DNA replication initiated by one-ended homologous recombination at a double-strand break is highly inaccurate, as it greatly stimulates frameshift mutations over the entire path of the replication fork.

The microtiter plate (also called 96-well plate) assay for studying biofilm formation is a method which allows for the observation of bacterial adherence to an abiotic surface. In this assay, bacteria are incubated in vinyl "U"-bottom or other types of 96-well microtiter plates. Following incubation, planktonic bacteria are rinsed away, and the remaining adherent bacteria (biofilms) are stained with crystal violet dye, thus allowing visualization of the biofilm. If quantitation is desired, the stained biofilms are solubilized and transferred to a 96-well optically clear flat-bottom plate for measurement by spectrophotometry.

In addition to regulating B cell development and activation, Bruton’s tyrosine kinase (Btk) functions downstream of multiple TLRs, including TLR7, to regulate innate immune responses in myeloid cells. Although critical for defense against RNA viruses such as influenza and Sendai virus, recognition of self-RNA by TLR7 also has been shown to be an important contributor to the pathophysiology of systemic lupus erythematosus. To date, the role of Btk in regulating TLR7-mediated responses is poorly understood. In the current study, we have demonstrated a hitherto undiscovered role for Btk in apoptotic cell uptake, identifying the molecular chaperone calreticulin (CRT) as a novel substrate for Btk in regulating this response. CRT together with the transmembrane receptor CD91 function at the cell membrane and regulate uptake of C1q-opsonised apoptotic cells. Our results show that Btk directly phosphorylates CRT and that in the absence of Btk, CRT fails to localize with CD91 at the cell surface and at the phagocytic cup. Critically, a blocking Ab against CRT in wild-type macrophages mimics the inability of Btk-deficient macrophages to phagocytose apoptotic cells efficiently, indicating the critical importance of Btk in regulating CRT-driven apoptotic cell uptake. Our data have revealed a novel regulatory role for Btk in mediating apoptotic cell clearance, with CRT identified as the critical component of the CRT/CD91/C1q system targeted by Btk. Given the importance of clearing apoptotic cell debris to prevent inappropriate exposure of TLRs to endogenous ligands, our results have important implications regarding the role of Btk in myeloid cell function.

Objective. To examine the role of 17␤-estradiol in the regulation of the autoantigen tripartite motifcontaining protein 21 (TRIM-21) in patients with systemic lupus erythematosus (SLE).Methods. Monocytes isolated from healthy control subjects and patients with SLE were stimulated with 17␤-estradiol and/or the estrogen receptor ␣ (ER␣) antagonist methyl-piperidino-pyrazole dihydrochloride. TRIM-21, ER␣, and CREM␣ expression was determined by real-time polymerase chain reaction (PCR) analysis. MatInspector software was used to identify putative binding sites within the TRIM-21 promoter. ER␣ binding to the TRIM-21 gene promoter region in monocytes was analyzed by chromatin immunoprecipitation (ChIP) assay. TRIM-21 and interferon regulatory factor 3 protein levels were analyzed by Western blotting. Conclusion. Our results demonstrate a role of estrogen in the regulation of TRIM-21 expression through an ER␣-dependent mechanism, a pathway that we observed to be overactive in SLE patients. Treatment of monocytes with an ER␣ antagonist abrogated estrogen-induced TRIM-21 expression and, as a consequence, decreased the expression of interleukin-23. These findings identify TRIM-21 as a novel ER␣-regulated gene and provide novel insights into the link between estrogen and the molecular pathogenesis of SLE. Results