MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of molecular species in tissue specimens. Numerous methodological improvements have been made to optimize tissue section preparation and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the number of signals observed. We systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize experimental variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and number of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystallization behavior as well as their ability to preserve tissue histology were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma

Lactobacillus-dominated vaginal microbiotas are associated with reproductive health and STI resistance in women, whereas altered microbiotas are associated with bacterial vaginosis (BV), STI risk and poor reproductive outcomes. Putative vaginal taxa have been observed in male first-catch urine, urethral swab and coronal sulcus (CS) specimens but the significance of these observations is unclear. We used 16 S rRNA sequencing to characterize the microbiota of the CS and urine collected from 18 adolescent men over three consecutive months. CS microbiotas of most participants were more stable than their urine microbiotas and the composition of CS microbiotas were strongly influenced by circumcision. BV-associated taxa, including Atopobium, Megasphaera, Mobiluncus, Prevotella and Gemella, were detected in CS specimens from sexually experienced and inexperienced participants. In contrast, urine primarily contained taxa that were not abundant in CS specimens. Lactobacilllus and Streptococcus were major urine taxa but their abundance was inversely correlated. In contrast, Sneathia, Mycoplasma and Ureaplasma were only found in urine from sexually active participants. Thus, the CS and urine support stable and distinct bacterial communities. Finally, our results suggest that the penis and the urethra can be colonized by a variety of BV-associated taxa and that some of these colonizations result from partnered sexual activity.

The rate of tumor recurrence post resection suggests that there are underlying molecular changes in nearby histologically normal tissue that go undetected by conventional diagnostic methods that utilize contrast agents and histochemistry. MALDI MS is a molecular technology that has the specificity and sensitivity to monitor and identify molecular species indicative of these processes. The current study utilizes this technology to assess molecular distributions within a tumor and adjacent normal tissue in clear cell renal cell carcinoma biopsies. Results indicate that the histologically normal tissue adjacent to the tumor expresses many of the molecular characteristics of the tumor. Proteins of the mitochondrial electron transport system are examples of such distributions. This work demonstrates the utility of MALDI MS for the analysis of tissue biopsies in the elucidation of molecular processes in the tumor microenvironment.

Purpose: To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies.Experimental Design: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR).Results: Proteomic and validation immunohistochemical analyses revealed that α-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxanebased treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery.Conclusion: We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane-based therapy. Clin Cancer Res; 16(2); 681-90.

Patients with melanoma metastatic to regional lymph nodes exhibit a range in tumor progression, survival, and treatment. Current approaches to stratify patients with this stage of disease predominantly involve clinical and histological methods. Molecular classification thus far has focused almost exclusively on genetic mutations. In this study, proteomic data from 69 melanoma lymph node metastases and 17 disease free lymph nodes acquired by histology-directed MALDI imaging mass spectrometry were used to classify tumor from control lymph node and to molecularly sub-classify patients with stage III disease. From these data, 12 survival associated protein signals and 3 recurrence associated signals in the acquired mass spectra were combined to generate a multiplex molecular signature to group patients into either poor or favorable groups for recurrence and survival. Proteins represented in the signature include cytochrome c, s100 A6, histone H4, and cleaved forms of thymosin β-4, thymosin β-10, and ubiquitin. In total over 40 protein signals from the tissue were identified.

Rationale: Previous work found the lung microbiome in healthy subjects infected with HIV was similar to that in uninfected subjects.

Background Emerging evidence suggests that CNS injury and neurocognitive impairment persist in the setting of chronic HIV infection and combination antiretroviral therapy (CART). Yet whether neurological injury can progress in this setting remains uncertain. Methods Magnetic resonance spectroscopy, neurocognitive and clinical assessments were performed over two years in 226 HIV-infected individuals on stable CART, including 138 individuals who were neurocognitively asymptomatic (NA). Concentrations of N-acetylaspartate (NAA), creatine (Cr), choline (Cho), myoinositol (MI), and glutamate/glutamine (Glx) were measured in the midfrontal cortex (MFC), frontal white matter (FWM) and basal ganglia (BG). Longitudinal changes in metabolite levels were determined using linear mixed effect models, as were metabolite changes in relation to global neurocognitive function. Results HIV-infected subjects showed significant annual decreases in brain metabolite levels in all regions examined, including NAA (2.95%), Cho (2.61%) in the FWM; NAA (1.89%), Cr (1.84%), Cho (2.19%) and Glx (6.05%) in the MFC; and Glx (2.80%) in the BG. Similar metabolite decreases were observed in the NA and subclinically impaired subgroups, including subjects with virologic suppression in plasma and CSF. Neurocognitive decline was associated with longitudinal decreases in Glx in the FWM and the BG, and in NAA in the BG. Conclusions Widespread progressive changes in the brain, including neuronal injury, occur in chronically HIV-infected persons despite stable antiretroviral treatment and virologic suppression and can lead to neurocognitive declines. The basis for these findings is poorly understood and warrants further study.

A reciprocal interaction between the implantation-competent blastocyst and receptive uterus is an absolute requirement for implantation, a process crucial for pregnancy success. A comprehensive understanding of this interaction has yet to be realized. One major difficulty in clearly defining this discourse is the complexity of the implantation process involving heterogeneous cell types of both the uterus and blastocyst, each endowed with unique molecular signatures that show dynamic changes during the course of pregnancy. Whereas gene expression studies by in situ hybridization or immunohistochemistry have shown differential expression patterns of specific genes during implantation, there is no report how numerous signaling proteins are spatially displayed at specific times and stages of implantation in the context of blastocyst-uterine juxtaposition. Using in situ imaging (matrix assisted laser desorption/ionization) mass spectrometry directly on uterine sections, here we provide molecular composition, relative abundance, and spatial distribution of a large number of proteins during the periimplantation period. This approach has allowed us for the first time to generate in situ proteome profiles of implantation and interimplantation sites in mice in a region- and stage-specific manner with the progression of implantation. This application is reliable because patterns of expression of several proteins displayed by in situ imaging mass spectrometry correlate well with in situ hybridization results. More interestingly, the use of this approach has provided new insights regarding uterine biology of cytosolic phospholipase A(2alpha) null females that show implantation defects.