Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma.

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Zinc-Supported Multiwalled Carbon Nanotube Nanocomposite: A Synergism to Micronutrient Release and a Smart Distributor To Promote the Growth of Onion Seeds in Arid Conditions.

In the current scenario, nanotechnological applications in the agriculture sector showing potential impacts on the improvement of plant growth in terms of protection and safety are at a very nascent stage. The present study deals with the synergistic role of zinc (Zn) and multiwalled carbon nanotubes (MWCNTs) synthesized as a zinc oxide (ZnO)/MWCNT nanocomposite, a prospective applicant to modulate the micronutrient supply and enhance the growth of onion seeds, thereby replacing harmful, unsafe chemical fertilizers. To the best of our knowledge, this is the first report wherein MWCNTs have been envisaged as a micronutrient distributor and a nutrient stabilizer enhancing the growth of onion plant under arid conditions. The growth trend of onion seeds was evaluated in an aqueous medium with varied concentrations of (i) MWCNTs, (ii) zinc oxide nanoparticles, and (iii) ZnO/MWCNT nanocomposites. ZnO/MWCNT nanocomposites with 15 µg/mL concentration displayed the best seedling growth with the maximum number of cells in telophase. A significant growth trend with increased concentration of ZnO/MWCNTs displayed no negative impact on plant growth in contrast to that with the use of MWCNTs. The synergistic impact of Zn nanoparticles and MWCNTs in ZnO/MWCNT nanocomposites on the rate of germination was explained via a mechanism supported by scanning transmission electron microscopy.

Development and Validation of a Novel Stability-Indicating Reversed-Phase Ion-Pair Chromatographic Method for the Quantitation of Impurities in Marbofloxacin Tablets.

A stability-indicating isocratic reversed-phase ion-pair chromatographic method was designed for the separation of impurities in the presence of degradation products. Marbofloxacin tablets and a placebo were exposed to the stress conditions of oxidative, acid, base, humidity, thermal, and photolytic degradation. Significant and moderate degradation was observed in acidic and oxidative stress conditions, respectively. The degradation products were well resolved from the main peak and its impurities, thus proving the stability-indicating analytical method. The method was developed by using an XTerra RP18 3.5 µm (150 × 4.6 mm) column, with the mobile phase containing a mixture of buffer (pH 2.5)-methanol-glacial acetic acid (77 + 23 + 0.5, v/v). The flow rate of the mobile phase was 1.2 mL/min, with a column oven temperature of 40°C and a detection wavelength of 315 nm. The proposed method met Veterinary International Conference on Harmonization requirements and was successfully used for impurity quantitation in marbofloxacin tablets.

Epstein-Barr virus-associated primary nodal T/NK-cell lymphoma shows a distinct molecular signature and copy number changes.

The molecular biology of primary nodal T- and NK-cell lymphoma and its relationship with extranodal NK/T-cell lymphoma, nasal type is poorly understood. In this study, we assessed the relationship between nodal and extranodal Epstein-Barr virus-positive T/NK-cell lymphomas using gene expression profiling and copy number aberration analyses. We performed gene expression profiling and copy number aberration analysis on 66 cases of Epstein-Barr virus-associated T/NK-cell lymphoma from nodal and extranodal sites, and correlated the molecular signatures with clinicopathological features. Three distinct molecular clusters were identified with one enriched for nodal presentation and loss of 14q11.2 (TCRA loci). T/NK-cell lymphomas with a nodal presentation (nodal-group) were significantly associated with older age, lack of nasal involvement, and T-cell lineage compared to those with an extranodal presentation (extranodal-group). On multivariate analysis, nodal presentation was an independent factor associated with short survival. Comparing the molecular signatures of the nodal and extranodal groups it was seen that the former was characterized by upregulation of PD-L1 and T-cell-related genes, including CD2 and CD8, and downregulation of CD56, consistent with the CD8+/CD56-immunophenotype. PD-L1 and CD2 protein expression levels were validated using multiplexed immunofluorescence. Interestingly, nodal group lymphomas were associated with 14q11.2 loss which correlated with loss of TCR loci and T-cell origin. Overall, our results suggest that T/NK-cell lymphoma with nodal presentation is distinct and deserves to be classified separately from T/NK-cell lymphoma with extranodal presentation. Upregulation of PD-L1 indicates that it may be possible to use anti-PD1 immunotherapy in this distinctive entity. In addition, loss of 14q11.2 may be a potentially useful diagnostic marker of T-cell lineage.