Rethinking Breast Cancer Diagnosis through Deep Learning Based Image Recognition.

This paper explored techniques for diagnosing breast cancer using deep learning based medical image recognition. X-ray (Mammography) images, ultrasound images, and histopathology images are used to improve the accuracy of the process by diagnosing breast cancer classification and by inferring their affected location. For this goal, the image recognition application strategies for the maximal diagnosis accuracy in each medical image data are investigated in terms of various image classification (VGGNet19, ResNet50, DenseNet121, EfficietNet v2), image segmentation (UNet, ResUNet++, DeepLab v3), and related loss functions (binary cross entropy, dice Loss, Tversky loss), and data augmentation. As a result of evaluations through the presented methods, when using filter-based data augmentation, ResNet50 showed the best performance in image classification, and UNet showed the best performance in both X-ray image and ultrasound image as image segmentation. When applying the proposed image recognition strategies for the maximal diagnosis accuracy in each medical image data, the accuracy can be improved by 33.3% in image segmentation in X-ray images, 29.9% in image segmentation in ultrasound images, and 22.8% in image classification in histopathology images.

SUPT4H1-edited stem cell therapy rescues neuronal dysfunction in a mouse model for Huntington's disease.

Huntington's disease (HD) is a severe inherited neurological disorder caused by a CAG repeat expansion in the huntingtin gene (HTT), leading to the accumulation of mutant huntingtin with polyglutamine repeats. Despite its severity, there is no cure for this debilitating disease. HTT lowering strategies, including antisense oligonucleotides (ASO) showed promising results very recently. Attempts to develop stem cell-based therapeutics have shown efficacy in preclinical HD models. Using an HD patient's autologous cells, which have genetic defects, may hamper therapeutic efficacy due to mutant HTT. Pretreating these cells to reduce mutant HTT expression and transcription may improve the transplanted cells' therapeutic efficacy. To investigate this, we targeted the SUPT4H1 gene that selectively supports the transcription of long trinucleotide repeats. Transplanting SUPT4H1-edited HD-induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) into the YAC128 HD transgenic mouse model improved motor function compared to unedited HD iPSC-NPCs. Immunohistochemical analysis revealed reduced mutant HTT expression without compensating wild-type HTT expression. Further, SUPT4H1 editing increased neuronal and decreased reactive astrocyte differentiation in HD iPSC-NPCs compared to the unedited HD iPSC-NPCs. This suggests that ex vivo editing of SUPT4H1 can reduce mutant HTT expression and provide a therapeutic gene editing strategy for autologous stem cell transplantation in HD.

Human iPSC-derived neural precursor cells differentiate into multiple cell types to delay disease progression following transplantation into YAC128 Huntington's disease mouse model.

OBJECTIVES: To investigate whether human HLA-homozygous induced pluripotent stem cell (iPSC)-derived neural precursor cells (iPSC-NPCs) can provide functional benefits in Huntington's disease (HD), we transplanted them into the YAC128 transgenic HD mouse model. MATERIALS AND METHODS: CHAi001-A, an HLA-homozygous iPSC line (A*33:03-B*44:03-DRB1*13:02), was differentiated into neural precursor cells, and then, they were transplanted into 6 months-old YAC128 mice. Various behavioural and histological analyses were performed for five months after transplantation. RESULTS: Motor and cognitive functions were significantly improved in transplanted animals. Cells transplanted in the striatum showed multipotential differentiation. Five months after transplantation, the donor cells had differentiated into neurons, oligodendrocytes and astrocytes. Transplantation restored DARPP-32 expression, synaptophysin density, myelin basic protein expression in the corpus callosum and astrocyte function. CONCLUSION: Altogether, these results strongly suggest that iPSC-NPCs transplantation induces neuroprotection and functional recovery in a mouse model of HD and should be taken forward for clinical trials in HD patients.

Two-photon probes for the endoplasmic reticulum: its detection in a live tissue by two-photon microscopy.

We report blue- and green-emitting two-photon probes derived from naphthalene and fluorene derivatives (as fluorophores) and an endoplasmic reticulum (ER) retrieval peptide (KDEL; as an ER-targeting moiety) that can detect the ER in a live cell by both one-photon and two-photon microscopy (TPM) and in a live tissue by TPM.

Two-Photon Probe for TNF-α. Assessment of the Transmembrane TNF-α Level in Human Colon Tissue by Two-Photon Microscopy.

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520-2830) × 10-50 cm4s/photon in RAW 264.7 cells. After this probe was labeled, it was possible to detect Pyr1-infliximab-transmembrane TNF-α complexes in a live cell and to determine the relative proportion of these complexes in human colon tissues. This proportion among healthy, possibly inflamed, and inflamed tissues of patients with ulcerative colitis was found to be 1.0/4.5/10. This probe may find useful applications for selective detection of transmembrane TNF-α in a live cell or tissue, for quantification of inflammation in human colon tissue or of antidrug antibodies in patients who stop responding to anti-TNF therapy, and for monitoring of the response to this therapy.

Two-Photon Probes for pH: Detection of Human Colon Cancer using Two-Photon Microscopy.

We have developed two-photon (TP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10-50cm4s/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl)/1,4-dioxane (7/3) solution. The TPM images of CCD-18co (a normal colon cell line) and HCT116 cells (a colon cancer cell line) labeled with BH-2 were too dim to be distinguished. When the same cells were labeled with BHEt-1, however, the TPM image of the HCT116 cells was much brighter than that of CCD-18co cells, and the relative proportion of the acidic vesicles (Pacid) of the former was 5-fold larger than that of latter. BHEt-1 could also differentiate HepG2 cells (a human liver cancer cell line) from LX-2 cells (a human hepatic stellate cell line) with a 6-fold larger Pacid value. Human colon cancer tissues labeled with BHEt-1 showed similar results, demonstrating much brighter TPM images and 6-fold larger Pacid values compared to normal tissue. These results suggest the potential utility of BHEt-1 for detecting colon cancer in human tissues using TPM.

Two-Photon Tracer for Human Epidermal Growth Factor Receptor-2: Detection of Breast Cancer in a Live Tissue.

We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 × 10-50 cm4s/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90-130 µm in live mouse tissue by TPM.

The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth.

Adenylate kinase 2 (AK2), which balances adenine nucleotide pool, is a multi-functional protein. Here we show that AK2 negatively regulates tumour cell growth. AK2 forms a complex with dual-specificity phosphatase 26 (DUSP26) phosphatase and stimulates DUSP26 activity independently of its AK activity. AK2/DUSP26 phosphatase protein complex dephosphorylates fas-associated protein with death domain (FADD) and regulates cell growth. AK2 deficiency enhances cell proliferation and induces tumour formation in a xenograft assay. This anti-growth function of AK2 is associated with its DUSP26-stimulating activity. Downregulation of AK2 is frequently found in tumour cells and human cancer tissues showing high levels of phospho-FADD(Ser194). Moreover, reconstitution of AK2 in AK2-deficient tumour cells retards both cell proliferation and tumourigenesis. Consistent with this, AK2(+/-) mouse embryo fibroblasts exhibit enhanced cell proliferation with a significant alteration in phospho-FADD(Ser191). These results suggest that AK2 is an associated activator of DUSP26 and suppresses cell proliferation by FADD dephosphorylation, postulating AK2 as a negative regulator of tumour growth.

The fragment structure of a putative HsdR subunit of a type I restriction enzyme from Vibrio vulnificus YJ016: implications for DNA restriction and translocation activity.

Among four types of bacterial restriction enzymes that cleave a foreign DNA depending on its methylation status, type I enzymes composed of three subunits are interesting because of their unique DNA cleavage and translocation mechanisms performed by the restriction subunit (HsdR). The elucidated N-terminal fragment structure of a putative HsdR subunit from Vibrio vulnificus YJ016 reveals three globular domains. The nucleolytic core within an N-terminal nuclease domain (NTD) is composed of one basic and three acidic residues, which include a metal-binding site. An ATP hydrolase (ATPase) site at the interface of two RecA-like domains (RDs) is located close to the probable DNA-binding site for translocation, which is far from the NTD nucleolytic core. Comparison of relative domain arrangements with other functionally related ATP and/or DNA complex structures suggests a possible translocation and restriction mechanism of the HsdR subunit. Furthermore, careful analysis of its sequence and structure implies that a linker helix connecting two RDs and an extended region within the nuclease domain may play a central role in switching the DNA translocation into the restriction activity.

Crystal structure of a minimal nitroreductase, ydjA, from Escherichia coli K12 with and without FMN cofactor.

Nitroreductases (NTR) are enzymes that reduce hazardous nitroaromatic compounds and are of special interest due to their potential use in bioremediation and their activation of prodrugs in directed anticancer therapies. We elucidated the crystal structures of ydjA from Escherichia coli (Ec_ydjA), one of the smallest NTRs, in its flavin mononucleotide (FMN)-bound and cofactor-free forms. The alpha+beta mixed monomeric Ec_ydjA forms a homodimeric structure through the interactions of the long central helices and the extended regions at both termini. Two FMN molecules are bound at the dimeric interface. The absence of the 30 internal amino acids in Ec_ydjA, which forms two helices and restricts the cofactor and substrate binding in other NTR family members, creates a wider and more flexible active site. Unlike the bent FMN ring structures present in most NTR complexes currently known, the flavin system in the Ec_ydjA structure maintains a flat ring conformation, which is sandwiched between a Trp and a His residue from each monomer. The analysis of our Ec_ydjA structure explains its specificity for larger substrates and provides structural information for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules.