[The value of conventional magnetic resonance imaging based radiomic model in predicting the texture of pituitary macroadenoma].

Objective: To investigate the value of conventional magnetic resonance imaging (MRI) based radiomic model in predicting the texture of pituitary macroadenoma. Methods: The complete data of 101 patients with pituitary macroadenoma confirmed by surgery and pathology in Yijishan Hospital of Wannan Medical College from December 2014 to December 2019 were retrospectively analyzed. According to the texture of the intraoperative pituitary tumor, patients were divided into soft group (n=58) and hard group (n=43). They were randomly divided into training group (n=72) and validation group (n=29) at a ratio of 7∶3. All patients underwent conventional MRI scan of the pituitary gland. Itk-snap software was used to manually outline the T(1)-weighted image (T(1)WI), T(2)-weighted image (T(2)WI) and enhanced T(1)WI image section by section on tumor area of interest (ROI) and perform three-dimensional fusion. Then AK software was imported to extract texture features. The regression analysis methods of minimum redundancy maximum relevance (mRMR) and least absolute shrinkage and selection operator (LASSO) were used for feature selection and radiomic signature establishment. The reliability of the model was verified by 100 leave-group-out cross validation (LGOCV), and the predictive ability of the model was evaluated by drawing the receiver operating characteristic (ROC) curve. The decision curve analysis (DCA) was used to evaluate the clinical application value of the model. Results: The AUC (Area Under the ROC Curve) (95%CI) values of T1WI, T2WI, enhanced T1WI, and the combined sequence model to predict the texture of pituitary macroadenomas in the training and validation groups were 0.91 (0.84-0.98) and 0.90 (0.78-1.00), 0.86 (0.78-0.95) and 0.83 (0.64-1.00), 0.90 (0.83-0.97) and 0.89 (0.77-1.00),0.92 (0.85-0.98) and 0.91 (0.79-1.00), respectively. DCA demonstrated that T(1)WI, T(2)WI, enhanced T(1)WI, and combined sequence model all had good net benefits in clinical practice. Conclusions: T(1)WI, T(2)WI, enhanced T(1)WI, and combined sequence model of conventional MRI all had high efficacy in predicting the texture of pituitary macroadenoma, which provided a new quantitative method for predicting the texture of pituitary macroadenoma.

Nephronophthisis and retinal degeneration in tmem218-/- mice: a novel mouse model for Senior-Løken syndrome?

Mice deficient in TMEM218 (Tmem218(-/-) ) were generated as part of an effort to identify and validate pharmaceutically tractable targets for drug development through large-scale phenotypic screening of knockout mice. Routine diagnostics, expression analysis, histopathology, and electroretinogram analyses completed on Tmem218(-/-) mice identified a previously unknown role for TMEM218 in the development and function of the kidney and eye. The major observed phenotypes in Tmem218(-/-) mice were progressive cystic kidney disease and retinal degeneration. The renal lesions were characterized by diffuse renal cyst development with tubulointerstitial nephropathy and disruption of tubular basement membranes in essentially normal-sized kidneys. The retinal lesions were characterized by slow-onset loss of photoreceptors, which resulted in reduced electroretinogram responses. These renal and retinal lesions are most similar to those associated with nephronophthisis (NPHP) and retinitis pigmentosa in humans. At least 10% of NPHP cases present with extrarenal conditions, which most often include retinal degeneration. Senior-Løken syndrome is characterized by the concurrent development of autosomal recessive NPHP and retinitis pigmentosa. Since mutations in the known NPHP genes collectively account for only about 30% of NPHP cases, it is possible that TMEM218 could be involved in the development of similar ciliopathies in humans. In reviewing all other reported mouse models of NPHP, we suggest that Tmem218(-/-) mice could provide a useful model for elucidating the pathogenesis of cilia-associated disease in both the kidney and the retina, as well as in developing and testing novel therapeutic strategies for Senior-Løken syndrome.

Cardiomyopathy in α-kinase 3 (ALPK3)-deficient mice.

Cardiomyopathy developed in mice deficient for α-kinase 3 (ALPK3), a nuclear kinase previously implicated in the differentiation of cardiomyocytes. Alpk3 (-/-) mice were produced according to normal Mendelian ratios and appeared normal except for a nonprogressive cardiomyopathy that had features of both hypertrophic and dilated forms of cardiomyopathy. Cardiac hypertrophy in Alpk3 (-/-) mice was characterized by increased thickness of both left and right ventricular (LV and RV) walls and by markedly increased heart weight and increased heart weight/body weight and heart weight/tibia length ratios. Magnetic resonance imaging studies confirmed the increased thickness in both septal and LV free walls at end-diastole, although there was no significant change in LV wall thickness at end-systole. Myocardial hypertrophy was the predominant feature in Alpk3 (-/-) mice, but several changes more typically associated with dilated cardiomyopathy included a marked increase in end-diastolic and end-systolic LV volume, as well as reduced cardiac output, stroke volume, and ejection fractions, suggesting LV chamber dilation. Magnetic resonance imaging showed a 50% reduction in both septal and free wall LV contractility in Alpk3 (-/-) mice. Interstitial fibrosis and inflammation were notably absent in Alpk3 (-/-) mice; however, light and electron microscopy revealed altered cardiomyocyte architecture, characterized by reduced numbers of abnormal intercalated discs being associated with mild disarray of myofibrils. These lesions could account for the impaired contractility of the myofibrillar apparatus and contribute to the pathogenesis of cardiomyopathy in Alpk3 (-/-) mice.

Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-selectin-knockout mice.

P-selectin and intercellular adhesion molecule-1 (ICAM-1) mediate early interaction and adhesion of neutrophils to coronary endothelial cells and myocytes after myocardial ischemia and reperfusion. In the present study, we examined the physiological consequences of genetic deletions of ICAM-1 and P-selectin in mice. In wild-type mice, after 1 h of ischemia followed by reperfusion, neutrophil influx into the area of ischemia was increased by 3 h with a peak at 24 h and a decline by 72 h. ICAM-1/P-selectin-deficient mice showed a significant reduction in neutrophils by immunohistochemistry or by myeloperoxidase activity at 24 h but no significant difference at 3 h. Infarct size (area of necrosis/area at risk) assessed 24 h after reperfusion was not different between wild-type and deficient mice after 30 min and 1 h of occlusion. Mice with a deficiency in both ICAM-1 and P-selectin have impaired neutrophil trafficking without a difference in infarct size due to myocardial ischemia-reperfusion.

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.

Tyrosine kinase inhibition decreases Muc-1 expression in mouse epithelial cells.

Mouse uterine epithelial cells (UEC) express high levels of both messenger RNA (mRNA) and protein encoding the polymorphic mucin glycoprotein, Muc-1, under most conditions in vivo and in vitro. Although steroid hormones modulate Muc-1 expression in vivo, it is not clear if these actions are mediated directly by steroid hormone receptors or indirectly by modulation of key intracellular signal transduction cascades. To address the latter issue, we examined the effects of a wide variety of modulators of signal transduction cascades on the expression of Muc-1 in primary cultures of polarized mouse UEC. Transient exposure of UEC to agents that inhibit tyrosine kinases by distinct mechanisms, i.e., tyrphostin, genistein, and staurosporine, consistently and significantly reduced Muc-1 expression. In contrast, a variety of agents that modulate protein kinase A- or C-dependent pathways had little or no effect on Muc-1. The effect of tyrphostin proved to be similar in magnitude at both the level of Muc-1 protein and mRNA expression. Transient transfection assays of mouse UEC and a murine mammary epithelial cell line, NMuMG, with mouse Muc-1 promoter-CAT reporter constructs demonstrated a similar (50-60%) degree of tyrphostin inhibition. These observations suggested an action at the level of Muc-1 gene expression. Levels of 100,000 g soluble tyrosine kinase activity in mouse UEC freshly isolated from estrous stage (high-level Muc-1 expression) and day 4 of pregnancy (low-level Muc-1 expression) correlated with Muc-1 expression. Furthermore, pretreatment of day 4 pregnant mice with the anti-progestin, RU486, an agent previously shown to restore or maintain high levels of Muc-1 expression, also restored soluble tyrosine kinase activity to levels similar to that observed in estrous stage mice. Collectively, these results indicate that tyrosine kinase activity is required to maintain high level Muc-1 expression in mice.

Malarial circumsporozoite protein is a novel gene delivery vehicle to primary hepatocyte cultures and cultured cells.

In this report we describe a novel gene delivery system using malaria circumsporozoite (CS) protein as a specific ligand. The CS protein covers the entire surface of sporozoites of malaria parasites. Previous studies have demonstrated that intravenously injected CS protein binds specifically to the basolateral surface of hepatocytes within minutes, indicating the high hepatocyte specificity of CS protein. This characteristic of CS protein prompted us to explore the possibility of using this protein as a liver-specific ligand for hepatic gene delivery vehicle. As an initial step, we investigated the efficacy of CS protein-mediated gene transfer into primary hepatocytes as well as established cell lines. Recombinant CS proteins were chemically conjugated to poly(L-lysine). The CS conjugates were complexed with recombinant plasmid DNA carrying a reporter gene. When the DNA complex was used to transfect primary hepatocytes, a very low level of expression of the reporter gene was observed. The level of expression was greatly enhanced when the cells were cotransfected with adenovirus, which presumably releases the internalized DNA from endosomal entrapment. The CS-mediated gene transfer into the cells required region II+, an evolutionarily conserved amino acid sequence conferring the binding of CS protein to its receptor. CS protein also efficiently mediated gene transfer into a number of cell lines, i.e. HepG2, HeLa, NIH3T3, and K562, but not HL-60, which contains low levels of receptor. Thus, the CS conjugate can be used to deliver DNA into many different cultured cells. Most importantly, the CS conjugate has a potential to be further developed into a liver-specific gene delivery vehicle in vivo.

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting.

Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90 degrees. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein--DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.

DNA interstrand cross-linking, DNA sequence specificity, and induced conformational changes produced by a dimeric analog of (+)-CC-1065.

U-77779 is a symmetrical dimer of the spirocyclopropyl alkylating subunit of (+)-CC-1065 in which the linker consists of two indole subunits separated by a ureido group. This compound was synthesized by scientists of the Upjohn Company and was found to be more active in both anti-tumor efficacy and cytotoxicity than its mono-alkylating analogs. Using three different 21-base pair DNA duplexes containing U-77779 reactive sequences, we have shown that U-77779 produces a stable interstrand cross-linked species that loses its internal self complementarity. A comparison of U-77779 with the mono-alkylating analogs of (+)-CC-1065 shows that it appears to have an increased sequence selectivity such that, while mono-alkylating compounds like (+)-CC-1065 react at more than one site, U-77779 reacts only at sites where there are two suitably positioned alkylation sites. Chemical footprinting with 1,10-phenanthroline-copper complex revealed a six base pair cross-linked region between the two covalently modified adenines with modulated cleavage outside this region. In the case of hydroxyl radical footprinting, considerable variability of the extent of cleavage within the cross-linked sequence was found. These results are discussed in terms of likely induced conformational changes in DNA. In contrast to (+)-CC-1065, non-denaturing gel electrophoresis did not reveal any net bending of DNA due to U-77779, which we believe is due to the 180 degrees out-of-phase bending produced on opposite strands of DNA by the cross-linker.