Ultrasound-based deep learning radiomics nomogram for differentiating mass mastitis from invasive breast cancer.

BACKGROUND: The purpose of this study is to develop and validate the potential value of the deep learning radiomics nomogram (DLRN) based on ultrasound to differentiate mass mastitis (MM) and invasive breast cancer (IBC). METHODS: 50 cases of MM and 180 cases of IBC with ultrasound Breast Imaging Reporting and Data System 4 category were recruited (training cohort, n = 161, validation cohort, n = 69). Based on PyRadiomics and ResNet50 extractors, radiomics and deep learning features were extracted, respectively. Based on supervised machine learning methods such as logistic regression, random forest, and support vector machine, as well as unsupervised machine learning methods using K-means clustering analysis, the differences in features between MM and IBC were analyzed to develop DLRN. The performance of DLRN had been evaluated by receiver operating characteristic curve, calibration, and clinical practicality. RESULTS: Supervised machine learning results showed that compared with radiomics models, especially random forest models, deep learning models were better at recognizing MM and IBC. The area under the curve (AUC) of the validation cohort was 0.84, the accuracy was 0.83, the sensitivity was 0.73, and the specificity was 0.83. Compared to radiomics or deep learning models, DLRN even further improved discrimination ability (AUC of 0.90 and 0.90, accuracy of 0.83 and 0.88 for training and validation cohorts), which had better clinical benefits and good calibratability. In addition, the information heterogeneity of deep learning features in MM and IBC was validated again through unsupervised machine learning clustering analysis, indicating that MM had a unique features phenotype. CONCLUSION: The DLRN developed based on radiomics and deep learning features of ultrasound images has potential clinical value in effectively distinguishing between MM and IBC. DLRN breaks through visual limitations and quantifies more image information related to MM based on computers, further utilizing machine learning to effectively utilize this information for clinical decision-making. As DLRN becomes an autonomous screening system, it will improve the recognition rate of MM in grassroots hospitals and reduce the possibility of incorrect treatment and overtreatment.

Preoperative magnetic resonance imaging-radiomics in cervical cancer: a systematic review and meta-analysis.

Background: The purpose of this systematic review and meta-analysis is to evaluate the potential significance of radiomics, derived from preoperative magnetic resonance imaging (MRI), in detecting deep stromal invasion (DOI), lymphatic vascular space invasion (LVSI) and lymph node metastasis (LNM) in cervical cancer (CC). Methods: A rigorous and systematic evaluation was conducted on radiomics studies pertaining to CC, published in the PubMed database prior to March 2024. The area under the curve (AUC), sensitivity, and specificity of each study were separately extracted to evaluate the performance of preoperative MRI radiomics in predicting DOI, LVSI, and LNM of CC. Results: A total of 4, 7, and 12 studies were included in the meta-analysis of DOI, LVSI, and LNM, respectively. The overall AUC, sensitivity, and specificity of preoperative MRI models in predicting DOI, LVSI, and LNM were 0.90, 0.83 (95% confidence interval [CI], 0.75-0.89) and 0.83 (95% CI, 0.74-0.90); 0.85, 0.80 (95% CI, 0.73-0.86) and 0.75 (95% CI, 0.66-0.82); 0.86, 0.79 (95% CI, 0.74-0.83) and 0.80 (95% CI, 0.77-0.83), respectively. Conclusion: MRI radiomics has demonstrated considerable potential in predicting DOI, LVSI, and LNM in CC, positioning it as a valuable tool for preoperative precision evaluation in CC patients.

Ablation of Fatty Acid Transport Protein-4 Enhances Cone Survival, M-cone Vision, and Synthesis of Cone-Tropic 9-cis-Retinal in rd12 Mouse Model of Leber Congenital Amaurosis.

The canonical visual cycle employing RPE65 as the retinoid isomerase regenerates 11-cis-retinal to support both rod- and cone-mediated vision. Mutations of RPE65 are associated with Leber congenital amaurosis that results in rod and cone photoreceptor degeneration and vision loss of affected patients at an early age. Dark-reared Rpe65-/- mouse has been known to form isorhodopsin that employs 9-cis-retinal as the photosensitive chromophore. The mechanism regulating 9-cis-retinal synthesis and the role of the endogenous 9-cis-retinal in cone survival and function remain largely unknown. In this study, we found that ablation of fatty acid transport protein-4 (FATP4), a negative regulator of 11-cis-retinol synthesis catalyzed by RPE65, increased the formation of 9-cis-retinal, but not 11-cis-retinal, in a light-independent mechanism in both sexes of RPE65-null rd12 mice. Both rd12 and rd12;Fatp4-/- mice contained a massive amount of all-trans-retinyl esters in the eyes, exhibiting comparable scotopic vision and rod degeneration. However, expression levels of M- and S-opsins as well as numbers of M- and S-cones surviving in the superior retinas of rd12;Fatp4-/ - mice were at least twofold greater than those in age-matched rd12 mice. Moreover, FATP4 deficiency significantly shortened photopic b-wave implicit time, improved M-cone visual function, and substantially deaccelerated the progression of cone degeneration in rd12 mice, whereas FATP4 deficiency in mice with wild-type Rpe65 alleles neither induced 9-cis-retinal formation nor influenced cone survival and function. These results identify FATP4 as a new regulator of synthesis of 9-cis-retinal, which is a "cone-tropic" chromophore supporting cone survival and function in the retinas with defective RPE65.

Comparison of the structure-function of five newly members of the calcin family.

Calcins are a group of scorpion toxin peptides specifically binding to ryanodine receptors (RyRs) with high affinity, and have the ability to activate and stabilize RyR in a long-lasting subconductance state. Five newly calcins synthesized compounds exhibit typical structural characteristics of a specific family through chemical synthesis and virtual analysis. As the calcins from the same species, Petersiicalcin1 and Petersiicalcin2, Jendekicalcin2 and Jendekicalcin3, have only one residue difference. Both Petersiicalcin1 and Petersiicalcin2 exhibited different affinities in stimulating [3H]ryanodine binding, but the residue mutation resulted in a 2.7 folds difference. Other calcins also exhibited a stimulatory effect on [3H]ryanodine binding to RyR1, however, their affinities were significantly lower than that of Petersiiicalcin1 and Petersiiicalcin2. The channel domain of RyR1 was found to be capable of binding with the basic residues of these calcins, which also exhibited interactions with the S6 helices on RyR1. Dynamic simulations were conducted for Petersiicalcin1 and Petersiicalcin2, which demonstrated their ability to form a highly stable conformation and resulting in an asymmetric tetramer structure of RyR1. The discovery of five newly calcins further enriches the diversity of the natural calcin family, which provides more native peptides for the structure-function analysis between calcin and RyRs.

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo-electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorders.

Inverse correlation between fatty acid transport protein 4 and vision in Leber congenital amaurosis associated with RPE65 mutation.

Fatty acid transport protein 4 (FATP4), a transmembrane protein in the endoplasmic reticulum (ER), is a recently identified negative regulator of the ER-associated retinal pigment epithelium (RPE)65 isomerase necessary for recycling 11-cis-retinal, the light-sensitive chromophore of both rod and cone opsin visual pigments. The role of FATP4 in the disease progression of retinal dystrophies associated with RPE65 mutations is completely unknown. Here we show that FATP4-deficiency in the RPE results in 2.8-fold and 1.7-fold increase of 11-cis- and 9-cis-retinals, respectively, improving dark-adaptation rates as well as survival and function of rods in the Rpe65 R91W knockin (KI) mouse model of Leber congenital amaurosis (LCA). Degradation of S-opsin in the proteasomes, but not in the lysosomes, was remarkably reduced in the KI mouse retinas lacking FATP4. FATP4-deficiency also significantly rescued S-opsin trafficking and M-opsin solubility in the KI retinas. The number of S-cones in the inferior retinas of 4- or 6-mo-old KI;Fatp4-/- mice was 7.6- or 13.5-fold greater than those in age-matched KI mice. Degeneration rates of S- and M-cones are negatively correlated with expression levels of FATP4 in the RPE of the KI, KI;Fatp4+/- , and KI;Fatp4-/- mice. Moreover, the visual function of S- and M-cones is markedly preserved in the KI;Fatp4-/- mice, displaying an inverse correlation with the FATP4 expression levels in the RPE of the three mutant lines. These findings establish FATP4 as a promising therapeutic target to improve the visual cycle, as well as survival and function of cones and rods in patients with RPE65 mutations.

Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis.

UNLABELLED: RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations. SIGNIFICANCE STATEMENT: LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations.

Interphotoreceptor Retinoid-Binding Protein Mitigates Cellular Oxidative Stress and Mitochondrial Dysfunction Induced by All-trans-Retinal.

PURPOSE: Point and null mutations in interphotoreceptor retinoid-binding protein (IRBP) cause retinal dystrophy in affected patients and IRBP-deficient mice with unknown mechanism. This study investigated whether IRBP protects cells from damages induced by all-trans-retinal (atRAL), which was increased in the Irbp(-/-) retina. METHODS: Wild-type and Irbp(-/-) mice retinal explants in buffer with or without purified IBRP were exposed to 800 lux light for different times and subjected to retinoid analysis by high-performance liquid chromatography. Purity of IRBP was determined by Coomassie Brilliant Blue staining and immunoblot analysis. Cellular damages induced by atRAL in the presence or absence of IRBP were evaluated in the mouse photoreceptor-derived 661W cells. Cell viability and death were measured by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and TUNEL assays. Expression and modification levels of retinal proteins were determined by immunoblot analysis. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were detected with fluorogenic dyes and confocal microscopy. Mitochondrial membrane potential was analyzed by using JC-1 fluorescent probe and a flow cytometer. RESULTS: Content of atRAL in Irbp(-/-) retinal explants exposed to light for 40 minutes was significantly higher than that in wild-type retinas under the same light conditions. All-trans-retinal caused increase in cell death, tumor necrosis factor activation, and Adam17 upregulation in 661W cells. NADPH oxidase-1 (NOX1) upregulation, ROS generation, NO-mediated protein S-nitrosylation, and mitochondrial dysfunction were also observed in 661W cells treated with atRAL. These cytotoxic effects were significantly attenuated in the presence of IRBP. CONCLUSIONS: Interphotoreceptor retinoid-binding protein is required for preventing accumulation of retinal atRAL, which causes inflammation, oxidative stress, and mitochondrial dysfunction of the cells.

Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis.

RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65.

Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration. However, the signaling pathway(s) leading to photoreceptor death in IRBP-deficient mice remains poorly understood. Here, we show that amounts of tumor necrosis factor-α (TNF-α) in the interphotoreceptor matrix and retinas of Irbp(-/-) mice were increased more than 10-fold and fivefold, respectively, compared with those in wild-type mice. Moreover, TNF-α receptor 1, an important membrane death receptor that mediates both programmed apoptosis and necrosis, was also significantly increased in Irbp(-/-) retina, and was colocalized with peanut agglutinin to the Irbp(-/-) cone outer segments. Although these death signaling proteins were increased, the caspase-dependent and independent apoptotic pathways were mildly activated in the Irbp(-/-) retinas, suggesting that other cell death mechanism(s) also contributes to the extensive photoreceptor degeneration in Irbp(-/-) retina. We found that receptor interacting protein 1 and 3 (RIP1 and RIP3) kinases, the intracellular key mediators of TNF-induced cellular necrosis, were elevated at least threefold in the Irbp(-/-) retinas. Moreover, pharmacological inhibition of RIP1 kinase significantly prevented cone and rod photoreceptor degeneration in Irbp(-/-) mice. These results reveal that RIP kinase-mediated necrosis strongly contributes to cone and rod degeneration in Irbp(-/-) mice, implicating the TNF-RIP pathway as a potential therapeutic target to prevent or delay photoreceptor degeneration in patients with retinitis pigmentosa caused by IRBP mutation.

Fatty acid transport protein 4 (FATP4) prevents light-induced degeneration of cone and rod photoreceptors by inhibiting RPE65 isomerase.

Although rhodopsin is essential for sensing light for vision, it also mediates light-induced apoptosis of photoreceptors in mouse. RPE65, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis-retinol (11cROL) in the visual cycle, controls the rhodopsin regeneration rate and photoreceptor susceptibility to light-induced degeneration. Mutations in RPE65 have been linked to blindness in affected children. Despite such importance, the mechanism that regulates RPE65 function remains unclear. Through unbiased expression screening of a bovine retinal pigment epithelium (RPE) cDNA library, we have identified elongation of very long-chain fatty acids-like 1 (ELOVL1) and fatty acid transport protein 4 (FATP4), which each have very long-chain fatty acid acyl-CoA synthetase (VLCFA-ACS) activity, as negative regulators of RPE65. We found that the VLCFA derivative lignoceroyl (C24:0)-CoA inhibited synthesis of 11cROL, whereas palmitoyl (C16:0)-CoA promoted synthesis of 11cROL. We further found that competition of FATP4 with RPE65 for the substrate of RPE65 was also involved in the mechanisms by which FATP4 inhibits synthesis of 11cROL. FATP4 was predominantly expressed in RPE, and the FATP4-deficient RPE showed significantly higher isomerase activity. Consistent with these results, the regeneration rate of 11-cis-retinaldehyde and the recovery rate for rod light sensitivity were faster in FATP4-deficient mice than wild-type mice. Moreover, FATP4-deficient mice displayed increased accumulation of the cytotoxic all-trans retinaldehyde and hypersusceptibility to light-induced photoreceptor degeneration. Our findings demonstrate that ELOVL1, FATP4, and their products comprise the regulatory elements of RPE65 and play important roles in protecting photoreceptors from degeneration induced by light damage.

Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65.

Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.

Filamin A-bound PEBP2beta/CBFbeta is retained in the cytoplasm and prevented from functioning as a partner of the Runx1 transcription factor.

The heterodimeric transcription factor PEBP2/CBF is composed of a DNA-binding subunit, called Runx1, and a non-DNA-binding subunit, called PEBP2beta/CBFbeta. The Runx1 protein is detected exclusively in the nuclei of most cells and tissues, whereas PEBP2beta is located in the cytoplasm. We addressed the mechanism by which PEBP2beta localizes to the cytoplasm and found that it is associated with filamin A, an actin-binding protein. Filamin A retains PEBP2beta in the cytoplasm, thereby hindering its engagement as a Runx1 partner. The interaction with filamin A is mediated by a region within PEBP2beta that includes amino acid residues 68 to 93. The deletion of this region or the repression of filamin A enables PEBP2beta to translocate to the nucleus. Based on these observations, we propose that PEBP2beta has two distinct domains, a newly defined regulatory domain that interacts with filamin A and the previously identified Runx1-binding domain.