Complex Mixtures

The fractional abundance matrix F can be determined for the bulk GEP matrix M, either through expression deconvolution as described above^{2 (link),3 (link)} or with prior empiric knowledge of the compositional representation of cell types within the bulk specimen (e.g., by an automated hematology analyzer, or by flow cytometry)^{25 (link)}. Once F is determined for a given M, a representative imputed GEP for each cell type in F can be estimated by solving the following system of linear equations:
$${\mathrm{H}}_{i,•}\times \mathrm{F}={\mathrm{M}}_{i,•},1\le i\le n$$
where H is a n × c expression matrix of n genes and c cell types, H_i,j≥ 0 for all i, j, and F is defined as above with the constraint that relative cell fractions sum to one for each mixture sample. Like Equation 1 above, the system should be overdetermined (k > c), with a greater difference between k and c generally leading to improved GEP estimation (Fig. 3e, Supplementary Fig. 6). To ensure biologically realistic estimates of gene expression, we employ non-negative least squares regression (NNLS), an optimization framework to solve the least squares problem with non-negativity constraints. Although NNLS is robust on simple mixtures and toy examples, its performance on more complex mixtures inherent within real tissue samples can be affected by noise, imprecision, and missing data in the linear system^{15 (link)}. We therefore developed a series of novel data normalization and filtering techniques to help mitigate these issues (Fig. 3b-d, see ‘Imputation of group-mode expression profiles’ in Supplementary Note 1).

Western blotting is a valuable tool to studies ranging from regulatory signaling processes to confirmatory serum diagnosis of HIV [68 (link)–70 (link)]. The evolution of western blot technique from identification of a specific protein in a complex mixture to the direct detection of protein in a single cell allows this technique to be an important analytical tool for clinical research. An advanced single cell western blotting technique was employed to study stem cell signaling and differentiation as well as drug response in tumor cells [69 (link)]. Through single cell western blotting it was possible to analyze cell-to-cell variations in approximately 2000 cells simultaneously within complex populations of cells [71 (link)]. With the integration of intact cell imaging, the technique allows the identification of protein expression changes of a single drug resistant tumor cell and its isoforms among heterogeneous population of tumor cells in human glioblastoma cells treated with chemotherapeutic daunomycin [69 (link)]. Identification of upregulated multidrug resistant protein, P-glycoprotein in living glioblastoma subpopulations was indicative of an active drug eflux pump as an underlying mechanism for drug resistance [69 (link),71 (link)]. With the application of 2-DE gel separation together with spotting of protein by peptide mass fingerprint, the analysis of clinically relevant Helicobacter pylori (H. pylori) in related gastric disease conditions (chronic gastritis, duodenal ulcer) was possible [72 (link)]. The database of H. pylori (low expressed and membrane proteins) was created through the application of one-dimensional or 2-DE/MALDI-mass spectrometry techniques [72 (link)]. In a similar manner, the Simple Western technique was employed for the analysis of 15-valent pneumococcal vaccine PCV15-CRM197 [73 (link)]. Due to its high sensitivity and automation, the Simple Western method may be extended to analyze serotypes of other polysaccharide protein conjugate vaccines [73 (link)].
Western blotting is commonly used for the clinical diagnosis of various parasitic and fungal diseases including echinococcosis [74 (link)], toxoplasmosis [75 (link)], and aspergillosis [76 (link)]. In a recent study, the assay was successfully used for the reliable serodiagnosis of Farmer’s lung disease (FLD), a pulmonary disorder caused by inhalation of antigenic particles [77 (link)]. Thus, this technique can be exploited for rapid routine diagnosis of FLD in clinics [77 (link)]. Similarly, for immunodiagnostic of tuberculosis meningitis which is a chronic disease of central nervous system different molecular and immunological methods were used for clinical diagnosis of the disease. However, each of these techniques has their own limitations [78 (link)]. To overcome diagnostic issues of lower sensitivity and specificity, the immunoreactivity to Mycobacterium tuberculosis antigens was performed by western blotting [78 (link)]. Furthermore, western blotting was performed for the early and sensitive diagnosis of congenital toxoplasmosis [79 (link)] and was employed for rapid and sensitive serological diagnosis of a serious infectious disease paracoccidioidomycosis (PCM) [80 (link)]. Using immunoblotting, a new subgroup of human lymphotropic retroviruses (HTLV), was detected in patients with the acquired immunodeficiency syndrome (AIDS) [81 (link)]. Antigens of HTLV-III, specifically detected by antibodies in serum from AIDS or pre-AIDS patients [81 (link)]. Western blotting has also been used as a test for variant Creutzfeldt-Jakob Disease [82 (link)], some forms of Lyme disease [83 (link)] and is sometimes used as a confirmatory test for Hepatitis B [84 ] and Herpes Type 2 [85 (link)] infections. Western blots have also been used to confirm feline immunodeficiency status in cats [86 (link)].
Recently, a commercial Aspergillus western blotting IgG kit was developed by LD Bio Diagnostics (France) to carry out immunoblotting for the clinical diagnosis of chronic aspergillosis. The commercial kit was found to be sensitive and can analyze hundreds of samples from patients with aspergillus disease [87 (link)]. Thus, the clinical applications of western blotting technique will continue to progress as further advancements are made to improve sensitivity and reproducibility of the western blot.