A novel deep intronic variant strongly associates with Alkaptonuria.

Alkaptonuria is a rare autosomal recessive inherited disorder of tyrosine metabolism, which causes ochronosis, arthropathy, cardiac valvular calcification, and urolithiasis. The epidemiology of alkaptonuria in East Asia is not clear. In this study, patients diagnosed with alkaptonuria from January 2010 to June 2020 were reviewed. Their clinical and molecular features were further compared with those of patients from other countries. Three patients were found to have alkaptonuria. Mutation analyses of the homogentisate 1,2-dioxygenase gene (HGD) showed four novel variants c.16-2063 A > C, p.(Thr196Ile), p.(Gly344AspfsTer25), and p.(Gly362Arg) in six mutated alleles (83.3%). RNA sequencing revealed that c.16-2063 A > C activates a cryptic exon, causing protein truncation p.(Tyr5_Ile6insValTer17). A literature search identified another 6 patients with alkaptonuria in East Asia; including our cases, 13 of the 18 mutated alleles have not been reported elsewhere in the world. Alkaptonuria is rare in Taiwan and East Asia, with HGD variants being mostly novel and private

Male Germ Cell-Specific RNA Binding Protein RBMY: A New Oncogene Explaining Male Predominance in Liver Cancer

Male gender is a risk factor for the development of hepatocellular carcinoma (HCC) but the mechanisms are not fully understood. The RNA binding motif gene on the Y chromosome (RBMY), encoding a male germ cell-specific RNA splicing regulator during spermatogenesis, is aberrantly activated in human male liver cancers. This study investigated the in vitro oncogenic effect and the possible mechanism of RBMY in human hepatoma cell line HepG2 and its in vivo effect with regards to the livers of human and transgenic mice. RBMY expression in HepG2 cells was knocked down by RNA interference and the cancer cell phenotype was characterized by soft-agar colony formation and sensitivity to hydrogen-peroxide-induced apoptosis. The results revealed that RBMY knockdown reduced the transformation and anti-apoptotic efficiency of HepG2 cells. The expression of RBMY, androgen receptor (AR) and its inhibitory variant AR45, AR-targeted genes insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) was analyzed by quantitative RT-PCR. Up-regulation of AR45 variant and reduction of IGF-1 and IGFBP-3 expression was only detected in RBMY knockdown cells. Moreover, RBMY positive human male HCC expressed lower level of AR45 as compared to RBMY negative HCC tissues. The oncogenic properties of RBMY were further assessed in a transgenic mouse model. Liver-specific RBMY transgenic mice developed hepatic pre-cancerous lesions, adenoma, and HCC. RBMY also accelerated chemical carcinogen-induced hepatocarcinogenesis in transgenic mice. Collectively, these findings suggest that Y chromosome-specific RBMY is likely involved in the regulation of androgen receptor activity and contributes to male predominance of HCC

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Immunohistochemical localization of key arachidonic acid metabolism enzymes during fracture healing in mice.

This study investigated the localization of critical enzymes involved in arachidonic acid metabolism during the initial and regenerative phases of mouse femur fracture healing. Previous studies found that loss of cyclooxygenase-2 activity impairs fracture healing while loss of 5-lipoxygenase activity accelerates healing. These diametric results show that arachidonic acid metabolism has an essential function during fracture healing. To better understand the function of arachidonic acid metabolism during fracture healing, expression of cyclooxygenase-1 (COX-1), cyclooxygenase -2 (COX-2), 5-lipoxygenase (5-LO), and leukotriene A4 hydrolase (LTA4H) was localized by immunohistochemistry in time-staged fracture callus specimens. All four enzymes were detected in leukocytes present in the bone marrow and attending inflammatory response that accompanied the fracture. In the tissues surrounding the fracture site, the proportion of leukocytes expressing COX-1, COX-2, or LTA4H decreased while those expressing 5-LO remained high at 4 and 7 days after fracture. This may indicate an inflammation resolution function for 5-LO during fracture healing. Only COX-1 was consistently detected in fracture callus osteoblasts during the later stages of healing (day 14 after fracture). In contrast, callus chondrocytes expressed all four enzymes, though 5-LO appeared to be preferentially expressed in newly differentiated chondrocytes. Most interestingly, osteoclasts consistently and strongly expressed COX-2. In addition to bone surfaces and the growth plate, COX-2 expressing osteoclasts were localized at the chondro-osseous junction of the fracture callus. These observations suggest that arachidonic acid mediated signaling from callus chondrocytes or from callus osteoclasts at the chondro-osseous junction regulate fracture healing

Immunolocalization of Enzyme Positive Cells at 6 hours after Fracture.

<p>The top image shows a fractured mouse femur stained with safranin-O (orange) and counter stained with fast green (green) and hematoxylin (black; CB: cortical bone; FX: fracture site; scale bar: 300 um). Bottom images show immunohistochemical staining of different cell types with rabbit IgG (Neg.) or with antibodies to COX-1, COX-2, 5-LO, or LTA4H (brown; scale bar: 50 um). Immunohistochemistry specimens were counter stained with methyl green. The higher magnification images are labeled with the primary antibody target (COX-1, COX-2, 5-LO, LTA4H, or Neg.) and with a letter indicating cell type and location as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088423#pone-0088423-g001" target="_blank">Figure 1</a>.</p

Immunolocalization of Enzyme Positive Cells at 1 day after Fracture.

<p>The top image shows a fractured mouse femur stained with safranin-O (orange) and counter stained with fast green (green) and hematoxylin (black; CB: cortical bone; FX: fracture site; scale bar: 300 um). Bottom images show immunohistochemical staining of different cell types with rabbit IgG (Neg.) or with antibodies to COX-1, COX-2, 5-LO, or LTA4H (brown; scale bar: 50 um). Immunohistochemistry specimens were counter stained with methyl green. The higher magnification images are labeled with the primary antibody target (COX-1, COX-2, 5-LO, LTA4H, or Neg.) and with a letter indicating cell type and location as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088423#pone-0088423-g001" target="_blank">Figure 1</a>.</p

Immunolocalization of Enzyme Positive Cells at 2 days after Fracture.

<p>The top image shows a fractured mouse femur stained with safranin-O (orange) and counter stained with fast green (green) and hematoxylin (black; CB: cortical bone; FX: fracture site; scale bar: 300 um). Bottom images show immunohistochemical staining of different cell types with rabbit IgG (Neg.) or with antibodies to COX-1, COX-2, 5-LO, or LTA4H (brown; scale bar: 50 um). Immunohistochemistry specimens were counter stained with methyl green. The higher magnification images are labeled with the primary antibody target (COX-1, COX-2, 5-LO, LTA4H, or Neg.) and with a letter indicating cell type and location as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088423#pone-0088423-g001" target="_blank">Figure 1</a>.</p

Immunolocalization of Enzyme Positive Cells before Fracture.

<p>Top image shows a mouse femur stained with safranin-O (orange) and counter stained with fast green (green) and hematoxylin (black; CB: cortical bone; scale bar: 300 um). Bottom images show immunohistochemical staining of different cell types with antibodies to COX-1, COX-2, 5-LO, or LTA4H (brown; scale bar: 50 um). Rabbit IgG was used as a negative control (Neg.). Immunohistochemistry specimens were counter stained with methyl green. The higher magnification images are labeled with the primary antibody target (COX-1, COX-2, 5-LO, LTA4H, or Neg.) and with a letter indicating cell type and location as listed in the bottom of the figure.</p