Complementation Group Research Articles

Replication protein A large subunit (RPA1a) limits chiasma formation during rice meiosis.

Replication protein A (RPA), a single-stranded DNA-binding protein, plays essential role in homologous recombination. However, because deletion of RPA causes embryonic lethality in mammals, the exact function of RPA in meiosis remains unclear. In this study, we generated an rpa1a mutant using CRISPR/Cas9 technology and explored its function in rice (Oryza sativa) meiosis. In rpa1a, 12 bivalents were formed at metaphase I, just like in wild-type, but chromosome fragmentations were consistently observed at anaphase I. Fluorescence in situ hybridization assays indicated that these fragmentations were due to the failure of the recombination intermediates to resolve. Importantly, the mutant had a highly elevated chiasma number, and loss of RPA1a could completely restore the 12 bivalent formations in the zmm (for ZIP1-4, MSH4/5, and MER3) mutant background. Protein-protein interaction assays showed that RPA1a formed a complex with the methyl methansulfonate and UV sensitive 81 (and the Fanconi anemia complementation group M-Bloom syndrome protein homologs (RECQ4A)-Topoisomerase3α-RecQ-mediated genome instability 1 complex to regulate chiasma formation and processing of the recombination intermediates. Thus, our data establish a pivotal role for RPA1a in promoting the accurate resolution of recombination intermediates and in limiting redundant chiasma formation during rice meiosis.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

MiRNA-218/FANCI is associated with metastasis and poor prognosis in lung adenocarcinoma: a bioinformatics analysis.

BackgroundIn this study, tumor microarray analysis was used to screen the key messenger RNAs (mRNAs) and microRNAs related to the progression of lung adenocarcinoma (LUAD), in order to provide a theoretical basis for early diagnosis, therapeutic targets, and prognosis evaluation of patients with LUAD.MethodsThe mRNA and miRNA expression datasets came from the Gene Expression Omnibus (GEO) project database. Differentially expressed genes (DEGs) and microRNAs (DEMs) between LUAD tissues and adjacent lung tissue were obtained using GEO2R. The Search Tool for the Retrieval of Interacting Genes website was also employed to construct and visualize the interactions of overlapped DEGs. The overall survival of DEMs was investigated using the Kaplan-Meier plotter. The TargetScan website (http://www.targetscan.org/) was used to verify the relationship between FA Complementation Group I (FANCI) and the expression of miRNA-218 (miR-218). The expression of FANCI was verified using the GEO and Human Protein Atlas databases, as well as Real Time Quantitative PCR using our own samples. Next, we analyzed the relationship between the expression of FANCI and the clinicopathological characteristics as well as the prognosis of patients with LUAD. We also explored whether the FANCI was related to immune cell infiltration in LUAD.ResultsFANCI was identified as a hub gene and associated with poor OS. We found that miR-218 negatively regulates FANCI mRNA expression. At the mRNA expression and protein level, FANCI was more highly expressed in LUAD tissues. The expression of FANCI in LUAD was related to tumor size (χ2=13.96, P<0.001), lymphatic metastasis (χ2=3.88, P<0.05), distant metastasis (χ2=45.39, P<0.001), and stage (χ2=11.03, P<0.05). In addition, the Cox regression model found that FANCI mRNA expression was an independent predictive factor of patient survival (P<0.05). FANCI expression was both weakly related to B cells and neutrophil infiltration in LUAD.ConclusionsmiR-218 may negatively regulate FANCI, and FANCI could promote metastasis via extracellular matrix (ECM) receptor interaction, leading to poor prognosis of LUAD. FANCI may be a key gene to the determine metastasis and poor prognosis in patients with LUAD. Changes in the immune microenvironment may be the mechanism through which FANCI leads to poor prognosis of LUAD.

Novel Lineage Depletion and Manufacturing Allows for Unprecedented Preservation of Autologous Blood Stem Cells for Gene Therapy of Fanconi Anemia Complementation Group Α

A hallmark of the rare, monogenic disorder called Fanconi anemia (FA) is an accelerated decline in hematopoietic stem cells (HSC) leading to bone marrow (BM) failure. Long-term treatment requires hematopoietic cell transplantation (HCT) from an unaffected donor, but success of this approach in ~70% of the patients lacking a matched sibling is associated with potentially severe side effects. Gene therapy could be an alternative, correcting the genetic defect in the patient's own HSC and negating the need for a HCT donor. Building on previous FA gene therapy studies, we developed an optimized protocol for lentivirus (LV)-mediated FANCA gene transfer into HSCs from FA-A patients [Becker, P. Human Gene Therapy, 2010]. This phase I clinical trial incorporates vector recommendations from the International FA Gene Therapy Working Group. Three patients have been treated on this trial to date (National Clinical Trials Registry ID: NCT01331018). This protocol collects unmanipulated HSC from BM or mobilized leukapheresis (mAPH) and does not include conditioning prior to cell infusion. In the first two patients enrolled, harvested BM demonstrated reduced CD34+ cell numbers and CD34 expression (Figure 1). In the first patient, direct enrichment of dimly CD34+ cells was very inefficient. Thus, for the second patient the enrichment step was eliminated. While this processing change preserved more of the CD34+ cells and improved transduction and viability, it required a prohibitive amount of LV vector. To reduce vector needs and preserve available CD34+ cells for future FA patients, we developed a novel strategy to deplete lineage+ (CD3+, CD14+, CD16+ and CD19+) cells from both mAPH and BM products. This process efficiently depletes >90% of lineage+ cells from healthy donor cell products while retaining ≥60% of the initial CD34+ cell fraction, reduced total nucleated cells (TNC) by 1-2 logs and maintained improved transduction and viability following gene transfer. Most importantly, transduced lineage- cell products engrafted in an immune deficient mouse model equivalent to that of purified CD34+ cells from the same donor when transplanted at matched CD34+ cell doses. This novel selection strategy has been approved by the regulatory agencies and a third patient was treated in July 2017 with this approach. Patient 3 is a 5-year old male with confirmed FA-A by complementation studies. Baseline ANC averaged 1.6 K/mcL and baseline platelet counts averaged 31 K/mcL in the 6 months prior to treatment, with declining ANC and platelets over a 4 year interval prior to gene therapy. Successful mobilization of CD34+ cells was achieved with combination filgrastim (16µg/kg BID or 32µg/kg/day) and plerixafor (240µg/kg). Two, successive apheresis collections resulted in 8.5×1010 TNC containing 1.6×108 CD34+ cells. A total of 5×1010 TNC were subjected to lineage depletion, resulting in a 94% reduction in TNC and a 55% retention of available CD34+ cells. A total of 52.4×106 CD34+ cells were transduced at 5 IU/cell, resulting in a final cell dose of 2.4×106 CD34+ cells per kg. We observed a VCN of 1.8 copies per cell and viability of the final cellular product was 99.3%. Infusion was well-tolerated and a fourth patient is expected to be treated in the Fall of 2017. This novel method of depleting lineage positive cells reduced TNC counts to feasible numbers for transduction while maintaining high numbers of CD34+ cells, a strategy which could have benefits in other disease targets for gene therapy. [Display omitted] DisclosuresAdair:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; CalImmune Inc.: Consultancy. Becker:GlycoMimetics, Inc.: Research Funding. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

TGF-β1 Production By Bone Marrow Mesenchymal Stem Cells of FANCD2 Differs from Other Fanconi Anemia Complementation Groups

TGF-β secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. As a component of the BM niche, BM- mesenchymal stem cells (BM-MSCs) may produce abnormal levels of TGF-β in Fanconi anemia (FA) as obtained for HSCs previously and may play a role in bone marrow failure.Herein, we present the molecular and cellular characterization of FA BM-MSCs by addressing their immunophenotypes, proliferation- and differentiation capacities, as well as expression and secretion levels of transforming growth factor beta (TGF-β) isoforms. Proliferative potency of FA cells was determined between P3 to P7 and compared with healthy donors by calculating cumulative population doubling (cPD) at each passage. Senescence of FA patient BM-MSCs was evaluated at P3 and P6 by SA-β-gal activity and compared with donor BM-MSCs. Diepoxybutane (DEB) effect on TGF-β1 expression of BM-MSCs from FA patients and donors was also assessed.In ten FA patients, mutations were classified as FANCA (n=7), FANCG (n=1) and FANCD2 (n=2). Of six were novel mutations, five in FANCA and one in FANCD2 . BM-MSCs immunophenotype and differentiation capacity of FA BM-MSCs were similar to healthy donors.The CD200 (OX-2) levels were slightly lower in FA MSCs compared to donors. The percentage of CD106 (VCAM-1) positive cells were slightly high especially in FANCD2 cells. FA BM-MSCs showed arrest in dip G2 following DEB treatment. BM-MSCs derived from FANCA- or FANCG -deficient patients displayed similar ex vivo proliferative capacity as healthy donor cells from P3 to P7. However, FANCD2-deficient cells presented lower proliferative capacity. FANCA BM-MSCs had notably increased β-gal positivity at P6, while cells displaying FANCD2 mutation had higher activity at P3 indicating premature aging of these cells. Relative gene expression of TGF-β1 was similar between donor and FA MSCs and did not change by DEB treatment. All cells did not secrete TGF-β2 and -β3. Since the variation of TGF-β1 secretion by FANCA and donor cells was high, a significant difference between the groups could not be obtained. On the other hand, FANCD2 -deficient cells were lack of TGF-β1 secretion. Our data suggest that absence of TGF-β1 secretion may lead to early onset of senescence of the FA-D2 BM-MSCs and may be of value to understand the Fanconi anaemia bone marrow dysfunction in humans. DisclosuresNo relevant conflicts of interest to declare.

Gene Heterogenicity Diagnosis on Children with Complicated Immunological and Hematological Abnormalities

Some abnormalities in peripheral blood of children coexist with abnormal immunological function,such as autoantibodies, humoral and /or cellular immunity disorders.It is difficult to diagnose in the past,especially in children suspected withImmune Thrombocytopenia and Evans syndrome.To explore accurate diagnostic method in children with complicated immunological and hematological abnormalities, 9 complicated cases with positive findings under 14 years old were studied. Gene mutations were screened with high-throughput sequencing, genetic data were analyzed using bioinformatics and clinical information analysis technology and the suspected pathogenic mutation were verified using Sanger sequencing technology. Interpretation of pathogenic mutation included biological significance, genetic significance and coincidence degree of clinical characteristics. In suspected cases , proband , parents and germline cells were examined as well.The initial diagnosis were: 3 cases of Immune Thrombocytopenia (the third was thalassemia with secondary ITP after hematopoietic stem cell transplantation), 4 cases of Evans syndrome, 1 EB virus-related Hemophagocytic syndrome , 1 case of unexplained hemolytic anemia and neutropenia. The main abnormal genes found were as follows respectively: WAS, MYH9, GP1BA,NF1+CBL, ITGA2,VWF, ABCG8, ERCC4,RPS19+LRBA.In the end ,they were diagnosed sequentially as Wiskott-Aldrich syndrome, Epstein syndrome/Fechtner syndrome/ Hemorrhagic disease platelet type 6, Von Willebrand disease/ Hemorrhagic disease platelet type 3, Juvenile Myelomonocytic Leukemia, Glycoprotein Ia deficiency / Hemorrhagic disease platelet type 6, II type Von Willebrand disease, Sitosterolemia, Fanconi anemia complementation group Q, Diamond-Blackfan anemia/Common variant immunodeficiency disease type 8.With multi-factor comprehensive analysis including the effect of mutation, mode of inheritance and clinical characteristics, mutations can be verified as causative agents.If gene sequencing of only hematological system diseases can not explain, it is suggested to do the whole exon mutation analysis of single gene genetic disease or CNVseq, mtDNAseq if necessary. Complex genetic defects exist in some complicated immunological and hematological abnormalities DisclosuresNo relevant conflicts of interest to declare.

Morten Harboe (1929‐2021), one of the founders of Scandinavian Journal of Immunology

Morten Harboe (1929‐2021), one of the founders of Scandinavian Journal of Immunology

Exploring Passenger Loyalty and Related Factors for Urban Railways in Thailand

The research investigates the relationships among indicators related to the loyalty of urban railways passenger in Thailand at three routes, which consisted of BTS Sky train, MRT, and Airport Rail Link. The research instrument was 600 questionnaires, and the purpose was to study the indicators that affected perceived service quality and passenger loyalty by using structural equation modeling. The analysis of influence information that affects passenger loyalty revealed that trust, satisfaction, appreciation, cost of service changing, and relationships have an effect on passenger loyalty, statistically significant at 0.01. The satisfied variable was an important variable that affected passenger loyalty and was directly influenced by trust, appreciation, and perceived service quality. The perceived service quality was measured using 36 indicators and grouped into five complement groups, which were station, news, services, staffs, and vehicle, respectively. The research finding was that the cooperation concern with urban railway service can apply the result to the marketing development strategy to be a sustainable method of standardized service and urban railways system improvement.

Polymorphism of XRCC1 Arg399Gln may predict for development of end-stage renal disease. A PCR confirmed case-control study

ObjectivesEvaluation of the frequency of X-ray Cross Complementing group 1 (XRCC1) Arg399Gln polymorphism among patients with end-stage renal disease (ESRD) patients and to determine if a possible association was present. Subjects & methodsBlood samples were obtained from 70 ESRD patients and 30 healthy volunteers as control group for routine laboratory investigations and genomic DNA was extracted using GeneJET Whole Blood DNA Purification Kit to detect XRCC1 codon 399 genotype Arg/Gln in exon 10. BCNL digestion resulted in two fragments of 389 and 121 bp for wild-type homozygous (Arg/Arg); one fragment of 510 bp for variant homozygous (Gln/Gln); and three fragments of 510, 389 and 121 bp for variant heterozygous (Arg/Gln). ResultsCompared to control subjects, ESRD patients showed significantly higher frequency of Arg/Gln genotype, but had significantly lower frequency of Arg/Arg genotype. Moreover, patients had significantly lower frequency of Arg allele in comparison to controls (57.1% vs. 83.3%, respectively). Furthermore, the frequency of recessive models; Gln/Gln + Arg/Gln was significantly higher, while the frequency of dominant models; Arg/Arg + Arg/Gln was non-significantly lower among patients than controls. Development of ESRD was positively correlated with the presence of XRCC1 Arg/Gln genotype, while was negatively correlated with the presence of XRCC1 Arg/Arg genotype. ROC curve analysis showed that presence of XRCC1 Arg/Arg genotype is a negative sensitive predictor, while XRCC1 Arg/Gln genotype is a positive significant predictor for development of ESRD and Regression analysis defined presence of XRCC1 Arg/Arg genotype as significant negative predictor for development of ESRD. ConclusionThe results of the current study indicated an intimate relation between XRCC1 gene polymorphism and development of ESRD and the predictive value of its presence for the possibility of progression of chronic kidney disease to ESRD stage.

Association of DNA repair gene XPC Ala499Val (rs2228000 C>T) and Lys939Gln (rs2228001 A>C) polymorphisms with the risk of chronic myeloid leukemia: A case-control study in a South Indian population.

Xeroderma pigmentosum complementation group C (XPC), a DNA repair protein, plays an important role in the maintenance of genomic integrity and is essential for the nucleotide excision repair pathway. Polymorphisms in the XPC gene may alter DNA repair leading to genetic instability and oncogenesis. The present study aimed to assess the relationship between the XPC Ala499Val (rs2228000 C>T) and Lys939Gln (rs2228001 A>C) non-synonymous polymorphisms and susceptibility to chronic myeloid leukemia (CML) pathogenesis, disease progression and the response to targeted therapeutic regimen, imatinib mesylate. This case-control study included 212 cases and 212 controls, and the genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism assays. Our results showed significant association of variant CT (odds ratio = 1.92, 95% confidence interval = 1.21-3.06, p =0.003) and TT (odds ratio = 2.84, 95% confidence interval = 1.22-6.71, p =0.007) genotypes in patients with the XPC Ala499Val polymorphism and CML risk. In addition, these genotypes were associated with CML progression to advanced phases (p =0.006), splenomegaly (p =0.017) and abnormal lactate dehydrogenase levels (p =0.03). XPC Lys939Gln was found to correlate with a poor response to therapy, showing borderline significant association with minor cytogenetic response (p =0.08) and a poor molecular response (p =0.06). Significant association of the Ala499Val and Lys939Gln polymorphisms with prognosis was observed (Hasford high risk, p = 0.031 and p = 0.019, respectively). Haplotype analysis showed a strong correlation of variant TC haplotype with poor therapy responses (minor cytogenetic response, p = 0.019; poor molecular response, p < 0.0001). In conclusion, our results suggest that XPC Ala499Val is a high-penetrance CML susceptibility polymorphism. Both polymorphisms studied are considered as genetic markers with respect to assessing disease progression, therapy response and prognosis in CML patients.

Progressive Ataxia and Neurologic Regression in RFXANK-Associated Bare Lymphocyte Syndrome.

ObjectiveTo identify the genetic cause of a late-onset immunodeficiency and subacute progressive neurodegenerative disease affecting cognition, motor, visual, and cerebellar systems in a patient with a family history of 2 younger siblings with an early-onset immunodeficiency disease.MethodsPhysical examinations, immunologic, brain MRI, whole-exome sequencing, and segregation studies were used to identify the genetic and neuroimmunologic etiology of disease in this family.ResultsWe identified a homozygous loss-of-function (LOF) mutation (c.271+1G>C) in the RFXANK gene in the index patient and one of his younger affected siblings. Biallelic mutations in the RFXANK gene are known to cause bare lymphocyte syndrome (BLS) type II, complementation group B. The clinical and immunologic investigations were consistent with a clinical diagnosis of BLS type II. MRI demonstrated global cerebral and cerebellar atrophy with white matter signal changes in the index case.ConclusionsIn addition to BLS type II, our study has expanded and further characterized the phenotype associated with the LOF of RFXANK to include progressive neurodegenerative disease. Our study also provides evidence for the impact of LOF on brain development and function. Thus, early bone marrow transplantation, as a standard of care for BLS, could prove to be protective against the neurologic phenotypes in this group of patients.

Diagnostic significance of stromal changes in biopsies of prostate adenocarcinoma

The diagnostic value of stromal changes in carcinomas, including prostate, is under debate; in terms of limited sample tissue of biopsy, in addition to glandular alterations, the stromal changes could have additional diagnostic value, but the results in clinical settings are controversial. The research aims to evaluate the potential of stromal changes as a supplementary tool to predict the presence of higher grade carcinomas in the prostate using Masson's trichrome and Fanconi anemia complementation group M (FANCM) antibody stainings.385 biopsies and corresponding radical prostatectomy specimens were analyzed to evaluate the rates of the diversity of ISUP grades. Of 128 upgraded prostatectomy cases, 82 were diagnosed with ISUP Gleason Grade 1 (GG1) in a biopsy. All 82 cancerous samples were stained with Masson's trichrome and FACNM antibody and compared with 82 samples without cancer to see if there was a difference in stromal composition. Additionally, 50 GG1 samples without the upgrade were stained to demonstrate if stromal changes can predict less differentiated carcinomas in the prostate.In FANCM stained samples, the average percentage of positively staining stroma over the total in non-upgraded GG1 biopsies was 36 % (13–59 %, SD = 11); 34 % (9–58, SD = 13) in samples from the upgraded cancerous group, and 44 % (22–69, SD = 11) in samples without cancer.In Masson's trichrome stained samples, with collagen quantified, the percentage in non-upgraded GG1 biopsies was 41 % (20–78 %, SD = 11); 44 % (23–89, SD = 15) in samples from upgraded cancerous group and 37 % (15–57, SD = 9) in samples without cancer.In both FANCM and Masson's trichrome, no statistical significance was found between upgraded and non-upgraded groups (p = 0.84 and p = 0.5, respectively), although some upgrades from GG1 to GG4 showed extreme values.The statistical significance was found in cancerous vs. benign samples with both FANCM (p < 0.01) and Masson's trichrome (p = 0.012). The main limiting factor is a significant overlap in staining intensity between cancerous and cancer-free groups.

Clinical characteristics and outcome of IgG4-related disease with hypocomplementemia: a prospective cohort study

BackgroundImmunoglobulin G4-related disease (IgG4-RD) is a newly recognized systemic, immune-mediated, and fibro-inflammatory disease. Hypocomplementemia was found in part of IgG4-RD patients especially in the setting of active disease.ObjectivesThis study aimed to clarify the clinical features, treatment efficacy, and outcome in IgG4-RD patients with hypocomplementemia.Methods312 IgG4-RD patients were recruited in our prospective cohort conducted in Peking Union Medical College Hospital. Patients were divided into hypocomplementemia group and normal complement group according to serum C3 and C4 levels measured at baseline before treatment. Low serum C3 levels (< 0.73 g/L) and/or C4 levels (< 0.10 g/L) were defined as hypocomplementemia. Demographic data, clinical characteristics, laboratory parameters, treatment, and outcome of two groups were analyzed and compared.ResultsHypocomplementemia was identified in 65 (20.8%) cases of untreated IgG4-RD patients at baseline. The average age of hypocomplementemia group was 55.85 ± 10.89 years, with male predominance (72.3%). Compared with normal complement group, patients with hypocomplementemia were likely to have more involved organs, higher IgG4-RD responder index (IgG4-RD RI), and higher laboratory parameters such as counts of eosinophils, inflammatory markers, immunoglobulin G (IgG), IgG1, IgG3, IgG4, and IgE. In addition, lymph nodes, lacrimal gland, submandibular gland, parotid gland, paranasal sinus, bile ducts, and prostate gland were more commonly affected (p < 0.05). Serum C3 and C4 showed a significant positively correlation with each other. Both C3 and C4 were negatively correlated with the number of involved organs, IgG, IgG3, IgG4, and IgG4-RD RI, as well as positively correlated with IgA and hypersensitive C reactive protein (hsCRP). 64 (98.5%) patients responded quickly to initial therapy at a 3-month follow-up. Fifteen (23.1%) patients relapsed during follow-up with mean recurrence time of 14.2 ± 13.8 months. Compared with normal complement group, there was no significant difference of relapse rate in two groups (P = 0.401).ConclusionsClinical characteristics of IgG4-related disease with hypocomplementemia differ from normal complement group. Serum C3 and C4 at baseline before treatment could be biological markers for disease activity. IgG4-RD with hypocomplementemia responded well to treatment and had no significant difference of relapse rate in IgG4-RD with normal complement.

Bordifications of hyperplane arrangements and their curve complexes

The complement of an arrangement of hyperplanes in $\mathbb C^n$ has a natural bordification to a manifold with corners formed by removing (or "blowing up") tubular neighborhoods of the hyperplanes and certain of their intersections. When the arrangement is the complexification of a real simplicial arrangement, the bordification closely resembles Harvey's bordification of moduli space. We prove that the faces of the universal cover of the bordification are parameterized by the simplices of a simplicial complex $\mathcal{C}$, the vertices of which are the irreducible "parabolic subgroups" of the fundamental group of the arrangement complement. So, the complex $\mathcal{C}$ plays a similar role for an arrangement complement as the curve complex does for moduli space. Also, in analogy with curve complexes and with spherical buildings, we prove that $\mathcal{C}$ has the homotopy type of a wedge of spheres.

Exploiting synthetic lethality to target BRCA1/2-deficient tumors: where we stand.

The principle of synthetic lethality, which refers to the loss of viability resulting from the disruption of two genes, which, individually, do not cause lethality, has become an attractive target approach due to the development and clinical success of Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this review, we present the most recent findings on the use of PARPi in the clinic, which are currently approved for second-line therapy for advanced ovarian and breast cancer associated with mutations of BRCA1 or BRCA2 (BRCA1/2) genes. PARPi efficacy, however, appears to be limited by acquired and inherent resistance, highlighting the need for alternative and synergistic targets to eliminate these tumors. Here, we explore other identified synthetic lethal interactors of BRCA1/2, including DNA polymerase theta (POLQ), Fanconi anemia complementation group D2 (FANDC2), radiation sensitive 52 (RAD52), Flap structure-specific endonuclease 1 (FEN1), and apurinic/apyrimidinic endodeoxyribonuclease 2 (APE2), as well as other protein and nonprotein targets, for BRCA1/2-mutated cancers and their implications for future therapies. A wealth of information now exists for phenotypic and functional characterization of these novel synthetic lethal interactors of BRCA1/2, and leveraging these findings can pave the way for the development of new targeted therapies for patients suffering from these cancers.

In Silico Drug Repurposing by Structural Alteration after Induced Fit: Discovery of a Candidate Agent for Recovery of Nucleotide Excision Repair in Xeroderma Pigmentosum Group D Mutant (R683W).

Xeroderma pigmentosum complementation group D (XPD) is a UV-sensitive syndrome and a rare incurable genetic disease which is caused by the genetic mutation of the excision repair cross-complementation group 2 gene (ERCC2). Patients who harbor only XPD R683W mutant protein develop severe photosensitivity and progressive neurological symptoms. Cultured cells derived from patients with XPD (XPD R683W cells) demonstrate a reduced nucleotide excision repair (NER) ability. We hope to ameliorate clinical symptoms if we can identify candidate agents that would aid recovery of the cells’ NER ability. To investigate such candidates, we created in silico methods of drug repurposing (in silico DR), a strategy that utilizes the recovery of ATP-binding in the XPD R683W protein after the induced fit. We chose 4E1RCat and aprepitant as the candidates for our in silico DR, and evaluated them by using the UV-induced unscheduled DNA synthesis (UDS) assay to verify the recovery of NER in XPD R683W cells. UDS values of the cells improved about 1.4–1.7 times after 4E1RCat treatment compared with solvent-only controls; aprepitant showed no positive effect. In this study, therefore, we succeeded in finding the candidate agent 4E1RCat for XPD R683W. We also demonstrated that our in silico DR method is a cost-effective approach for drug candidate discovery.

Nitric oxide synthase-2 (NOS2) gene polymorphism c.1832C>T (Ser608Leu) associated with nitrosative stress in Fanconi anaemia.

Fanconi anemia (FA) occurs due to genomic instability with predisposition to bone marrow failure, phenotypic abnormalities and cancers. Though mutations in 22 genes leading to DNA repair defect have been identified, the cellular factor such as oxidative stress has also shown to be associated with FA. Nitrosative Stress (NS) is biochemically correlated to many oxidative stress related disorders and the NS as a pathological hallmark in FA has been so far overlooked. We carried out the study first time in Indian patients with FA with an objective to understand the role of NS in the pathogenesis of FA. The study was carried out in 70 FA subjects. The FA subjects were diagnosed by chromosomal breakage analysis. Molecular study was carried out by Next Generation Sequencing and Sanger sequencing. The 3-nitrotyrosine [3-NT] levels were estimated through enzyme-linked immuno-sorbent assay (ELISA) and the nitric oxide synthase genes- NOS1 (c.-420-34221G>A (rs1879417), c.-420-10205C>T (rs499776), c.4286+720G>C (rs81631)) and NOS2 (c.1823C>T (p. Ser608Leu) (rs2297518)) polymorphism were studied by direct sequencing. Chromosomal breakage analysis revealed a high frequency of chromosomal breaks (Mean chromosomal breakage-4.13 ± 1.5 breaks/metaphase) in 70 FA patients as compared to the control. Molecular studies revealed FANCA (58.34%), FANCG (18.34%) and FANCL (16.6%) complementation groups. The 3-nitrotyrosine [3-NT] levels showed to be significantly (p < 0.05) elevated in FA subjects when compared to the age match controls. Genotyping of the NOS2 gene c.1823C>T (p. Ser608Leu) (rs2297518), showed statistically significant (P < 0.05) association with FA. Elevated level of 3-NT is one of the cause of NS and NOS2 gene polymorphism associated with FA is an important target in the treatment regimen.

The Association of Single-Nucleotide Polymorphism rs13181 in ERCC2 with Risk and Prognosis of Nasopharyngeal Carcinoma in an Endemic Chinese Population.

ObjectiveWe examined whether the single-nucleotide polymorphism (SNP) rs13181 in the gene encoding excision repair cross complementation group 2 (ERCC2) is associated with the risk and prognosis of nasopharyngeal carcinoma (NPC).MethodsSNPs at rs13181 were genotyped in 439 NPC patients (NPC group) and 431 age- and gender-matched cancer-free controls (control group) from a region of China where NPC is endemic, and frequencies of GG, GT and TT genotypes were compared between the two groups in the case–control study. In a subset of 365 NPC cases, SNPs were examined for potential correlation with tumor-free survival time (TFS) and overall survival (OS).ResultsRelative to NPC risk with a TT genotype, NPC risk was similar with GT + GG genotypes (OR 1.052, 95% CI 0.656–1.688), after adjusting for gender, age, smoking history, and immunoglobin A against Epstein-Barr virus capsid antigen (EBV-VCA-IgA) status. Univariate analysis showed that the GG or GT genotype was associated with significantly worse TFS (p<0.001) and OS (p=0.010) than the TT genotype. Prognosis was significantly worse for men than for women (TFS, p=0.045; OS, p=0.031), for T3–T4 classification than for T1–T2 (TFS, p=0.009; OS, p=0.007), for N3 than for N0+N1+N2 (TFS, p<0.001; OS, p<0.001). Based on multivariate analysis, independent risk factors for poor TFS were GG or GT genotype (HR 2.629, 95% CI 1.625–4.254, p<0.001), T3–T4 classification (HR 2.146, 95% CI 1.244–3.701, p=0.006) and N3 (HR 2.527, 95% CI 1.574–4.059, p<0.001). GG or GT genotype (HR 2.217, 95% CI 1.283–3.832, p=0.004), gender (HR 1.989, 95% CI 1.046–3.785, p=0.036), T3–T4 (HR 2.431, 95% CI 1.306–4.526, p=0.005) and N3 (HR 2.693, 95% CI 1.637–4.432, p<0.001) were independent risk factors for poor OS.ConclusionThe rs13181 SNP in ERCC2 does not appear to be associated with NPC risk, but it may serve as an independent prognostic factor for NPC recurrence and death.

KERA: analysis tool for multi-process, multi-state single-molecule data.

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA’s utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.