Regulatory challenges and investigational device exemption protocols for fenestrated and branched EVAR in the United States.

The US Food and Drug Administration (FDA) was created with the intent to ensure the safety of consumers in the United States. During the 1970s, this mandate was expanded to include medical devices. Aortic endografts, implanted since the 1990s, fall under this purview. Industry-sponsored and physician-sponsored investigation of the efficacy and safety of these devices is critical to the ongoing development of interventions for complex aortic anatomy. This research, in accordance with federal rules and regulation, must be conducted in coordination and with the approval of the FDA. The FDA has designed a process for which manufacturers or investigators may conduct efficacy and safety research using new or modified devices with oversight by the federal government. To conduct this type of research, an investigational device exemption must be obtained from the FDA. An investigational device exemption, although useful from a regulatory perspective, places a large time and financial responsibility on the investigator. As it stands now, the regulatory environment limits research into fenestrated and branched endovascular aortic repair to only those physicians and manufacturers able to provide the significant number of resources required to complete the laborious pre-application, application, and ongoing reports associated with obtaining and maintaining an investigational device exemption.

Genome-wide promoter assembly in E. coli measured at single-base resolution.

When detected at single-base-pair resolution, the genome-wide location, occupancy level, and structural organization of DNA-binding proteins provide mechanistic insights into genome regulation. Here we use ChIP-exo to provide a near-base-pair resolution view of the epigenomic organization of the Escherichia coli transcription machinery and nucleoid structural proteins at the time when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We examined the site specificity of three sigma factors (RpoD/σ70, RpoH/σ32, and RpoN/σ54), RNA polymerase (RNAP or RpoA, -B, -C), and two nucleoid proteins (Fis and IHF). We suggest that DNA shape at the flanks of cognate motifs helps drive site specificity. We find that although RNAP and sigma factors occupy active cognate promoters, RpoH and RpoN can occupy quiescent promoters without the presence of RNAP. Thus, promoter-bound sigma factors can be triggered to recruit RNAP by a mechanism that is distinct from an obligatory cycle of free sigma binding RNAP followed by promoter binding. These findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape, and further define mechanistic steps in regulated genome-wide assembly of RNAP at promoters in E. coli.

Transcarotid Arterial Revascularization Adoption Should not Be Hindered by a Concern for a Long Learning Curve.

BACKGROUND: Transcarotid arterial revascularization (TCAR) offers a novel technique for carotid artery stenting (CAS) that provides flow reversal in the carotid artery and avoids aortic arch manipulation, thus, potentially lowering ipsilateral and contralateral periprocedural stroke rates. As a new technology, adoption may be limited by concern for learning a new technique. This study seeks to examine the number of cases needed for a surgeon to reach technical proficiency. METHODS: Retrospective analysis was performed using a prospectively collected database of all TCAR procedures performed in a tertiary health care system between 2016 and 2020. Patient demographics and anatomic characteristics were collected. Intraoperative variables and perioperative outcomes were examined. These variables were collated into groups for the first 4 procedures, procedures 5-8, and after 8. Independent Samples t test, 1-way ANOVA, and logarithmic regression were used to statistically analyze the data. RESULTS: One-hundred and eighty-seven TCARs were performed by 14 surgeons. One hundred and twenty-two (65%) were male, 59 (32%) were older than 75 years, and 83 (44%) were symptomatic. The most common indications were high-lesions in 87 patients (47%) and recurrent stenosis after CEA in 37 patients (20%). Significant differences were found between the first and second groups of 4 cases when comparing mean operative time (71 vs. 58 min; P = 0.001) and flow reversal time (10.8 vs. 7.9 min; P= 0.004). similar significant differences were found between the first and third groups of 4 cases but not between the second and third groups. There was a reduction in contrast usage and fluoroscopy time after the first 4 cases, however, this did not reach statistical significance. There was no ipsilateral perioperative strokes. One patient had a contralateral stroke on postoperative day 2 due to intracranial atherosclerosis, and there was one perioperative mortality that occurred on postoperative day 3 after discharge. CONCLUSIONS: Procedural and flow reversal times significantly shorten after 4 TCAR procedures are performed. Other metrics, such as fluoroscopy time and contrast usage, are also decreased. Complications, in general, are minimal. Proficiency in TCAR, as measured by these metrics, is met after performing only 4 procedures.

RAD52: Paradigm of Synthetic Lethality and New Developments.

DNA double-strand breaks and inter-strand cross-links are the most harmful types of DNA damage that cause genomic instability that lead to cancer development. The highest fidelity pathway for repairing damaged double-stranded DNA is termed Homologous recombination (HR). Rad52 is one of the key HR proteins in eukaryotes. Although it is critical for most DNA repair and recombination events in yeast, knockouts of mammalian RAD52 lack any discernable phenotypes. As a consequence, mammalian RAD52 has been long overlooked. That is changing now, as recent work has shown RAD52 to be critical for backup DNA repair pathways in HR-deficient cancer cells. Novel findings have shed light on RAD52's biochemical activities. RAD52 promotes DNA pairing (D-loop formation), single-strand DNA and DNA:RNA annealing, and inverse strand exchange. These activities contribute to its multiple roles in DNA damage repair including HR, single-strand annealing, break-induced replication, and RNA-mediated repair of DNA. The contributions of RAD52 that are essential to the viability of HR-deficient cancer cells are currently under investigation. These new findings make RAD52 an attractive target for the development of anti-cancer therapies against BRCA-deficient cancers.

A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.

A high-resolution protein architecture of the budding yeast genome.

The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.

Systematic review and meta-analysis of endovascular interventions for Stanford type A aortic dissection.

OBJECTIVE: The standard surgical approach to Stanford type A aortic dissection is open repair. However, up to one in four patients will be declined surgery because of prohibitive risk. Patients who are treated nonoperatively have an unacceptably high mortality. Endovascular repair of the ascending aorta is emerging as an alternative treatment for a select group of patients. The reported rates of technical success, mortality, stroke, and reintervention have varied. The objective of the study was to systematically report outcomes for acute type A dissections repaired using an endovascular approach. METHODS: The systematic review and meta-analysis was conducted in accordance with the PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines. We performed online literature database searches through April 2020. The demographic and procedural characteristics of the individual studies were tabulated. Data on technical success, short-term mortality, stroke, and reintervention were extracted and underwent meta-analysis using a random effects model. RESULTS: Fourteen studies with 80 cases of aortic dissection (55 acute and 25 subacute) were included in the final analysis. A wide variation was found in technique and device design across the studies. The outcomes rates were estimated at 17% (95% confidence interval [CI], 10%-26%) for mortality, 15% (95% CI, 8%-23%) for technical failure, 11% (95% CI, 6%-19%) for stroke and 18% (95% CI, 9%-31%) for reintervention. The mean Downs and Black quality assessment score was 13.9 ± 3.2. CONCLUSIONS: The technique for endovascular repair of type A aortic dissection is feasible and reproducible. The results of our meta-analysis demonstrate an acceptable safety profile for inoperable patients who otherwise would have an extremely poor prognosis. Data from clinical trials are required before the technique can be introduced into routine clinical practice.

Acute stress drives global repression through two independent RNA polymerase II stalling events in Saccharomyces.

In multicellular eukaryotes, RNA polymerase (Pol) II pauses transcription ~30-50 bp after initiation. While the budding yeast Saccharomyces has its transcription mechanisms mostly conserved with other eukaryotes, it appears to lack this fundamental promoter-proximal pausing. However, we now report that nearly all yeast genes, including constitutive and inducible genes, manifest two distinct transcriptional stall sites that are brought on by acute environmental signaling (e.g., peroxide stress). Pol II first stalls at the pre-initiation stage before promoter clearance, but after DNA melting and factor acquisition, and may involve inhibited dephosphorylation. The second stall occurs at the +2 nucleosome. It acquires most, but not all, elongation factor interactions. Its regulation may include Bur1/Spt4/5. Our results suggest that a double Pol II stall is a mechanism to downregulate essentially all genes in concert.

Alignment and quantification of ChIP-exo crosslinking patterns reveal the spatial organization of protein-DNA complexes.

The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. Applying our approach to collections of ChIP-exo data, we demonstrate that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to analyze Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.

Simplified ChIP-exo assays.

ChIP-seq and ChIP-exo identify where proteins bind along any genome in vivo. Although ChIP-seq is widely adopted in academic research, it has inherently high noise. In contrast, ChIP-exo has relatively low noise and achieves near-base pair resolution. Consequently, and unlike other genomic assays, ChIP-exo provides structural information on genome-wide binding proteins. Construction of ChIP-exo libraries is technically difficult. Here we describe greatly simplified ChIP-exo methods, each with use-specific advantages. This is achieved through assay optimization and use of Tn5 tagmentation and/or single-stranded DNA ligation. Greater library yields, lower processing time, and lower costs are achieved. In comparing assays, we reveal substantial limitations in other ChIP-based assays. Importantly, the new ChIP-exo assays allow high-resolution detection of some protein-DNA interactions in organs and in as few as 27,000 cells. It is suitable for high-throughput parallelization. The simplicity of ChIP-exo now makes it a highly appropriate substitute for ChIP-seq, and for broader adoption.

Genome-wide determinants of sequence-specific DNA binding of general regulatory factors.

General regulatory factors (GRFs), such as Reb1, Abf1, Rap1, Mcm1, and Cbf1, positionally organize yeast chromatin through interactions with a core consensus DNA sequence. It is assumed that sequence recognition via direct base readout suffices for specificity and that spurious nonfunctional sites are rendered inaccessible by chromatin. We tested these assumptions through genome-wide mapping of GRFs in vivo and in purified biochemical systems at near-base pair (bp) resolution using several ChIP-exo-based assays. We find that computationally predicted DNA shape features (e.g., minor groove width, helix twist, base roll, and propeller twist) that are not defined by a unique consensus sequence are embedded in the nonunique portions of GRF motifs and contribute critically to sequence-specific binding. This dual source specificity occurs at GRF sites in promoter regions where chromatin organization starts. Outside of promoter regions, strong consensus sites lack the shape component and consequently lack an intrinsic ability to bind cognate GRFs, without regard to influences from chromatin. However, sites having a weak consensus and low intrinsic affinity do exist in these regions but are rendered inaccessible in a chromatin environment. Thus, GRF site-specificity is achieved through integration of favorable DNA sequence and shape readouts in promoter regions and by chromatin-based exclusion from fortuitous weak sites within gene bodies. This study further revealed a severe G/C nucleotide cross-linking selectivity inherent in all formaldehyde-based ChIP assays, which includes ChIP-seq. However, for most tested proteins, G/C selectivity did not appreciably affect binding site detection, although it does place limits on the quantitativeness of occupancy levels.

Widespread and precise reprogramming of yeast protein-genome interactions in response to heat shock.

Gene expression is controlled by a variety of proteins that interact with the genome. Their precise organization and mechanism of action at every promoter remains to be worked out. To better understand the physical interplay among genome-interacting proteins, we examined the temporal binding of a functionally diverse subset of these proteins: nucleosomes (H3), H2AZ (Htz1), SWR (Swr1), RSC (Rsc1, Rsc3, Rsc58, Rsc6, Rsc9, Sth1), SAGA (Spt3, Spt7, Ubp8, Sgf11), Hsf1, TFIID (Spt15/TBP and Taf1), TFIIB (Sua7), TFIIH (Ssl2), FACT (Spt16), Pol II (Rpb3), and Pol II carboxyl-terminal domain (CTD) phosphorylation at serines 2, 5, and 7. They were examined under normal and acute heat shock conditions, using the ultrahigh resolution genome-wide ChIP-exo assay in Saccharomyces cerevisiae Our findings reveal a precise positional organization of proteins bound at most genes, some of which rapidly reorganize within minutes of heat shock. This includes more precise positional transitions of Pol II CTD phosphorylation along the 5' ends of genes than previously seen. Reorganization upon heat shock includes colocalization of SAGA with promoter-bound Hsf1, a change in RSC subunit enrichment from gene bodies to promoters, and Pol II accumulation within promoter/+1 nucleosome regions. Most of these events are widespread and not necessarily coupled to changes in gene expression. Together, these findings reveal protein-genome interactions that are robustly reprogrammed in precise and uniform ways far beyond what is elicited by changes in gene expression.

RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions.

In eukaryotes, RAD54 catalyzes branch migration (BM) of Holliday junctions, a basic process during DNA repair, replication, and recombination. RAD54 also stimulates RAD51 recombinase and has other activities. Here, we investigate the structural determinants for different RAD54 activities. We find that the RAD54 N-terminal domain (NTD) is responsible for initiation of BM through two coupled, but distinct steps; specific binding to Holliday junctions and RAD54 oligomerization. Furthermore, we find that the RAD54 oligomeric state can be controlled by NTD phosphorylation at S49, a CDK2 consensus site, which inhibits RAD54 oligomerization and, consequently, BM. Importantly, the effect of phosphorylation on RAD54 oligomerization is specific for BM, as it does not affect stimulation of RAD51 recombinase by RAD54. Thus, the transition of the oligomeric states provides an important control of the biological functions of RAD54 and, likely, other multifunctional proteins.

Polarity and bypass of DNA heterology during branch migration of Holliday junctions by human RAD54, BLM, and RECQ1 proteins.

Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3'→5' direction, whereas RuvAB drives it in the 5'→3' direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.

Reconstituting the key steps of the DNA double-strand break repair in vitro.

Double-stranded DNA breaks (DSB), the most harmful type of DNA lesions, cause cell death and genome instability. Homologous recombination repairs DSB using homologous DNA sequences as templates. Here we describe a set of reactions that lead to reconstitution of the double-stranded DNA break repair process in vitro employing purified human homologous recombination proteins and DNA polymerase η. Reconstitution of critical steps of DSB repair in vitro may help to better understand the mechanisms of recombinational DNA repair and the role of various human homologous recombination proteins in this process.

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.

Cooperation of RAD51 and RAD54 in regression of a model replication fork.

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the 'template-strand switch' mechanism. This mechanism involves replication fork regression into a Holliday junction ('chicken foot structure'), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.

Analyzing the branch migration activities of eukaryotic proteins.

The Holliday junction is a key intermediate of DNA repair, recombination, and replication. Branch migration of Holliday junctions is a process in which one DNA strand is progressively exchanged for another. Branch migration of Holliday junctions may serve several important functions such as affecting the length of genetic information transferred between homologous chromosomes during meiosis, restarting stalled replication forks, and ensuring the faithful repair of double strand DNA breaks by homologous recombination. Several proteins that promote branch migration of Holliday junctions have been recently identified. These proteins, which function during DNA replication and repair, possess the ability to bind Holliday junctions and other branched DNA structures and drive their branch migration by translocating along DNA in an ATPase-dependent manner. Here, we describe methods employing a wide range of DNA substrates for studying proteins that catalyze branch migration of Holliday junctions.