Proteomic Analysis of Individual Drosophila Hemolymph

Developing analysis methods and tools to understand the chemical information of the proteome of an organism of limited volume are challenging and vital for modern proteomic studies. Drosophila melanogaster (also called fruit fly) is one of the ideal animal models for the study of proteomic composition because of the high degree of genome homology with the human genome. Nevertheless, it has less than 50 nL blood (hemolymph) available for collection due to its small size (about 3 mm). The goal of this thesis is to present sample preparation and analysis methods to improve the identification of proteins in volume-limited biological samples using a series of chromatography and mass spectrometry methods. A hyphenated nano-reverse phase liquid chromatography chip column-mass spectrometry (nano-RPLC chip column-MS) method was developed to obtain proteomic information of hemolymph from an individual fruit fly. This is the first study to report a qualitative analysis of proteomic composition of hemolymph from individual adult female fruit flies. A microliter-scale protein digestion protocol was also developed to assist the digestion efficiency of limited volume sample. With the improved sample preparation method, six novel proteins were identified for the first time at the translation level. Detection of 13 proteins that are well-known in the literature speaks to the method’s validity and demonstrates the ability to reproducibly analyze volume-limited samples from individual fruit flies for protein content. Further, comprehensive prefractionation methods were developed by fabricating 2-cm long chromatography columns for individual fruit fly hemolymph samples. Detection of lower abundance proteins was enhanced with reverse phase and ion exchange prefractionation methods when followed by high mass resolution and high mass accuracy fourier transform ion cyclotron resonance-mass spectrometry (FT-ICR) and Orbitrap mass spectrometers. CE was brought on-line connected with a portable ion trap mass spectrometer by developing an interface. The sheathless CE-MS hyphenated instrument, built in house, enables efficient separation and detection of a list of amino acid standards and provides an alternative fast separation and detection method for small molecule analysis. This coupled CE-MS instrument compliments the complicated FT-ICR tandem mass spectrometry identification for larger molecular weight protein samples

Engineered Nanovesicles Expressing Bispecific Single Chain Variable Fragments to Protect against SARS-CoV‑2 Infection

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in high morbidity and mortality rates worldwide. Although the epidemic has been controlled in many areas and numerous patients have been successfully treated, the risk of reinfection persists due to the low neutralizing antibody titers and weak immune response. To provide long-term immune protection for infected patients, novel bispecific CB6/dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (SIGN) nanovesicles (NVs) were constructed to target both the SARS-CoV-2 spike protein (S) and the DC receptors for virus neutralization and immune activation. Herein, we designed NVs expressing both CB6 and DC-SIGN single chain variable fragments (scFvs) on the surface to block SARS-CoV-2 invasion and activate DC function. Monophosphoryl lipid A (MPLA) was loaded into the CB6/DC-SIGN NVs as an adjuvant to promote this process. The CB6/DC-SIGN NVs prevented a pseudovirus expressing the S protein from infecting the target cells expressing high levels of angiotensin-converting enzyme 2 in vitro. Additionally, CB6/DC-SIGN NVs admixed with S-expressing pseudoviruses activated the DCs, which was promoted by the adjuvant MPLA loaded in the NVs. Using a mouse model, we also confirmed that the CB6/DC-SIGN NVs effectively improved the neutralizing antibody titer and inhibited the growth of tumors expressing the S protein after 3 weeks of treatment. This potential NV-based treatment not only exerts a blocking effect by binding the S protein in the short term but may also provide patients with long-term protection against secondary infections

Comparison of locoregional failure-free survival between the combined chemo-radiotherapy and radiotherapy groups.

<p>Footnote: RT, radiotherapy; CRT, standard chemo-radiotherapy.</p

Additional file 1: Figure S1. of Critical weight loss predicts poor prognosis in nasopharyngeal carcinoma

Comparison of overall survival among patients with ≥5 % weight loss, patients with <5 % weight loss, and patients with weight gain and without weight loss. (TIF 34 kb

Additional file 2: of Induction Chemotherapy Followed by Radiotherapy versus Concurrent Chemoradiotherapy in elderly patients with nasopharyngeal carcinoma: finding from a propensity-matched analysis

List of elderly patients with nasopharyngeal carcinoma in our study. (XLSX 49 kb

The morphology and histology of cv. 9311 calli.

<p>The light yellowish and vigorously growing calli of cv. 9311 after two weeks subculture (A, B); histological view of cv. 9311 calli (C), the embryogenic cells which exhibited dense cytoplasm and small vacuoles were indicated by the red arrow at the surface of calli; the browning calli of cv. 9311 (D).</p

Changes of nitrite level and lignin content in cv. 9311 calli during the process of culture.

<p>The cv. 9311 calli were inoculated on the M1 (control, without nitrite) or M2 (with nitrite) for 14 days duration, and sampled at the indicated periods for the measurement of nitrite level and lignin content. Data were derived from 3 independent replicates. Asterisks indicate a statistical difference between calli grown on medium with nitrite (M1) and without (M2, control) at the same inoculated time (*<i>P <0.05</i>, **<i>P <0.001</i>).</p

Nitrogen source of medium used in this study.

<p>Nitrate was deprived from the N6 medium and 3.5 mM (NH4)₂SO₄ was used as the nitrogen source in M1, M2, M3, M4 medium. 2 mM KNO₂ or 20 mM glutamine was added in these medium if necessary. The other components composed of N6 basal medium (deprive of nitrogen source), 20 mM KCl, 800 mg/L casein hydrolysate, 600 mg/L proline, 2.0 mg/L 2,4-D, 3% sucrose, and 0.3% phytagel at pH 5.8. ‘―’ indicates the component was not added. The standard N6 medium was used as a control in this experiment.</p

Histogram presentation of enriched gene ontology (GO) classification.

<p>All the DEGs were mapped to GO terms in the database, and counted the frequency of genes in every class. The normalized frequency (y-axis) were calculated as frequency of the class in the input data set divided by the frequency of the class in the genome, and the significant enriched GO terms were presented with <i>p</i> value <0.05.</p