Endoscopic mastectomy followed by immediate breast reconstruction with fat grafting for breast cancer.

BACKGROUND: Although endoscopic mastectomy has been associated with good tolerance and enhanced patient satisfaction, limitations such as the implant or flap size for reconstruction with small incisions remain unresolved. Fat grafting (FG) can expand tissue volume with pinhole skin incisions. Herein, we evaluated the safety and efficacy of endoscopic mastectomy followed by immediate FG. METHODS: Patients who underwent endoscopic mastectomy with immediate FG reconstruction from 2015 to 2021 were retrospectively evaluated to establish surgical outcomes and prognosis. RESULTS: Twenty-three patients with clinical stage 0 or I breast cancer underwent unilateral endoscopic mastectomy with immediate FG. The median age was 45 years (41-55), and the median body mass index was 19.3 kg/m2 (15.8-26.6). Endoscopically performed procedures included skin-sparing mastectomies in 18 patients (78%) and nipple-sparing mastectomies in five patients (22%). The median procedure duration was 295 min (242-346). The median specimen weight was 133 g (71-334), and the median grafted fat volume was 200 mL (136-320). No patient required reoperation or additional procedures for complications. One patient experienced recurrence at a median follow-up of 56.1 months and underwent resection; the patient was alive without recurrence 54 months post-resection. CONCLUSION: To the best of our knowledge, this is the first report of endoscopic mastectomy with immediate FG for reconstruction. When compared with other immediate autologous reconstructions, our strategy could minimize the skin incision and procedure duration, as well as limit complications. Further prospective investigations are needed to evaluate oncological safety, surgical outcomes, and patient satisfaction.

Clinicopathological Characteristics and Prognosis of Triple-Negative Apocrine Carcinoma: A Case-Control Study.

Background: With a prevalence of only 1% among all breast cancers in Japan, apocrine carcinoma (AC) is a rare type of breast cancer, and its clinicopathological characteristics remain unclear. The aim of this study was to evaluate the characteristics and prognosis of AC, in relation to the presence or absence of androgen receptor (AR). Methods: We conducted a retrospective multi-center case-control study (Yokohama Clinical Oncology Group (YCOG): YCOG1701 study) in Japan. A total of 53 patients were registered who were diagnosed with AC between 2000 and 2017 in YCOG-affiliated hospitals. Results: The median age of the patients was 67 (43 - 94) years, and the median observation time was 6.1 years. Among the 53 cases, 24 had triple-negative pure AC (TN-PAC; AR-positive), whereas 29 had other types of AC (other-AC; estrogen receptor-positive and/or human epidermal growth factor receptor 2-positive or AR-negative). Tumor size was smaller (1.4 vs. 2.1 cm, P = 0.024) and metastasis occurred in fewer nodes (12.5% vs. 37.9%, P = 0.036) in the TN-PAC group than in the other-AC group. The number of patients who were administered perioperative adjuvant chemotherapy did not significantly differ between the two groups (TN-PAC/other-AC = 50.0%/55.2%, P = 0.525); however, there was no recurrence in the TN-PAC group, compared to five cases with relapse in the other-AC group. Conclusions: AR-positive AC patients showed a favorable prognosis without adjuvant chemotherapy, even with the TN subtype. A clinical trial exploring the possibility of treatment de-escalation is anticipated.

BRCA2 reversion mutation confers resistance to olaparib in breast cancer.

Key Clinical Message: A rare missense mutation was identified as a reversion mutation using cancer genomic profiling and a suspected mechanism underlying resistance to olaparib in breast cancer. Abstract: A 34-year-old woman with breast cancer and BRCA2: p.Gln3047Ter was treated with olaparib. After tumor progression, cancer genomic profiling testing using liquid biopsy revealed BRCA2 p.Gln3047Ter and p.Gln3047Tyr, with 48.9% and 0.37% allele frequency, respectively. These findings shed light on reversion mutation as a mechanism of resistance to olaparib in breast cancer.

Granulomatous mastitis in a male breast: A case report and review of literature.

Granulomatous mastitis (GM) is a rare disease, particularly among men. Herein, we present a case of GM diagnosed in a 63-year-old male patient who showed reduction in the tumor size during 3 months of observation.

Identification of Potent and Selective Inhibitors of Fat Mass Obesity-Associated Protein Using a Fragment-Merging Approach.

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO-selective inhibitors.

Correction: Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0204160.].

Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5-10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection. Mycobacterial DNA-binding protein 1 (MDP1) is a major protein in persistent Mycobacterium tuberculosis and has potential for diagnostic use in detecting asymptomatic infection. However, a previous ELISA-based study revealed a specificity problem; IgGs against MDP1 were detected in both M. tuberculosis-infected and uninfected individuals. Although the tertiary structures of an antigen are known to influence antibody recognition, the MDP1 structural details have not yet been investigated. The N-terminal half of MDP1, homologous to bacterial histone-like protein HU, is predicted to be responsible for DNA-binding, while the C-terminal half is assumed as totally intrinsically disordered regions. To clarify the relationship between the MDP1 tertiary structure and IgG recognition, we refined the purification method, which allow us to obtain a recombinant protein with the predicted structure. Furthermore, we showed that an IgG-ELISA using MDP1 purified by our refined method is indeed useful in the detection of asymptomatic tuberculosis.

A modified single-cell electroporation method for molecule delivery into a motile protist, Euglena gracilis.

Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga. Single-cell electroporation introduced molecules into individual living E. gracilis cells after a negative pressure was applied through a syringe connected to the micropipette to the target cell. The new method achieves high transfection efficiency and viability after electroporation. With the new technique, we successfully introduced a variety of molecules such as GFP, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) RNA probes into individual motile E. gracilis cells. We demonstrate imaging of endogenous mRNA in living E. gracilis without interfering with their physiological functions, such as swimming or division, over an extended period of time. Thus the modified single-cell electroporation technique is suitable for delivering versatile functional molecules into individual motile protists.

Integrated molecular mechanism directing nucleosome reorganization by human FACT.

Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular ß structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Crystal structure of afadin PDZ domain-nectin-3 complex shows the structural plasticity of the ligand-binding site.

Afadin, a scaffold protein localized in adherens junctions (AJs), links nectins to the actin cytoskeleton. Nectins are the major cell adhesion molecules of AJs. At the initial stage of cell-cell junction formation, the nectin-afadin interaction plays an indispensable role in AJ biogenesis via recruiting and tethering other components. The afadin PDZ domain (AFPDZ) is responsible for binding the cytoplasmic C-terminus of nectins. AFPDZ is a class II PDZ domain member, which prefers ligands containing a class II PDZ-binding motif, X-Φ-X-Φ (Φ, hydrophobic residues); both nectins and other physiological AFPDZ targets contain this class II motif. Here, we report the first crystal structure of the AFPDZ in complex with the nectin-3 C-terminal peptide containing the class II motif. We engineered the nectin-3 C-terminal peptide and AFPDZ to produce an AFPDZ-nectin-3 fusion protein and succeeded in obtaining crystals of this complex as a dimer. This novel dimer interface was created by forming an antiparallel ß sheet between ß2 strands. A major structural change compared with the known AFPDZ structures was observed in the α2 helix. We found an approximately 2.5 Å-wider ligand-binding groove, which allows the PDZ to accept bulky class II ligands. Apparently, the last three amino acids of the nectin-3 C-terminus were sufficient to bind AFPDZ, in which the two hydrophobic residues are important.

Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism.

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.

Structure and function of the N-terminal nucleolin binding domain of nuclear valosin-containing protein-like 2 (NVL2) harboring a nucleolar localization signal.

The N-terminal regions of AAA-ATPases (ATPase associated with various cellular activities) often contain a domain that defines the distinct functions of the enzymes, such as substrate specificity and subcellular localization. As described herein, we have determined the solution structure of an N-terminal unique domain isolated from nuclear valosin-containing protein (VCP)-like protein 2 (NVL2(UD)). NVL2(UD) contains three α helices with an organization resembling that of a winged helix motif, whereas a pair of ß-strands is missing. The structure is unique and distinct from those of other known type II AAA-ATPases, such as VCP. Consequently, we identified nucleolin from a HeLa cell extract as a binding partner of this domain. Nucleolin contains a long (∼300 amino acids) intrinsically unstructured region, followed by the four tandem RNA recognition motifs and the C-terminal glycine/arginine-rich domain. Binding analyses revealed that NVL2(UD) potentially binds to any of the combinations of two successive RNA binding domains in the presence of RNA. Furthermore, NVL2(UD) has a characteristic loop, in which the key basic residues RRKR are exposed to the solvent at the edge of the molecule. The mutation study showed that these residues are necessary and sufficient for nucleolin-RNA complex binding as well as nucleolar localization. Based on the observations presented above, we propose that NVL2 serves as an unfoldase for the nucleolin-RNA complex. As inferred from its RNA dependence and its ATPase activity, NVL2 might facilitate the dissociation and recycling of nucleolin, thereby promoting efficient ribosome biogenesis.

Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana.

Human DNA polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents.

A common substrate recognition mode conserved between katanin p60 and VPS4 governs microtubule severing and membrane skeleton reorganization.

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.

Functional significance of octameric RuvA for a branch migration complex from Thermus thermophilus.

The RuvAB complex promotes migration of Holliday junction at the late stage of homologous recombination. The RuvA tetramer specifically recognizes Holliday junction to form two types of complexes. A single tetramer is bound to the open configuration of the junction DNA in complex I, while the octameric RuvA core structure sandwiches the same junction in complex II. The hexameric RuvB rings, symmetrically bound to both sides of RuvA on Holliday junction, pump out DNA duplexes, depending upon ATP hydrolysis. We investigated functional differences between the wild-type RuvA from Thermus thermophilus and mutants impaired the ability of complex II formation. These mutant RuvA, exclusively forming complex I, reduced activities of branch migration and ATP hydrolysis, suggesting that the octameric RuvA is essential for efficient branch migration. Together with our recent electron microscopic analysis, this finding provides important insights into functional roles of complex II in the coordinated branch migration mechanism.

Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex.

During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.

Characterization of the binding of distamycin A to damaged DNA: a comparison with the DNA recognition of the human DDB protein.

Distamycin A binds to DNA containing the (6-4) photoproduct, a major UV lesion that is recognized by the damaged DNA-binding (DDB) protein in human cells. We analyzed the binding properties of distamycin A and compared the results with those of the DDB protein. Structural change of the DNA duplex was not observed for distamycin A in two types of experiments, whereas the protein induced a large bending of the helix. Although the substrate specificity was different between the drug and the protein, thymine glycol was recognized by both of them, and inhibition of the DDB protein binding to the (6-4) photoproduct-containing DNA by distamycin A was tested.

Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay.

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.

Synthesis and characterization of oligonucleotides containing 2'-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction.

Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.

Right- and left-sided colorectal cancers display distinct expression profiles and the anatomical stratification allows a high accuracy prediction of lymph node metastasis.

Accurate preoperative prediction of lymph node metastasis and degree of tumor invasion would facilitate an appropriate decision of the extent of surgical resection of cancers to reduce unnecessary complication or to minimize the risk of recurrence in patients. We analyzed gene expression profiles characteristic of the invasiveness of colorectal carcinoma in a total of 89 cases, using a cDNA array and pattern classification algorithms. We set binary classes for a panel of clinicopathologic parameters, each of which was divided at different levels for categories (discrete) or values (continuous). We searched an optimal combination of genes to discriminate the classes by using of a feature subset selection algorithm, which was applied to a set of genes preselected on the basis of statistical difference in expression (two-sided t test, P < or = 0.05). We used a sequential forward feature selection which additively searched a combination of genes, giving a minimal leave-one-out classification error rate of a k-nearest neighbor classifier. In the process of gene preselection, we found a remarkable difference in the expression pattern of genes according to the anatomical location of cancers. The difference was most prominent when the classes were set for cecum, ascending colon, transverse colon, and descending colon (CATD) versus sigmoid colon and rectum (SR). By stratifying these two locations, we were able to extract gene expression profiles characteristic of the classes of the presence versus absence of lymph node metastasis, lymphatic invasion, vascular invasion and degree of mural invasion, and pathological stages, with an accuracy of more than 90%. These results suggest that colorectal cancers harbor distinct molecular pathophysiological statuses according to their right-to-left locations, of which stratification is important for pattern classification of cDNA array data.