption. In this respect, high-speed atomic force microscopy (HS-AFM) (12–15) offers the advantage of probing membrane proteins in liquid in their lipidic environment with high spatial and temporal resolution (16, 17). Uptake experiments performed on CitS report an apparent turnover rate of roughly once every second at saturating conditions (SI Appendix, Table S1) (18). This rate is within the scanning capabilities of HS-AFM, and here, we report HS-AFM experiments directly visualizing the dynamics of citrate transport, at the single-molecule level, to unveil the molecular mechanism behind transport.     

Enhancing the chloramphenicol sensing performance of Cu–MoS2 nanocomposite-based electrochemical nanosensors: roles of phase composition and copper loading amount

The rational design of nanomaterials for electrochemical nanosensors from the perspective of structure–property–performance relationships is a key factor in improving the analytical performance toward residual antibiotics in food. We have investigated the effects of the crystalline phase and copper loading amount on the detection performance of Cu–MoS₂ nanocomposite-based electrochemical sensors for the antibiotic chloramphenicol (CAP). The phase composition and copper loading amount on the MoS₂ nanosheets can be controlled using a facile electrochemical method. Cu and Cu₂O nanoparticle-based electrochemical sensors showed a higher CAP electrochemical sensing performance as compared to CuO nanoparticles due to their higher electrocatalytic activity and conductivity. Moreover, the design of Cu–MoS₂ nanocomposites with appropriate copper loading amounts could significantly improve their electrochemical responses for CAP. Under optimized conditions, Cu–MoS₂ nanocomposite-based electrochemical nanosensor showed a remarkable sensing performance for CAP with an electrochemical sensitivity of 1.74 μA μM⁻¹ cm⁻² and a detection limit of 0.19 μM in the detection range from 0.5–50 μM. These findings provide deeper insight into the effects of nanoelectrode designs on the analytical performance of electrochemical nanosensors.

In this work, we clarify the roles of phase composition and copper loading amount on the CAP sensing performance of Cu–MoS₂ nanocomposite-based electrochemical nanosensors.     

Chemical synthesis of Nd2Fe14B/Fe–Co nanocomposite with high magnetic energy product

Nd₂Fe₁₄B is one of the most popular permanent magnets (PMs) possessing the best energy product (BH)_max among the common PM materials. However, exchange-coupled nanocomposite magnets fabricated by embedding nanostructures of soft-phase magnetic materials into a hard-phase magnetic matrix manifest higher remanence and a higher energy product. Here we present the fabrication of exchange coupled Nd₂Fe₁₄B/Fe–Co magnetic nanocomposites using gel-combustion and diffusion–reduction processes. Pre-fabricated CoFe₂O₄ nanoparticles (NPs) of ∼5 nm diameter were incorporated into a Nd–Fe–B oxide matrix during its synthesis by gel-combustion. The obtained mixed oxide was further processed with oxidative annealing at 800 °C for 2 h and reductive annealing at 900 °C for 2 h to form a Nd₂Fe₁₄B/Fe–Co nanocomposite. Nanocomposites with different mol% of soft-phase were prepared and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and physical property measurement system (PPMS) to study their crystalline phase, morphology and magnetic behavior. Addition of 7.7 mol% of soft-phase was found to be optimum, producing a coercivity (H_c) of 5.6 kOe and remanence (M_r) of 54 emu g⁻¹ in the nanocomposite.

Nd₂Fe₁₄B is one of the most popular permanent magnets (PMs) possessing the best energy product (BH)_max among the common PM materials.