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In many anoxic environments, methanogenesis is the predominant terminal electron accepting process involved in the mineralization of organic matter, which is catalyzed by methanogenic Archaea. These organisms represent a unique but phylogenetically diverse guild of prokaryotes, which can be conveniently tracked in the environment by targeting the mcrA gene as a functional marker. This gene encodes the alpha subunit of the methyl-coenzyme M reductase (MCR), which catalyzes the last step in methanogenesis and is present in all methanogens. Cultivation-independent analysis of methanogenic communities involves the polymerase chain reaction (PCR) amplification of the mcrA gene from extracted community DNA, comparative analysis of mcrA clone libraries, or PCR-based fingerprinting analysis by terminal restriction fragment polymorphism analysis (T-RFLP). It has also been suggested that anaerobic methane-oxidizing Archaea possess MCR, which facilitates detection of this novel group of "reverse methanogens" as well using the mcrA gene as a functional marker.

This chapter discusses the role of immunohistochemical expression of 1,25-dihyroxyvitamin-D 3 -receptors in ovarion carcinoma. Ovarian carcinoma is a leading cause of death in gynecologic cancer. The immunohistochemical expression of Vitamin D receptor (VDR) in normal and carcinomatous ovarian tissue is analyzed to evaluate the potential target of ovarian tissue for biologically active vitamin D analogs. VDR expression is compared immunohistochemically, with the staining pattern of Ki-67, for analyzing the VDR expression in ovarian tissue for a function of proliferation. Estrogen receptor (ER) and progesterone receptor (PR) are immunohistochemically analyzed to evaluate the expression of VDR that correlates with the expression of these steroid hormone receptors. The percentage of positive tumor cells, the intensity of staining, and a resulting immuno reactivity score are determined for the semi-quantitative evaluation of VDR, ER, and PR expressions. Both the intensity of VDR immunostaining and the number of VDR-positive cells are statistically significantly increased in ovarian carcinomas (OCs) as compared to the normal ovarian tissue. By analyzing the co-expression of VDR with the proliferation marker Ki-67 or with the estrogen and progesterone receptors no statistically significant correlation is found.

Sulfate‐reducing prokaryotes (SRPs) exploit sulfate as an electron acceptor for anaerobic respiration and exclusively catalyze this essential step of the world's sulfur cycle. Because SRPs are found in many prokaryotic phyla and are often closely related to non‐SRPs, 16S rRNA gene‐based analyses are inadequate to identify novel lineages of this guild in a cultivation‐independent manner. This problem can be solved by comparative sequence analysis of environmentally retrieved gene fragments of the dissimilatory (bi)sulfite (dsrAB) and adenosine‐5′‐phosphosulfate reductases (apsA), which encode key enzymes of the SRP energy metabolism. This chapter provides detailed protocols for the application of these functional marker molecules for SRP diversity surveys in the environment. Data from the analysis of dsrAB sequence diversity in water samples from the Mariager Fjord in northeast Denmark are presented to illustrate the different steps of the protocols. Furthermore, this chapter describes a novel gel retardation‐based technique, suitable for fingerprinting of the approximately 1.9‐kb‐large dsrAB polymerase chain reaction amplification products, which efficiently increases the chance of retrieving rare and novel dsrAB sequence types from environmental samples.