The role of SLFN11 in DNA replication stress response and its implications for the Fanconi anemia pathway.

Fanconi anemia (FA) is a hereditary disorder characterized by a deficiency in the repair of DNA interstrand crosslinks and the response to replication stress. Endogenous DNA damage, most likely caused by aldehydes, severely affects hematopoietic stem cells in FA, resulting in progressive bone marrow failure and the development of leukemia. Recent studies revealed that expression levels of SLFN11 affect the replication stress response and are a strong determinant in cell killing by DNA-damaging cancer chemotherapy. Because SLFN11 is highly expressed in the hematopoietic system, we speculated that SLFN11 may have a significant role in FA pathophysiology. Indeed, we found that DNA damage sensitivity in FA cells is significantly mitigated by the loss of SLFN11 expression. Mechanistically, we demonstrated that SLFN11 destabilizes the nascent DNA strands upon replication fork stalling. In this review, we summarize our work regarding an interplay between SLFN11 and the FA pathway, and the role of SLFN11 in the response to replication stress.

Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices.

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4-dinitrochlorobenzene (DNCB-PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB-PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB-PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum-containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB-PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety.

Prognostic impact of radiological tumor burden in patients with metastatic urothelial carcinoma treated with pembrolizumab.

PURPOSE: Radiological tumor burden has been reported to be prognostic in many malignancies in the immunotherapy era, yet whether it is prognostic in patients with metastatic urothelial carcinoma (mUC) treated with pembrolizumab remains uninvestigated. We sought to assess the predictive and prognostic value of radiological tumor burden in patients with mUC. METHODS: We performed a retrospective analysis of 308 patients with mUC treated with pembrolizumab. Radiological tumor burden was represented by baseline tumor size (BTS) and baseline tumor number (BTN). Optimal cut-off value of BTS was determined as 50 mm using the Youden index (small BTS: n = 194, large BTS: n = 114). Overall (OS), cancer-specific (CSS), progression-free survival (PFS), and objective response rate (ORR) were compared. Non-linear associations between BTS and OS and CSS were evaluated using restricted cubic splines. RESULTS: Patients with large BTS were less likely to have undergone the surgical resection of the primary tumor (P = 0.01), and more likely to have liver metastasis (P < 0.001) and more metastatic lesions (P < 0.001). On multivariable analyses controlling for the effects of confounders (resection of primary tumor, metastatic site, number of metastases and lactate dehydrogenase level), large BTS and high BTN were independently associated with worse OS (HR 1.52; P = 0.015, and HR 1.69; P = 0.018, respectively) and CSS (HR 1.59; P = 0.01, and HR 1.66; P = 0.031, respectively), but not PFS. Restricted cubic splines revealed BTS was correlated with OS and CSS in linear relationships. Additionally, large BTS was significantly predictive of lower ORR and complete response rate on univariable analyses (P = 0.041 and P = 0.032, respectively), but its association disappeared on multivariable analyses. CONCLUSION: Radiological tumor burden has independent prognostic value with a linear relationship in pembrolizumab-treated patients with mUC and might help drive the earlier introduction of second-line pembrolizumab and/or switching to subsequent therapies.

Mouse Slfn8 and Slfn9 genes complement human cells lacking SLFN11 during the replication stress response.

The Schlafen (SLFN)11 gene has been implicated in various biological processes such as suppression of HIV replication, replication stress response, and sensitization of cancer cells to chemotherapy. Due to the rapid diversification of the SLFN family members, it remains uncertain whether a direct ortholog of human SLFN11 exists in mice. Here we show that mSLFN8/9 and hSLFN11 were rapidly recruited to microlaser-irradiated DNA damage tracks. Furthermore, Slfn8/9 expression could complement SLFN11 loss in human SLFN11-/- cells, and as a result, reduced the growth rate to wild-type levels and partially restored sensitivity to DNA-damaging agents. In addition, both Slfn8/9 and SLFN11 expression accelerated stalled fork degradation and decreased RPA and RAD51 foci numbers after DNA damage. Based on these results, we propose that mouse Slfn8 and Slfn9 genes may share an orthologous function with human SLFN11. This notion may facilitate understanding of SLFN11's biological role through in vivo studies via mouse modeling.

Fanconi anemia and Aldehyde Degradation Deficiency Syndrome: Metabolism and DNA repair protect the genome and hematopoiesis from endogenous DNA damage.

We have identified a set of Japanese children with hypoplastic anemia caused by combined defects in aldehyde degrading enzymes ADH5 and ALDH2. Their clinical characteristics overlap with a hereditary DNA repair disorder, Fanconi anemia. Our discovery of this disorder, termed Aldehyde Degradation Deficiency Syndrome (ADDS), reinforces the notion that endogenously generated aldehydes exert genotoxic effects; thus, the coupled actions of metabolism and DNA repair are required to maintain proper hematopoiesis and health.

Is the Preoperative Prognostic Nutritional Index a Useful Marker for the Decision to Perform Limited Resection in High-risk Patients With Stage I Non-small Cell Lung Cancer?

BACKGROUND/AIM: The indications for limited resection in high-risk patients with stage I non-small cell lung cancer (NSCLC) remain controversial. The purpose of this study was to evaluate the prognostic impact of the preoperative prognostic nutritional index (PNI) in high-risk patients undergoing limited resection. PATIENTS AND METHODS: High-risk patients undergoing limited resection for stage I NSCLC in our institution from 2005 to 2020 were retrospectively reviewed. Patients with clinical/pathological Tis/minimally invasive adenocarcinoma and multiple NSCLC were excluded. A multivariate Cox regression analysis was conducted to identify factors associated with overall survival (OS). RESULTS: Ninety eligible patients were included in this study. Grade ≥2 postoperative complications were significantly more frequent in the low-PNI group (6 cases, 16.6% vs. 7 cases, 12.9%; p=0.03). The rate of death due to other diseases was significantly higher in the low-PNI group than in the high-PNI group (14 cases, 50.0% vs. 11 cases, 25.0%; p=0.002). The multivariate analysis showed that male sex, Brinkman index ≥400, preoperative low PNI and pathological T factor ≥T1c/T2a were independent prognostic factors for OS. CONCLUSION: In high-risk patients undergoing limited resection for stage I NSCLC, low PNI was a poor prognostic factor, especially in relation to death from other diseases and lung cancer. The results may support thoracic surgeons in decision-making in relation to the indications for surgery.

Lack of impact of the ALDH2 rs671 variant on breast cancer development in Japanese BRCA1/2-mutation carriers.

The aldehyde degrading function of the ALDH2 enzyme is impaired by Glu504Lys polymorphisms (rs671, termed A allele), which causes alcohol flushing in east Asians, and elevates the risk of esophageal cancer among habitual drinkers. Recent studies suggested that the ALDH2 variant may lead to higher levels of DNA damage caused by endogenously generated aldehydes. This can be a threat to genome stability and/or cell viability in a synthetic manner in DNA repair-defective settings such as Fanconi anemia (FA). FA is an inherited bone marrow failure syndrome caused by defects in any one of so far identified 22 FANC genes including hereditary breast and ovarian cancer (HBOC) genes BRCA1 and BRCA2. We have previously reported that the progression of FA phenotypes is accelerated with the ALDH2 rs671 genotype. Individuals with HBOC are heterozygously mutated in either BRCA1 or BRCA2, and the cancer-initiating cells in these patients usually undergo loss of the wild-type BRCA1/2 allele, leading to homologous recombination defects. Therefore, we hypothesized that the ALDH2 genotypes may impact breast cancer development in BRCA1/2 mutant carriers. We genotyped ALDH2 in 103 HBOC patients recruited from multiple cancer centers in Japan. However, we were not able to detect any significant differences in clinical stages, histopathological classification, or age at clinical diagnosis across the ALDH2 genotypes. Unlike the effects in hematopoietic cells of FA, our current data suggest that there is no impact of the loss of ALDH2 function in cancer initiation and development in breast epithelium of HBOC patients.

A turn-on hydrazide oxidative decomposition-based fluorescence probe for highly selective detection of Cu2+ in tap water as well as cell imaging.

Copper (II) is one of the most important metal ions for the human body that act as a catalytic cofactor for many metalloenzymes and proteins, and its homeostasis disruption could lead to many neurological diseases. The reported probes for Cu (II) determination are mostly based on fluorescence quenching mechanism, which provides low precision and reliability. In the present work, a turn-on fluorescence probe, (Z)-1-[2-oxo-2-[2-[1-oxoaceanthrylen-2 (1H)-ylidene]hydrazinyl]ethyl]-pyridinium (OAHP), for highly selective detection of Cu2+ was developed. Hydrazide moiety of OAHP quenches probe fluorescence; however, upon its reaction with Cu, oxidative cleavage of the hydrazide moiety and intramolecular cyclization occurs, forming oxadiazole derivative with strong fluorescent properties. In this context, OAHP displayed significant fluorescence enhancement with increasing levels of Cu2+. OAHP could detect Cu2+ selectively with a detection limit of 18 nM (1.1 ppb). This is the first report for a probe that uses the ability of Cu2+ to induce oxidative decomposition of hydrazide with intramolecular cyclization, and it showed exceptional selective performance and exquisite sensitivity. Next, the method was applied successfully for monitoring Cu2+ in tap water samples with good accuracy (found% of 95.8-101.5%) and precisions (RSD<10%). Finally, OAHP was successfully applied for imaging Cu2+ in living cells, and this result indicates the potential of OAHP for selective detection of Cu2+ in complicated matrices.

Comparison of quantification of selenocyanate and thiocyanate in cultured mammalian cells between HPLC-fluorescence detector and HPLC-inductively coupled plasma mass spectrometer.

The simultaneous detection of cyanide (CN), thiocyanate (SCN), and selenocyanate (SeCN) by a HPLC-fluorescence detector (FLD) with the post-column König reaction was recently reported. SCN and SeCN are also detectable by HPLC-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) because sulfur and selenium can be detected, respectively, without any pre- or post-treatment. ICP-MS has high sensitivity for selenium and sulfur detection and is robust to sample matrices. In this study, we compared HPLC-FLD with the post-column König reaction and HPLC-ICP-MS in terms of SCN and SeCN detection sensitivity and linearity. The limit of detection (LOD) for SCN indicated that HPLC-FLD with the post-column König reaction was 354 times more sensitive than HPLC-ICP-MS. Likewise, the LOD for SeCN indicated that HPLC-FLD was 51 times more sensitive than HPLC-ICP-MS. These results demonstrated that HPLC-FLD was a more suitable technique for SeCN and SCN detection than HPLC-ICP-MS. We previously reported that SeCN was generated in selenite-exposed mammalian cells to detoxify excess selenite. HPLC-FLD with the post-column König reaction enabled good separation and detection for quantifying SCN and SeCN in mammalian cell lines exposed to selenite. The intracellular SCN and SeCN concentrations determined by this technique suggested differences in the metabolic capacity for selenite to form SeCN among the cell lines. In addition, since the amount of intracellular SCN and SeCN were significantly decreased by pretreatment of myeloperoxidase (MPO) inhibitors, SCN and SeCN were resulted from the interaction of sulfur and selenium with endogenous CN, respectively, generated with MPO.

Structure and Immunomodulatory Activity of Glycogen Derived from Honeybee Larvae (Apis mellifera).

Honeybee larvae have been recognized as nutrient-rich food in many countries. Although glycogen, a storage form of glucose in animals, is synthesized in honeybee larvae, there is no information on the structure of glycan and its biological activity. In this study, we successfully extracted glycogen from honeybee larvae using hot water extraction and investigated the structure and biological activity of glycan. It was found that the molecular weight of glycogen from honeybee larvae is higher than that of glycogen from bovine liver and oysters. In addition, treatment of RAW264.7 cells with glycogen from honeybee larvae resulted in a much higher production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than treatment with glycogen from either bovine liver or oysters. These results suggest that the high molecular weight glycogen from honeybee larvae is a functional food ingredient with immunomodulatory activity.

Analysis of disease model iPSCs derived from patients with a novel Fanconi anemia-like IBMFS ADH5/ALDH2 deficiency.

We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.

Quantification of progesterone in beef with marbling using liquid chromatography-tandem mass spectrometry with stable isotope-labelled standards.

Progesterone (P4) is contained naturally in animal tissue, and it is also used as a veterinary drug in cattle for treatment purposes. To assess the risk from P4 residues in beef derived from treated cattle, it is essential to quantify the P4 contained naturally in cattle tissue (endogenous P4). Therefore, we performed a method validation for the quantification of endogenous P4 (method quantification limit = 0.06 ng g-1) by using isotope-labelled P4s, and investigated the P4 contents in Japanese beef (n= 112; 0.07 to 121 ng g-1). The P4 contents in cattle muscle ranged from 0.07 to 54.3 ng g-1 in males, and from 0.27 to 121 ng g-1 in females. Our investigation also indicated that the developed method using both 13C- and deuterium-labelled P4 standards could be used to certify the recovery of P4 from cattle muscle containing various amounts of intramuscular fat, and enabled the determination of the P4 content in all Japanese beef samples that exceeded the method quantification limit.

SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells.

Fanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2-/-SLFN11-/- HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2-/- cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2-/- cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.

Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

C-terminal of ABCA1 separately regulates cholesterol floppase activity and cholesterol efflux activity.

ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for cholesterol homeostasis. We recently reported that ABCA1 not only exports excess cholesterol in an apoA-I dependent manner, but that it also flops cholesterol from the inner to the outer leaflet of the plasma membrane. However, the relationship between these two activities of ABCA1 is still unclear. In this study, we analyzed the subcellular localization of ABCA1 by using a newly generated monoclonal antibody against its extracellular domain and the functions of eleven chimera proteins, in which the C-terminal domain of ABCA1 was replaced with those of the other ABCA subfamily members. We identified two motifs important for the functions of ABCA1. Three periodically repeated leucine residues were necessary for the cholesterol floppase activity but not the cholesterol efflux activity, while a VFVNFA motif was essential for both activities of ABCA1. These results suggest that the C-terminal of ABCA1 separately regulates the cholesterol floppase activity and the cholesterol efflux activity.

Detection of Selenocyanate in Biological Samples by HPLC with Fluorescence Detection Using König Reaction.

We developed a simple and sensitive HPLC method for the determination of selenocyanate (SeCN-). The König reaction, which is generally used for the determination of cyanide and thiocyanate, was applied for the post-column detection, and using barbituric acid as a fluorogenic reagent made it possible to detect SeCN- with high sensitivity. The limits of detection (LOD) and quantification (LOQ) were 73.5 fmol and 245.1 fmol, respectively. Subsequently, the amounts of SeCN- in human blood and in cultured cell samples were analyzed, and no SeCN- was detected in human whole blood. Interestingly, we have found that some of the spiked SeCN- decomposed to cyanide in human whole blood. Ascorbic acid suppressed the decomposition of SeCN- to cyanide by reducing the ferric ion, which is typically involved in SeCN- decomposition. Then, SeCN- was detected in cultured HEK293 cells exposed to selenite. The established HPLC method with fluorescence detection of SeCN- is useful for investigating small amounts of SeCN- in biological samples.

Regulation of R-loops and genome instability in Fanconi anemia.

Fanconi anemia (FA) is a devastating hereditary disorder with impaired genome stability resulting in physical abnormalities, gradual loss of hematopoietic stem cells and development of tumours and leukaemia. It has been suggested that functions of FA genes are required to maintain genome stability by counteracting endogenous metabolites, such as aldehydes, that damage DNA and stall replication forks. Recent studies have implicated co-transcriptional R-loops, consisting of a DNA:RNA hybrid and displaced single-stranded DNA, as one of the potential endogenous sources that induce genome instability and the FA phenotype. This review focuses on recent literature, including our own, regarding the interplay between FA proteins and R-loops, and will provide readers with a concise summary of this rapidly evolving field.

FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R-loops during mild replication stress.

R-loops, which consist of DNA : RNA hybrids and displaced single-strand DNA, are a major threat to genome stability. We have previously reported that a key Fanconi anemia protein, FANCD2, accumulates on large fragile genes during mild replication stress in a manner depending on R-loops. In this study, we found that FANCD2 suppresses R-loop levels. Furthermore, we identified FANCD2 interactions with RNA processing factors, including hnRNP U and DDX47. Our data suggest that FANCD2, which accumulates with R-loops in chromatin, recruits these factors and thereby promotes efficient processing of long RNA transcripts. This may lead to a reduction in transcription-replication collisions, as detected by PLA between PCNA and RNA Polymerase II, and hence, lowered R-loop levels. We propose that this mechanism might contribute to maintenance of genome stability during mild replication stress.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

Comprehensive analysis of glycosaminoglycans from the edible shellfish.

We have previously reported that the keratan sulfate (KS) disaccharide was branched to the C-3 position of glucuronate in chondroitin sulfate (CS)-E derived from the Mactra chinensis. We carried out the comprehensive disaccharide analysis of GAGs from 10 shellfish, Ruditapes philippinarum, Scapharca broughtonii, Mizuhopecten yessoensis, Turbo cornutus, Crassostrea nippona, Corbicula japonica, Mytilus galloprovincialis, Neptunea intersculpta, Pseudocardium sachalinense and Crassostrea gigas, to better understand the glycan structures in marine organisms. The contents of CS, heparan sulfate and hyaluronic acid and their compositions depend on the species of shellfish. Interestingly, a peak corresponding to a pentasaccharide containing KS disaccharide was observed when GAGs from T. cornutus was treated with chondroitinase (Chase) ACII but not Chase ABC. In addition, unidentified peaks were also observed when CS derived from R. philippinarum, S. broughtonii were treated with Chase ACII. These results suggest the presence of additional unidentified structure of CS in these shellfish.