Combination of Physicochemical Tropism and Affinity Moiety Targeting of Lipid Nanoparticles Enhances Organ Targeting.

Two camps have emerged for targeting nanoparticles to specific organs and cell types: affinity moiety targeting and physicochemical tropism. Here we directly compare and combine both using intravenous (IV) lipid nanoparticles (LNPs) designed to target the lungs. We utilized PECAM antibodies as affinity moieties and cationic lipids for physicochemical tropism. These methods yield nearly identical lung uptake, but aPECAM LNPs show higher endothelial specificity. LNPs combining these targeting methods had >2-fold higher lung uptake than either method alone and markedly enhanced epithelial uptake. To determine if lung uptake is because the lungs are the first organ downstream of IV injection, we compared IV vs intra-arterial (IA) injection into the carotid artery, finding that IA combined-targeting LNPs achieve 35% of the injected dose per gram (%ID/g) in the first-pass organ, the brain, among the highest reported. Thus, combining the affinity moiety and physicochemical strategies provides benefits that neither targeting method achieves alone.

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

G-quadruplex landscape and its regulation revealed by a new antibody capture method.

Our understanding of DNA G-quadruplexes (G4s) from in vitro studies has been complemented by genome-wide G4 landscapes from cultured cells. Conventionally, the formation of G4s is accepted to depend on G-repeats such that they form tetrads. However, genome-wide G4s characterized through high-throughput sequencing suggest that these structures form at a large number of regions with no such canonical G4-forming signatures. Many G4-binding proteins have been described with no evidence for any protein that binds to and stabilizes G4s. It remains unknown what fraction of G4s formed in human cells are protein-bound. The G4-chromatin immunoprecipitation (G4-ChIP) method hitherto employed to describe G4 landscapes preferentially reports G4s that get crosslinked to proteins in their proximity. Our current understanding of the G4 landscape is biased against representation of G4s which escape crosslinking as they are not stabilized by protein-binding and presumably transient. We report a protocol that captures G4s from the cells efficiently without any bias as well as eliminates the detection of G4s formed artifactually on crosslinked sheared chromatin post-fixation. We discover that G4s form sparingly at SINEs. An application of this method shows that depletion of a repeat-binding protein CGGBP1 enhances net G4 capture at CGGBP1-dependent CTCF-binding sites and regions of sharp interstrand G/C-skew transitions. Thus, we present an improved method for G4 landscape determination and by applying it we show that sequence property-specific constraints of the nuclear environment mitigate G4 formation.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up or down-regulate any protein of interest. LNPs have mostly been targeted to specific cell types or organs by physicochemical targeting in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. Here lung-tropic LNPs are examined, whose organ tropism derives from containing either a cationic or ionizable lipid conferring a positive zeta potential. Surprisingly, these LNPs are found to induce massive thrombosis. Such thrombosis is shown in the lungs and other organs, and it is shown that it is greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles, and even by lung-tropic ionizable lipids that do not have a permanent cationic charge. The mechanism depends on the LNPs binding to and then changing the conformation of fibrinogen, which then activates platelets and thrombin. Based on these mechanisms, multiple solutions are engineered that enable positively charged LNPs to target the lungs while ameliorating thrombosis. The findings illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

PSIP1/LEDGF reduces R-loops at transcription sites to maintain genome integrity.

R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up- or down-regulate any protein of interest. LNPs have been targeted to specific cell types or organs by physicochemical targeting, in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. In a popular approach, physicochemical targeting is accomplished by formulating with charged lipids. Negatively charged lipids localize LNPs to the spleen, and positively charged lipids to the lungs. Here we found that lung-tropic LNPs employing cationic lipids induce massive thrombosis. We demonstrate that thrombosis is induced in the lungs and other organs, and greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles. The mechanism depends on the LNPs binding to fibrinogen and inducing platelet and thrombin activation. Based on these mechanisms, we engineered multiple solutions which enable positively charged LNPs to target the lungs while not inducing thrombosis. Our findings implicate thrombosis as a major barrier that blood erects against LNPs with cationic components and illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

H4K16ac activates the transcription of transposable elements and contributes to their cis-regulatory function.

Mammalian genomes harbor abundant transposable elements (TEs) and their remnants, with numerous epigenetic repression mechanisms enacted to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here, we demonstrate that the male-specific lethal (MSL)-complex-mediated histone H4 acetylation at lysine 16 (H4K16ac) is enriched at TEs in human embryonic stem cells (hESCs) and cancer cells. This in turn activates transcription of subsets of full-length long interspersed nuclear elements (LINE1s, L1s) and endogenous retrovirus (ERV) long terminal repeats (LTRs). Furthermore, we show that the H4K16ac-marked L1 and LTR subfamilies display enhancer-like functions and are enriched in genomic locations with chromatin features associated with active enhancers. Importantly, such regions often reside at boundaries of topologically associated domains and loop with genes. CRISPR-based epigenetic perturbation and genetic deletion of L1s reveal that H4K16ac-marked L1s and LTRs regulate the expression of genes in cis. Overall, TEs enriched with H4K16ac contribute to the cis-regulatory landscape at specific genomic locations by maintaining an active chromatin landscape at TEs.

Meta-analysis of material properties influencing nanoparticle plasma pharmacokinetics.

Thorough characterization of the plasma pharmacokinetics (PK) is a critical step in clinical development of novel therapeutics and is routinely performed for small molecules and biologics. However, there is a paucity of even basic characterization of PK for nanoparticle-based drug delivery systems. This has led to untested generalizations about how nanoparticle properties govern PK. Here, we present a meta-analysis of 100 nanoparticle formulations following IV administration in mice to identify any correlations between four PK parameters derived by non-compartmental analysis (NCA) and four cardinal properties of nanoparticles: PEGylation, zeta potential, size, and material. There was a statistically significant difference between the PK of particles stratified by nanoparticle properties. However, single linear regression between these properties and PK parameters showed poor predictability (r2 < 0.10 for all analyses), while multivariate regressions showed improved predictability (r2 > 0.38, except for t1/2). This suggests that no single nanoparticle property alone is even moderately predictive of PK, while the combination of multiple nanoparticle features does provide moderate predictive power. Improved reporting of nanoparticle properties will enable more accurate comparison between nanoformulations and will enhance our ability to predict in vivo behavior and design optimal nanoparticles.

Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone-marrow-failure disorder characterized by a triad of mucocutaneous features that include abnormal skin pigmentation, nail dystrophy, and oral leucoplakia. Despite the identification of several genetic variants that cause DC, a significant proportion of probands remain without a molecular diagnosis. In a cohort of eight independent DC-affected families, we have identified a remarkable series of heterozygous germline variants in the gene encoding thymidylate synthase (TYMS). Although the inheritance appeared to be autosomal recessive, one parent in each family had a wild-type TYMS coding sequence. Targeted genomic sequencing identified a specific haplotype and rare variants in the naturally occurring TYMS antisense regulator ENOSF1 (enolase super family 1) inherited from the other parent. Lymphoblastoid cells from affected probands have severe TYMS deficiency, altered cellular deoxyribonucleotide triphosphate pools, and hypersensitivity to the TYMS-specific inhibitor 5-fluorouracil. These defects in the nucleotide metabolism pathway resulted in genotoxic stress, defective transcription, and abnormal telomere maintenance. Gene-rescue studies in cells from affected probands revealed that post-transcriptional epistatic silencing of TYMS is occurring via elevated ENOSF1. These cell and molecular abnormalities generated by the combination of germline digenic variants at the TYMS-ENOSF1 locus represent a unique pathogenetic pathway for DC causation in these affected individuals, whereas the parents who are carriers of either of these variants in a singular fashion remain unaffected.

Synthesis of cyclic α-pinane carbonate - a potential monomer for bio-based polymers.

This work reports the first known synthesis of α-pinane carbonate from an α-pinene derivative. Pinane carbonate is potentially useful as a monomer for poly(pinane carbonate), which would be a sustainable bio-based polymer. α-Pinene is a major waste product from the pulp and paper industries and the most naturally abundant monoterpene in turpentine oil. α-Pinene is routinely converted to pinene oxide and pinanediol, but no study has yet demonstrated the conversion of pinanediol into α-pinane carbonate. Here, α-pinane carbonate was synthesised via carboxylation of α-pinanediol with dimethyl carbonate under base catalysis using triazabicyclodecene guanidine (TBD). 81.1 ± 2.8% α-pinane carbonate yield was achieved at 98.7% purity. The produced α-pinane carbonate was a white crystalline solid with a melting point of 86 °C. It was characterised using FTIR, NMR, GCMS and a quadrupole time-of-flight (QTOF) mass spectrometer. The FTIR exhibited a C[double bond, length as m-dash]O peak at 1794 cm-1 confirming the presence of a cyclic carbonate. GCMS showed that the α-pinane carbonate fragments with loss of CO2, forming pinene epoxide. Base hydrolysis of the α-pinane carbonate using NaOH/ethanol/water regenerated the pinanediol with formations of Na2CO3.

Chimeric chromosome landscapes of human somatic cell cultures show dependence on stress and regulation of genomic repeats by CGGBP1.

Genomes of somatic cells in culture are prone to spontaneous mutations due to errors in replication and DNA repair. Some of these errors, such as chromosomal fusions, are not rectifiable and subject to selection or elimination in growing cultures. Somatic cell cultures are thus expected to generate background levels of potentially stable chromosomal chimeras. A description of the landscape of such spontaneously generated chromosomal chimeras in cultured cells will help understand the factors affecting somatic mosaicism. Here we show that short homology-associated non-homologous chromosomal chimeras occur in normal human fibroblasts and HEK293T cells at genomic repeats. The occurrence of chromosomal chimeras is enhanced by heat stress and depletion of a repeat regulatory protein CGGBP1. We also present evidence of homologous chromosomal chimeras between allelic copies in repeat-rich DNA obtained by methylcytosine immunoprecipitation. The formation of homologous chromosomal chimeras at Alu and L1 repeats increases upon depletion of CGGBP1. Our data are derived from de novo sequencing from three different cell lines under different experimental conditions and our chromosomal chimera detection pipeline is applicable to long as well as short read sequencing platforms. These findings present significant information about the generation, sensitivity and regulation of somatic mosaicism in human cell cultures.

CGGBP1-dependent CTCF-binding sites restrict ectopic transcription.

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from the SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones that show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in the synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand-specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription barrier seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. RNA-sequencing reveals that CGGBP1-regulated CTCF-binding sites act as barriers to transcription at multiple locations genome-wide. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1.

BACKGROUND: Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins. METHODS: We have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen. RESULTS: Our primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding. CONCLUSIONS: The assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

CGGBP1-regulated cytosine methylation at CTCF-binding motifs resists stochasticity.

BACKGROUND: The human CGGBP1 binds to GC-rich regions and interspersed repeats, maintains homeostasis of stochastic cytosine methylation and determines DNA-binding of CTCF. Interdependence between regulation of cytosine methylation and CTCF occupancy by CGGBP1 remains unknown. RESULTS: By analyzing methylated DNA-sequencing data obtained from CGGBP1-depleted cells, we report that some transcription factor-binding sites, including CTCF, resist stochastic changes in cytosine methylation. By analysing CTCF-binding sites we show that cytosine methylation changes at CTCF motifs caused by CGGBP1 depletion resist stochastic changes. These CTCF-binding sites are positioned at locations where the spread of cytosine methylation in cis depends on the levels of CGGBP1. CONCLUSION: Our findings suggest that CTCF occupancy and functions are determined by CGGBP1-regulated cytosine methylation patterns.

A Qualitative Analysis of Malpractice Litigation in Cardiology Using Case Summaries Through a National Legal Database Analysis.

Introduction Physicians are increasingly practicing defensive medicine as a response to society's litigious climate. This study sought to characterize cardiology malpractice claims and elucidate the allegations underlying the use of defensive medicine. Methods The WestlawNext™ database was queried to obtain state and federal jury verdicts and settlements related to medical malpractice and cardiology that occurred in the United States between 2010 and 2015. Cardiology cases were identified using the search terms "medical malpractice" and "cardiology" and reviewed by two individuals utilizing available case documents. Duplicate and nonpertinent cases were excluded. Binary logistic regression models were created to predict the likelihood of defendant verdict, plaintiff verdict, and settlement based on the various reasons for litigation cited. Results Inclusion criteria were met in 166 cases. The plaintiffs were predominantly male (94 cases; 56.6%), and the average patient age was 53.3±17.5 years. More than half of the cases involved a cardiologist as a defendant. The most common reasons for litigation were: failure to treat (129; 77.7%), failure to diagnose (115; 69.3%), failure to refer/order diagnostic tests (107; 64.5%), and patient death (118; 71.1%). Among cases tried for failure to diagnose, the most commonly missed diagnosis was myocardial infarction. Cases most commonly resulted in a defendant verdict (94; 56.6%). However, odds of a plaintiff verdict were significantly higher when failure to diagnose was alleged with an odds ratio (OR) of 7.60 (95% confidence interval 1.14 - 50.87, p = 0.0365). Conclusions Failure to diagnose remains a commonly alleged base for litigation. In conclusion, our analysis suggests increased training for non-cardiologists in the recognition of the acute coronary syndrome and enhanced awareness of inherent biases among all physicians may facilitate reducing missed diagnoses.

CGGBP1 regulates CTCF occupancy at repeats.

BACKGROUND: CGGBP1 is a repeat-binding protein with diverse functions in the regulation of gene expression, cytosine methylation, repeat silencing and genomic integrity. CGGBP1 has also been identified as a cooperator of histone-modifying enzymes and as a component of CTCF-containing complexes that regulate the enhancer-promoter looping. CGGBP1-CTCF cross talk in chromatin regulation has been hitherto unknown. RESULTS: Here, we report that the occupancy of CTCF at repeats depends on CGGBP1. Using ChIP-sequencing for CTCF, we describe its occupancy at repetitive DNA. Our results show that endogenous level of CGGBP1 ensures CTCF occupancy preferentially on repeats over canonical CTCF motifs. By combining CTCF ChIP-sequencing results with ChIP sequencing for three different kinds of histone modifications (H3K4me3, H3K9me3 and H3K27me3), we show that the CGGBP1-dependent repeat-rich CTCF-binding sites regulate histone marks in flanking regions. CONCLUSION: CGGBP1 affects the pattern of CTCF occupancy. Our results posit CGGBP1 as a regulator of CTCF and its binding sites in interspersed repeats.

A Readability Analysis of Online Cardiovascular Disease-Related Health Education Materials.

BACKGROUND: Online cardiovascular health materials are easily accessible with an Internet connection, but the readability of its content may limit practical use by patients. OBJECTIVE: The goal of our study was to assess the readability of the most commonly searched Internet health education materials for cardiovascular diseases accessed via Google. METHODS: We selected 20 commonly searched cardiovascular disease terms: aneurysm, angina, atherosclerosis, cardiomyopathy, congenital heart disease, coronary artery disease, deep vein thrombosis, heart attack, heart failure, high blood pressure, pericardial disease, peripheral arterial disease, rheumatic heart disease, stroke, sudden death, valvular heart disease, mini-stroke, lower extremity edema, pulmonary embolism, and exertional dyspnea. Terms were selected on Google and selected up to 10 results in order of presentation in the search results by reviewing a maximum of 15 pages of Google search results specifically providing education toward patients to yield 196 total patient education articles. KEY RESULTS: All readability measures assessing grade level measures found the 196 articles were written at a mean 10.9 (SD = 1.8) grade reading level. Moreover, 99.5% of the articles were written beyond the 5th- to 6th-grade level recommended by the American Medical Association. CONCLUSIONS: Given the prominent use of online patient education material, we consider readability as a quality metric that should be evaluated prior to online publication of any health education materials. Further study of how to improve the readability of online materials may enhance patient education, engagement, and health outcomes.

Distinct DNA Sequence Preference for Histone Occupancy in Primary and Transformed Cells.

Genome-wide occupancy of several histone modifications in various cell types has been studied using chromatin immunoprecipitation (ChIP) sequencing. Histone occupancy depends on DNA sequence features like inter-strand symmetry of base composition and periodic occurrence of TT/AT. However, whether DNA sequence motifs act as an additional effector of histone occupancy is not known. We have analyzed the presence of DNA sequence motifs in publicly available ChIP-sequence datasets for different histone modifications. Our results show that DNA sequence motifs are associated with histone occupancy, some of which are different between primary and transformed cells. The motifs for primary and transformed cells showed different levels of GC-richness and proximity to transcription start sites (TSSs). The TSSs associated with transformed or primary cell-specific motifs showed different levels of TSS flank transcription in primary and transformed cells. Interestingly, TSSs with a motif-linked occupancy of H2AFZ, a component of positioned nucleosomes, showed a distinct pattern of RNA Polymerase II (POLR2A) occupancy and TSS flank transcription in primary and transformed cells. These results indicate that DNA sequence features dictate differential histone occupancy in primary and transformed cells, and the DNA sequence motifs affect transcription through regulation of histone occupancy.

Role of de novo DQ donor-specific antibody in antibody-mediated rejection in renal transplant recipient: A case study.

The human leukocyte antigen (HLA) matching plays an important role in determining the clinical outcome of renal transplantation. The development of donor specific antibodies (DSA) against HLA is associated with antibody mediated allograft tissue injury, poor outcome and rejection. The DQ-DSA develops in a denovo pattern and its unfavorable impact on renal transplantation has not yet been widely reported. We investigated the clinical significance of DQ-DSA in a patient diagnosed with hypertension, CKD stage V on maintenance hemodialysis (MHD) for second renal transplant. The histocompatibility workup before the first transplant included low resolution HLA-A, B, DR typing of both patient and donor. HLA type of the patient was HLA-A*29, 68, HLAB*44, 44, DRB1*07, 11. HLA type of the donor was HLA-A*03, 68, HLA-B*39, 44, DRB1*07, 10 with a 3/6 match. The HLA antibody screen and complement dependent cytotoxicity crossmatch (CDC) were found to be negative. No therapeutic plasma exchanges (TPE) were done during stay and post-transplant the patient was on triple immunosuppressant therapy. After four years the patient was diagnosed with recurrent membranoproliferative glomerulonephritis and second renal transplant was planned, therefore, histocompatibility workup was initiated. HLA antibody screen was found to be positive for HLA class II. Initially only HLA-A, B, DR typing was performed and that too only low resolution, further, high resolution HLA typing was done for HLA-DR and DQ to rule out if these antibodies are de-novo DQ/DR DSA. We analyzed that the patient had developed de-novo DSA against HLA-DRB1* 10:01 (DR10), MFI-2374 and DQB1*06:01 (DQ6), MFI-15315. This study suggests the role of DQ antibodies in determining the graft survival and to highlight the need of HLA DQ typing as a routine of the diagnostic work-up in a solid organ transplant.

Dynamic bimodal changes in CpG and non-CpG methylation genome-wide upon CGGBP1 loss-of-function.

OBJECTIVES: Although CpG methylation is well studied, mechanisms of non-CpG methylation in mammals remains elusive. Studying proteins with non-CpG cytosine methylation-sensitive DNA-binding, such as human CGGBP1, can unveil cytosine methylation regulatory mechanisms. Here we have resequenced a published genome-wide bisulfite sequencing library and analyzed it at base level resolution. CpG, CHG and CHH (where H is any nucleotide other than G) methylation states in non-targeting or CGGBP1-targeting shmiR lentivirus-transduced cells have been analyzed to identify how CGGBP1 regulates CpG and non-CpG methylation. RESULTS: We report that CGGBP1 acts as a dynamic bimodal balancer of methylation. Both gain and loss of methylation observed upon CGGBP1 depletion were spatially overlapping at annotated functional regions and not identifiable with any sequence motifs but clearly associated with GC-skew. CGGBP1 depletion caused clustered methylation changes in cis, upstream of R-loop forming promoters. This was complemented by clustered occurrences of methylation changes in proximity of transcription start sites of known cytosine methylation regulatory genes, altered expression of which can regulate cytosine methylation in trans. Despite low coverage, our data provide reliable estimates of the spectrum of methylation changes regulated by CGGBP1 in all cytosine contexts genome-wide through a combination of cis and trans-acting mechanisms.