Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis

Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. The regulon that it governs is comprised of over 120 genes based on microarray analysis, although it is not known how many of these are under direct Spx control. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products that are conserved in low %GC Gram-positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as poor growth in minimal medium and sensitivity to methyglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methyglyoxal. The ytpQ mutant showed elevated oxidative protein damage when exposed to methylglyoxal, and reduced growth rate in liquid culture. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons of B. subtilis. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in iron homeostasis. The ytpQ mutant phenotype mimics that of a fur mutation, suggesting a condition of low cellular iron. In vitro transcription analysis indicated that Spx stimulates transcription from the ytpPQR operon within which the ytpQ gene resides. The work uncovers a link between Spx and control of iron homeostasis

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

CT perfusion for early response evaluation of radiofrequency ablation of focal liver lesions: first experience

PURPOSE To investigate the value of perfusion CT (P-CT) for early assessment of treatment response in patients undergoing radiofrequency ablation (RFA) of focal liver lesions. METHODS AND MATERIALS 20 consecutive patients (14 men; mean age 64 ± 14) undergoing P-CT within 24 h after RFA of liver metastases (n = 10) or HCC (n = 10) were retrospectively included. Two readers determined arterial liver perfusion (ALP, mL/min/100 mL), portal liver perfusion (PLP, mL/min/100 mL), and hepatic perfusion index (HPI, %) in all post-RFA lesions by placing a volume of interest in the necrotic central (CZ), the transition (TZ), and the surrounding parenchymal (PZ) zone. Patients were classified into complete responders (no residual tumor) and incomplete responders (residual/progressive tumor) using imaging follow-up with contrast-enhanced CT or MRI after a mean of 57 ± 30 days. Prediction of treatment response was evaluated using the area under the curve (AUC) from receiver operating characteristic analysis. RESULTS Mean ALP/PLP/HPI of both readers were 4.8/15.4/61.2 for the CZ, 9.9/16.8/66.3 for the TZ and 20.7/29.0/61.8 for the PZ. Interreader agreement of HPI was fair for the CZ (intraclass coefficient 0.713), good for the TZ (0.813), and excellent for the PZ (0.920). For both readers, there were significant differences in HPI of the CZ and TZ between responders and nonresponders (both, P < 0.05). HPI of the TZ showed the highest AUC (0.911) for prediction of residual tumor, suggesting a cut-off value of 76 %. CONCLUSION Increased HPI of the transition zone assessed with P-CT after RFA might serve as an early quantitative biomarker for residual tumor in patients with focal liver lesions

Computed tomography perfusion imaging for monitoring transarterial chemoembolization of hepatocellular carcinoma

PURPOSE: To prospectively monitor changes in tumor perfusion of hepatocellular carcinoma (HCC) in response to doxorubicin-eluted bead based transarterial chemoembolization (DEB-TACE) using perfusion-CT (P-CT). METHODS AND MATERIALS: 24 patients (54-79 years) undergoing P-CT before and shortly after DEB-TACE of HCC were prospectively included in this dual-center study. Two readers determined arterial-liver-perfusion (ALP, mL/min/100mL), portal-venous-perfusion (PLP, mL/min/100mL) and the hepatic-perfusion-index (HPI, %) by placing matched regions-of-interests within each HCC before and after DEB-TACE. Imaging follow-up was used to determine treatment response and to distinguish complete from incomplete responders. Performance of P-CT for prediction and early response assessment was determined using receiver-operating-characteristics curve analysis. RESULTS: Interreader agreement was fair to excellent (ICC, 0.716-0.942). PLP before DEB-TACE was significantly higher in pre-treated vs non-treated lesions (P<0.05). Mean changes of ALP, PLP and HPI from before to after DEB-TACE were -55%, +24% and -27%. ALP and HPI after DEB-TACE were correlating with response-grades (r=0.45/0.48; both, p<0.04), showing an area-under-the-curve (AUC) of 0.74 and 0.80 respectively for identification of complete response. CONCLUSION: High arterial and low portal-venous perfusion of HCC early after DEB-TACE indicates incomplete response with good diagnostic accuracy

Arterio-portal shunts in the cirrhotic liver: perfusion computed tomography for distinction of arterialized pseudolesions from hepatocellular carcinoma

OBJECTIVES: To determine perfusion computed tomography (P-CT) findings for distinction of arterial pseudolesions (APL) from hepatocellular carcinoma (HCC) in the cirrhotic liver. METHODS: 32 APL and 21 HCC in 20 cirrhotic patients (15 men; 65 ± 10 years), who underwent P-CT for evaluation of HCC pre- (N = 9) or post- (N = 11) transarterial chemoembolization, were retrospectively included using CT follow-up as the standard of reference. All 53 lesions were qualitatively (visual) and quantitatively (perfusion parameters) analysed according to their shape (wedge, irregular, nodular), location (not-/adjunct to a fistula), arterial liver perfusion (ALP), portal venous liver perfusion (PLP), hepatic perfusion index (HPI). Accuracy for diagnosis of HCC was determined using receiver operating characteristics. RESULTS: 18/32 (56 %) APL were wedge shaped, 10/32 (31 %) irregular and 4/32 (12 %) nodular, while 11/21 (52 %) HCC were nodular or 10/21 (48 %) irregular, but never wedge shaped. Significant difference between APL and HCC was seen for lesion shape in pretreated lesions (P < 0.001), and for PLP and HPI in both pre- and post-treated lesions (all, P < 0.001). Diagnostic accuracy for HCC was best for combined assessment of lesion configuration and PLP showing an area under the curve of 0.901. CONCLUSION: Combined assessment of lesion configuration and portal venous perfusion derived from P-CT allows best to discriminate APL from HCC with high diagnostic accuracy. KEY POINTS: • Arterio-portal shunting is common in the cirrhotic liver, especially after local treatment. • Arterial pseudolesions (APL) due to shunting might mimic hepatocellular carcinoma (HCC). • Perfusion-CT allows for qualitative and quantitative assessment of liver lesions. • Lesion configuration fails to discriminate APL from HCC in locally treated patients. • Integration of quantitative perfusion analysis improves accuracy for diagnosis of HCC

MRI texture analysis for differentiation of malignant and benign hepatocellular tumors in the non-cirrhotic liver

Purpose: To find potentially diagnostic texture analysis (TA) features and to evaluate the diagnostic accuracy of two-dimensional (2D) magnetic resonance (MR) TA for differentiation between hepatocellular carcinoma (HCC) and benign hepatocellular tumors in the non-cirrhotic liver in an exploratory MR-study. Materials and methods: 108 non-cirrhotic patients (62 female; 41.5 ± 18.3 years) undergoing preoperative contrast-enhanced MRI were retrospectively included in this multi-center-study. TA including gray-level histogram, co-occurrence and run-length matrix features (total 19 features) was performed by two independent readers. Native fat-saturated-T1w and T2w as well as arterial and portal-venous post contrast-enhanced 2D-image-slices were assessed. Conventional reading was performed by two separate independent readers. Differences in TA features between HCC and benign lesions were investigated using independent sample t-tests. Logistic regression analysis was performed to obtain the optimal number/combination of TA-features and diagnostic accuracy of TA analysis. Sensitivity and specificity of the better performing radiologist were compared to TA analysis. Results: The highest number of significantly differing TA-features (n = 5) was found using the arterial-phase images including one gray-level histogram (skewness, p = 0.018) and four run-length matrix features (all, p < 0.02). The optimal binary logistic regression model for TA-features of the arterial-phase images contained 13 parameters with an accuracy of 84.5% (sensitivity 84.1%, specificity 84.9%) and area-under-the-curve of 0.92 (95%-confidence-interval 0.85–0.98) for diagnosis of HCC. Conventional reading yielded a significantly lower sensitivity (63.6%, p = 0.027) and no significant difference in specificity (94.6%, p = 0.289) at best. Conclusion: 2D-TA of MR images is a feasible objective method that may help to distinguish HCC from benign hepatocellular tumors in the non-cirrhotic liver. Most promising results were found in TA features in the arterial phase images