A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium's own F1Fo ATP synthase.

Several intracellular pathogens, including Salmonella enterica and Mycobacterium tuberculosis, require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm, whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein.

Brain decoding of spontaneous thought: Predictive modeling of self-relevance and valence using personal narratives.

The contents and dynamics of spontaneous thought are important factors for personality traits and mental health. However, assessing spontaneous thoughts is challenging due to their unconstrained nature, and directing participants' attention to report their thoughts may fundamentally alter them. Here, we aimed to decode two key content dimensions of spontaneous thought-self-relevance and valence-directly from brain activity. To train functional MRI-based predictive models, we used individually generated personal stories as stimuli in a story-reading task to mimic narrative-like spontaneous thoughts (n = 49). We then tested these models on multiple test datasets (total n = 199). The default mode, ventral attention, and frontoparietal networks played key roles in the predictions, with the anterior insula and midcingulate cortex contributing to self-relevance prediction and the left temporoparietal junction and dorsomedial prefrontal cortex contributing to valence prediction. Overall, this study presents brain models of internal thoughts and emotions, highlighting the potential for the brain decoding of spontaneous thought.

A bacterial mRNA leader that employs different mechanisms to sense disparate intracellular signals.

Bacterial mRNAs often contain leader sequences that respond to specific metabolites or ions by altering expression of the associated downstream protein-coding sequences. Here we report that the leader RNA of the Mg(2+) transporter gene mgtA of Salmonella enterica, which was previously known to function as a Mg(2+)-sensing riboswitch, harbors an 18 codon proline-rich open reading frame-termed mgtL-that permits intracellular proline to regulate mgtA expression. Interfering with mgtL translation by genetic, pharmacological, or environmental means was observed to increase the mRNA levels from the mgtA coding region. Substitution of the mgtL proline codons by other codons abolished the response to proline and to hyperosmotic stress but not to Mg(2+). Our findings show that mRNA leader sequences can consist of complex regulatory elements that utilize different mechanisms to sense separate signals and mediate an appropriate cellular response.

Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response.

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.

Swd2/Cps35 determines H3K4 tri-methylation via interactions with Set1 and Rad6.

BACKGROUND: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. RESULTS: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region. CONCLUSIONS: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level.

Mitochondria and Endoplasmic Reticulum Stress in Retinal Organoids from Patients with Vision Loss.

Activating transcription factor 6 (ATF6), a key regulator of the unfolded protein response, plays a key role in endoplasmic reticulum function and protein homeostasis. Variants of ATF6 that abrogate transcriptional activity cause morphologic and molecular defects in cones, clinically manifesting as the human vision loss disease achromatopsia (ACHM). ATF6 is expressed in all retinal cells. However, the effect of disease-associated ATF6 variants on other retinal cell types remains unclear. Herein, this was investigated by analyzing bulk RNA-sequencing transcriptomes from retinal organoids generated from patients with ACHM, carrying homozygous loss-of-function ATF6 variants. Marked dysregulation in mitochondrial respiratory complex gene expression and disrupted mitochondrial morphology in ACHM retinal organoids were identified. This indicated that loss of ATF6 leads to previously unappreciated mitochondrial defects in the retina. Next, gene expression from control and ACHM retinal organoids were compared with transcriptome profiles of seven major retinal cell types generated from recent single-cell transcriptomic maps of nondiseased human retina. This indicated pronounced down-regulation of cone genes and up-regulation in Müller glia genes, with no significant effects on other retinal cells. Overall, the current analysis of ACHM patient retinal organoids identified new cellular and molecular phenotypes in addition to cone dysfunction: activation of Müller cells, increased endoplasmic reticulum stress, disrupted mitochondrial structure, and elevated respiratory chain activity gene expression.

Developing and Validating the Senior Meaning in Life Evaluation (SMiLE) Scale.

The Senior Meaning in Life Evaluation scale encompasses not only older adults' personal motivation and growth but also the meaning for them in society and in their relationships: With this scale, we aimed to present their voices. A three-phase process was followed: The scale's items were developed empirically from interviews of older adults; exploratory factor analysis (EFA) was conducted to test convergent and concurrent validity; and finally, confirmatory factor analysis (CFA) was performed. EFA resulted in 18 items grouped into 4 factors (i.e., proactive on life, overcoming emptiness, acceptance in life, and social contribution), which was supported by the CFA.

uORF-mediated riboregulation controls transcription of whiB7/wblC antibiotic resistance gene.

WhiB7/WblC is a transcriptional factor of actinomycetes conferring intrinsic resistance to multiple translation-inhibitory antibiotics. It positively autoregulates its own transcription in response to the same antibiotics. The presence of a uORF and a potential Rho-independent transcription terminator in the 5' leader region has suggested a possibility that the whiB7/wblC gene is regulated via a uORF-mediated transcription attenuation. However, experimental evidence for the molecular mechanism to explain how antibiotic stress suppresses the attenuator, if any, and induces transcription of the whiB7/wblC gene has been lacking. Here we report that the 5' leader sequences of the whiB7/wblC genes in sub-clades of actinomycetes include conserved antiterminator RNA structures. We confirmed that the putative antiterminator in the whiB7/wblC leader sequences of both Streptomyces and Mycobacterium indeed suppresses Rho-independent transcription terminator and facilitates transcription readthrough, which is required for WhiB7/WblC-mediated antibiotic resistance. The antibiotic-mediated suppression of the attenuator can be recapitulated by amino acid starvation, indicating that translational inhibition of uORF by multiple signals is a key to induce whiB7/wblC expression. Our findings of a mechanism leading to intrinsic antibiotic resistance could provide an alternative to treat drug-resistant mycobacteria.

Recovery of dopaminergic amacrine cells after strobe light stimulation in the developing rat retina.

Concerns regarding the impact of strobe light on human health and life have recently been raised. Sources of strobe light include visual display terminals, light-emitting diodes, and computer monitors. Strobe light exposure leads to visual discomfort, headaches, and poor visual performance and affects the number of dopaminergic amacrine cells (DACs) in the developing retina, as well as retinal dopamine levels in animals. DACs serve as the sole source of retinal dopamine, and dopamine release from the retina is activated by light exposure following a circadian rhythm. Using a Sprague-Dawley rat model, this study sought to determine whether changes in DACs caused by strobe light are recoverable after ceasing strobe light exposure during retinal development. From eye opening (postnatal 2 weeks), rats in the control group were reared under normal light (an unflickering 150 lux incandescent lamp with a 12 h light/dark cycle), whereas those in the experimental group (i.e., strobe-recovery group) were reared under strobe light (2 Hz for 12 h/day) exposure for 2 weeks. After postnatal week 4, normal light was provided to all animals to observe the reversibility of the effect of strobe light. Immunohistochemistry and immunoblot analysis for the rate limiting enzyme for dopamine synthesis, tyrosine hydroxylase (TH), as well as high-pressure liquid chromatography for measuring dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were performed at postnatal weeks 4, 6, 8, and 10. The number of type I and type II TH-immunoreactive (TH-IR) cells across the entire retina was counted to evaluate whether changes in DACs induced by strobe light could recover after ceasing strobe light exposure. The number of type I TH-IR cells slightly decreased but remained at a constant level in the control group. In contrast, the number of type I TH-IR cells rapidly decreased up to postnatal week 6, but then increased after postnatal week 8 in the strobe-recovery group. Subsequently, the number of type I TH-IR cells eventually reached a number similar to that in the control group. In addition, the number of intermediate-sized TH-IR cells were increased at postnatal weeks 8 and 10 and the dopamine level was decreased at postnatal week 8 in the strobe-recovery group. However, the levels of DOPAC and TH proteins did not differ between the two groups. This suggests that changes in DACs caused by strobe light are reversible and that type II TH-IR cells may play a key role in this recovery.

Subgroups of self-neglect and effects on suicidal ideation among the older adults.

The issue of self-neglect among older adults is receiving attention in modern societies where aging is accelerating. To help expand our understanding of this phenomenon, this study identified its different types using latent profile analysis and verified the main variables that distinguish these types from each other. The three profiles that were identified are high self-neglect (HSN: 28.8%), low self-neglect (LSN: 35.6%), and poor personal hygiene (PPH: 35.6%). Interestingly, PPH showed a high rate and was identified as a noticeable type of elder self-neglect. Gender, age group, SES, support size, and suicidal ideation were significant in classifying the types of self-neglect. Men were more likely to be within the HSN group, and late elderly were more likely to be within the PPH group. The higher SES and social support, the higher the probability of being within the LSN group. The higher the suicidal ideation, the higher the possibility of falling under the HSN group. To reduce self-neglect among older adults, this study suggests to older adults vulnerable to self-neglect, expansion of the social support available to them, and provision of mental health services to this population.

Gene Expression of Clusterin, Tissue Inhibitor of Metalloproteinase-1, and Their Receptors in Retinal Pigment Epithelial Cells and Müller Glial Cells Is Modulated by Inflammatory Stresses.

Balanced activities of matrix metalloproteinases (MMPs) and their inhibitors are essential for photoreceptor (PR) cell survival. PR rod cell survival in rodent models of inherited retinitis pigmentosa (RP) is prolonged by recombinant tissue inhibitor of metalloproteinase (TIMP)-1 or clusterin (CLU) proteins. Retinal pigment epithelial cells (RPE) and Müller glia (MG) cells support PR cells. In human RPE and MG cell lines, we measured their mRNA levels of the two genes with quantitative real-time PCR (qRT-PCR) with interleukin (IL)-1ß treatment, a key pathological component in retinal degeneration. Endogenous CLU gene expression was significantly downregulated by IL-1ß in both cell types, whereas TIMP-1 expression was upregulated in MG cells, suggesting the transcriptional control of CLU is potentially more sensitive to inflammatory conditions. The expression levels of CLU endocytic receptors revealed that the low-density lipoprotein receptor-related protein 2 (LRP2) was upregulated only in MG cells by the treatment with no detectable change in RPE cells. Like LRP2, IL-1ß upregulated TIMP-1 receptor LRP1 expression in MG cells; however, it was decreased in the expression of RPE cells. These data suggest that the gene expression of CLU and TIMP-1 and their receptors may be dynamically modulated in inflammatory conditions.

Lysine Ubiquitylation Drives Rhodopsin Protein Turnover.

Rhodopsin is a G-protein-coupled receptor that is specifically and abundantly expressed in rod photoreceptors. Over 150 rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP). The most common mutation in the United States is the conversion of proline to histidine at position 23 (P23H) in the N-terminal domain of rhodopsin. We previously found that P23H rhodopsin was misfolded, ubiquitinylated, and rapidly degraded. Here, we investigated the role of lysine residues on P23H rhodopsin ubiquitinylation and turnover. We transfected HEK293 cells with a P23H human rhodopsin construct where all 11 lysine residues were mutated to arginine (K-null P23H). We found that the K-null P23H rhodopsin was significantly less ubiquitylated than intact P23H rhodopsin. We found that K-null P23H protein turnover was significantly slower compared to P23H rhodopsin through cycloheximide chase analysis. Finally, we also generated a wild-type rhodopsin construct where all lysines were converted to arginine and found significantly reduced ubiquitylation. Our findings identify ubiquitinylation of lysine residues as an important posttranslational modification involved in P23H rhodopsin protein degradation.

A new brain-cutting device and ultraviolet resin-mounted human brain slices as a teaching adjunct for neuroanatomy education.

Although the level of neuroscience research is rapidly developing with the introduction of new technologies, the method of neuroanatomy education remains at the traditional level and requires improvement to meet the needs of educators and trainees. We developed a new three-dimensional (3D) printed device (human brain-cutting mold, HBCM) for creating human brain slices; moreover, we demonstrated a simple method for creating semi-permanent ultraviolet (UV) resin-mounted brain slice specimens for neuroanatomy education. We obtained brain slices of uniform thickness (3 mm) through the HBCM; the resultant brain slices were optimal for assessing morphological details of the human brain. Furthermore, we used an agar-embedding method for brain-slicing with the HBCM, which minimized geometrical distortions of the brain slices. Also, we prepared semi-permanent brain serial specimens using an acrylic brain slice frame and UV-curable resin, which was highly compatible with moist bio-specimens. During UV resin curing, neither air bubble formation nor color change occurred. The resultant UV resin-mounted brain slices produced definite coronal sections with high transparency and morphological accuracy. We also performed 3D modeling by stacking brain slice images that differentiated the cortical area and nine subcortical regions via manual segmentation. This method could be a reliable alternative for displaying high-quality human brain slices and would be helpful for students and trainee to understand anatomical orientation from 2D images to 3D structures. Also, this may present an innovative approach for preparing and preserving coronal sections of the normal or pathological human brain.

Identification of Antibacterial Sterols from Korean Wild Mushroom Daedaleopsis confragosa via Bioactivity- and LC-MS/MS Profile-Guided Fractionation.

As part of an ongoing natural product chemical research for the discovery of bioactive secondary metabolites with novel structures, wild fruiting bodies of Daedaleopsis confragosa were collected and subjected to chemical and biological analyses. We subjected the fractions derived from the methanol extract of the fruiting bodies of D. confragosa to bioactivity-guided fractionation because the methanol extract of D. confragosa showed antibacterial activity against Helicobacter pylori strain 51, according to our bioactivity screening. The n-hexane and dichloromethane fractions showed moderate to weak antibacterial activity against H. pylori strain 51, and the active fractions were analyzed for the isolation of antibacterial compounds. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the n-hexane fraction contains several compounds which are absent in the other fractions, so the fraction was prioritized for further fractionation. Through chemical analysis of the active n-hexane and dichloromethane fractions, we isolated five ergosterol derivatives (1-5), and their chemical structures were determined to be demethylincisterol A3 (1), (20S,22E,24R)-ergosta-7,22-dien-3ß,5α,6ß-triol (2), (24S)-ergosta-7-ene-3ß,5α,6ß-triol (3), 5α,6α-epoxy-(22E,24R)-ergosta-7,22-dien-3ß-ol (4), and 5α,6α-epoxy-(24R)-ergosta-7-en-3ß-ol (5) by NMR spectroscopic analysis. This is the first report on the presence of ergosterol derivatives (1-5) in D. confragosa. Compound 1 showed the most potent anti-H. pylori activity with 33.9% inhibition, rendering it more potent than quercetin, a positive control. Compound 3 showed inhibitory activity comparable to that of quercetin. Distribution analysis of compound 1 revealed a wide presence of compound 1 in the kingdom Fungi. These findings indicate that demethylincisterol A3 (1) is a natural antibiotic that may be used in the development of novel antibiotics against H. pylori.

Bacterial Mg2+ homeostasis, transport, and virulence.

Organisms must maintain physiological levels of Mg(2+) because this divalent cation is critical for the stabilization of membranes and ribosomes, for the neutralization of nucleic acids, and as a cofactor in a variety of enzymatic reactions. In this review, we describe the mechanisms that bacteria utilize to sense the levels of Mg(2+) both outside and inside the cytoplasm. We examine how bacteria achieve Mg(2+) homeostasis by adjusting the expression and activity of Mg(2+) transporters and by changing the composition of their cell envelope. We discuss the connections that exist between Mg(2+) sensing, Mg(2+) transport, and bacterial virulence. Additionally, we explore the logic behind the fact that bacterial genomes encode multiple Mg(2+) transporters and distinct sensing systems for cytoplasmic and extracytoplasmic Mg(2+). These analyses may be applicable to the homeostatic control of other cations.

A trans-acting leader RNA from a Salmonella virulence gene.

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection.

Molecular alterations and poziotinib efficacy, a pan-HER inhibitor, in human epidermal growth factor receptor 2 (HER2)-positive breast cancers: Combined exploratory biomarker analysis from a phase II clinical trial of poziotinib for refractory HER2-positive breast cancer patients.

We aimed to investigate the impact of genetic alterations on the efficacy of poziotinib in a phase II clinical trial of patients with heavily treated HER2-positive metastatic breast cancer (BC). We performed targeted ultra-deep sequencing with a customized cancer gene panel and RNA expression assay using BC specimens. Of 106 patients, biomarker data were available for 85. Copy number (CN) amplifications of HER2 were observed in 72 patients (85%), and CN >8 in 50 (59%). Single nucleotide variants (SNVs) of HER2 were found in 16 patients (19%). Genetic alterations of PIK3CA pathway were found in 40 patients (47%). Median progression free survival (PFS) of the biomarker analysis group was 3.61 months. In terms of PFS, HER2 with CN >8 prolonged (hazard ratio (HR) 0.61, 95% CI: 0.38, 0.97, p = 0.037) and alteration of PIK3CA pathway shortened the duration of survival (HR 2.25, 95% CI: 1.39, 3.63, p = 0.001). SNVs of HER2 increased survival duration, but the effect was not significant (HR: 0.58, 95% CI: 0.31, 1.08, p = 0.085). In addition, SNVs in the ERBB3 cytoplasmic domain decreased poziotinib response (HR: 4.58, 95% CI: 2.02, 10.37, p < 0.001). In multigene analysis, BC with HER2 CN >8 and intact PIK3CA pathway had significantly longer PFS compared to others (HR: 0.37, 95% CI: 0.21, 0.66, p = 0.001), while SNVs in the ERBB3 cytoplasmic domain predicted poor prognosis (HR: 4.28, 95% CI: 1.71, 10.71, p < 0.001). In conclusion, HER2 CN amplification, PIK3CA pathway alteration, and ERBB3 cytoplasmic mutation showed predictive roles on clinical outcomes of HER2-positive MBC treated with poziotinib.

MoS2 Field-Effect Transistor-Amyloid-ß1-42 Hybrid Device for Signal Amplified Detection of MMP-9.

The detection of circulating protein (CP) is very important for the diagnosis and therapeutics of cancer. Conventional techniques based on a specific antibody-antigen interaction are still lacking because of a shortage of cost effectiveness, complicated sandwich structure and tagging process, and inconsistent detection of CP due to the inherent instability of antibodies. Herein, we demonstrate a hybrid device consisting of two-dimensional (2D) nanoscale molybdenum disulfide (MoS2) field-effect transistor (FET) with an amyloid-ß1-42 (Aß1-42) functionalized surface, which amplifies electric signals of the FET in order to detect matrix metalloproteinase-9 (MMP-9), which is a certain type of CP that degrades Aß1-42. With the hybrid device, we detected the concentrations of MMP-9 in the range from 1 pM to 10 nM. Moreover, using tapping-mode atomic force microscopy and Kelvin probe force microscopy, we verified that the signal amplification corresponding to the MMP-9 concentrations was caused by the reduced length and the decreased surface potential of degraded Aß1-42 due to MMP-9. The hybrid device studied in this paper can be very useful for monitoring MMP-9 activity, as well as serving as a sensing platform for the electrical signal amplification of 2D MoS2 FET-biosensors.

Exploratory biomarker analysis from a phase II clinical trial of eribulin plus gemcitabine versus paclitaxel plus gemcitabine for HER2-negative metastatic breast cancer patients (KCSG BR13-11).

PURPOSE: We conducted an exploratory biomarker study from a phase II clinical trial of eribulin plus gemcitabine (EG) versus paclitaxel plus gemcitabine (PG) in HER2-negative metastatic breast cancer (BC) patients. METHODS: We performed targeted deep sequencing with a customized cancer gene panel and RNA expression assay. Tumor mutation burden (TMB) and mutation signatures were determined based on genetic alteration in targeted regions. Gene set variation analysis was performed with PanCancer Immune Profiling and PanCancer Pathway Panels. Statistical analyses were conducted to identify the associations between genetic alterations and clinical outcomes. RESULTS: Of 119 patients, 40 had available biomarker data. Among the 40 patients, 4 supported their post-treatment tissues. In targeted deep sequencing, FAT3 (48%) was the most frequently mutated gene, followed by PKHD1, TP53, GATA3, PARP4, and PIK3CA. In terms of gene expression, low expression of epithelial-mesenchymal transition (EMT) pathway genes was associated with prolonged progression-free survival (PFS) in the EG group, while high expression of the EMT pathway was associated with good prognosis in the PG group. Median TMB was 6.5 (range 2.44-46.34) and there was no relationship between TMB and patient prognosis. Analysis of mutation signatures showed that signatures 3, 20, and 26 were frequently observed in our cohort. Further survival analysis according to mutation signature showed that mutation signature 3, as a homologous recombinant deficiency-related signature, was highly associated with disease progression (hazard ratio (log2 scale) 8.21, 95% confidence interval 2.93-13.48, p = 0.002). Kaplan-Meier plot also showed that BCs with signature 3 had short PFS compared to those without these signatures (median PFS (months) for signature 3 (low vs. high): 17.2 vs. 8.1, p = 0.0026). CONCLUSIONS: Mutation signature 3, found in about 30% of MBCs regardless of hormone receptor status, was associated with short PFS for patients with cytotoxic chemotherapy. TRIAL REGISTRY: ClinicalTrials.gov number: NCT02263495.

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation.

Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models. Some studies reported long-term survival and successful function of porcine islets in diabetic monkeys. Genetic engineering techniques may improve the survival and function of porcine islets. A recent study reported the generation of transgenic pigs expressing human insulin rather than porcine insulin by changing one amino acid at the end of the ß-chain in insulin. However, C-peptide from pigs still existed. In this study, we generated transgenic pigs expressing human proinsulin to express human insulin and C-peptide using fibroblasts from proinsulin knockout pigs as donor cells for somatic cell nuclear transfer. Eleven live piglets were delivered from three surrogates and characterized to confirm the genotype and phenotype of the generated piglets. Genotype analysis of the generated piglets showed that five of the eleven piglets contained the human proinsulin gene. Insulin expression was confirmed in the serum and pancreas in two of the five piglets. C-peptide derived from human proinsulin was also confirmed by liquid chromatography tandem mass spectrometry. Non-fasting blood glucose level was measured to verify the function of the insulin derived from the human proinsulin. Two piglets expressing insulin showed normal glucose levels similar to that in the wild-type control. In conclusion, human insulin- and C-peptide-expressing pigs without porcine insulin and C-peptide were successfully established. These pigs can be used as a source of islets for islet xenotransplantation.