Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer.

Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.

BRCA1 and RNAi factors promote repair mediated by small RNAs and PALB2-RAD52.

Strong connections exist between R-loops (three-stranded structures harbouring an RNA:DNA hybrid and a displaced single-strand DNA), genome instability and human disease1-5. Indeed, R-loops are favoured in relevant genomic regions as regulators of certain physiological processes through which homeostasis is typically maintained. For example, transcription termination pause sites regulated by R-loops can induce the synthesis of antisense transcripts that enable the formation of local, RNA interference (RNAi)-driven heterochromation6. Pause sites are also protected against endogenous single-stranded DNA breaks by BRCA17. Hypotheses about how DNA repair is enacted at pause sites include a role for RNA, which is emerging as a normal, albeit unexplained, regulator of genome integrity8. Here we report that a species of single-stranded, DNA-damage-associated small RNA (sdRNA) is generated by a BRCA1-RNAi protein complex. sdRNAs promote DNA repair driven by the PALB2-RAD52 complex at transcriptional termination pause sites that form R-loops and are rich in single-stranded DNA breaks. sdRNA repair operates in both quiescent (G0) and proliferating cells. Thus, sdRNA repair can occur in intact tissue and/or stem cells, and may contribute to tumour suppression mediated by BRCA1.

Dollars and Sense: The Financial Argument for Dedicated Posttrauma Center Care.

OBJECTIVE: To demonstrate that the creation of a Center for Trauma Survivorship (CTS) is not cost-prohibitive but is a revenue generator for the institution. BACKGROUND: A dedicated CTS has been demonstrated to increase adherence with follow-up visits and improve overall aftercare in severely injured patients discharged from the trauma center. A potential impediment to the creation of similar centers is its assumed prohibitive cost. METHODS: This pre and post-cohort study examines the financial impact of patients treated by the CTS. Patients in the PRE cohort were those treated in the year before CTS inception. Eligibility criteria are trauma patients admitted who are ≥18 years of age and have a New Injury Severity Score ≥16 or intensive care unit stay ≥2 days. Financial data were obtained from the hospital's billing and cost accounting systems for a 1-year time period after discharge. RESULTS: There were 176 patients in the PRE and 256 in the CTS cohort. The CTS cohort generated 1623 subsequent visits versus 748 in the PRE cohort. CTS patients underwent more follow-up surgery in their first year of recovery as compared with the PRE cohort (98 vs 26 procedures). Each CTS patient was responsible for a $7752 increase in net revenue with a positive contribution margin of $4558 compared with those in the PRE group. CONCLUSIONS: A dedicated CTS increases subsequent visits and necessary procedures and is a positive revenue source for the trauma center. The presumptive financial burden of a CTS is incorrect and the creation of dedicated centers will improve patients' outcomes and the institution's bottom line.

Straight Leg Raise Cannot Replace Computed Tomography in the Detection of Spinal Column Fractures.

INTRODUCTION: An active straight leg raise (SLR) is a weight bearing test which assesses pain upon movement and a patient's ability to load their pelvis, lumbar, and thoracic spine. Since many stable patients undergo computed tomography (CT) scanning solely for spinal tenderness, our hypothesis is that performing active straight leg raising could effectively rule out lumbar and thoracic vertebral fractures. METHODS: Blunt trauma patients ≥18 years of age with Glasgow Coma Scale 15 presenting in hemodynamically stable condition were screened. Patients remaining in the supine position were asked to perform SLR at 12, 18, and 24 inches above the bed. The patient's ability to raise the leg, baseline pain, and pain at each level were assessed. Patients also underwent standard CT scanning of the chest, abdomen and pelvis. The clinical examination results were then matched post hoc with the official radiology reports. RESULTS: 99 patients were screened, 65 males and 34 females. Spinal fractures were present in 15/99 patients (16%). Mechanisms of injury included motor vehicle collision 51%, pedestrian struck 25%, fall1 9%, and other 4%. The median pain score of patients with and without significant spinal fractures at 12, 18, 24 inches was 7.5, 7, 6 and 5, 5, 4, respectively. At 24 inches, active SLR had sensitivity of 0.47, a specificity of 0.59, a positive predictive value of 0.17, and an negative predictive value of 0.86. CONCLUSIONS: Although SLR has been discussed as a useful adjunct to secondary survey and physical exam following blunt trauma, its positive and more importantly negative predictive value are insufficient to rule out spinal column fractures. Liberal indications for CT based upon mechanism and especially pain and tenderness are necessary to identify all thoraco-lumbar spine fractures.

BRCA1/FANCD2/BRG1-Driven DNA Repair Stabilizes the Differentiation State of Human Mammary Epithelial Cells.

An abnormal differentiation state is common in BRCA1-deficient mammary epithelial cells, but the underlying mechanism is unclear. Here, we report a convergence between DNA repair and normal, cultured human mammary epithelial (HME) cell differentiation. Surprisingly, depleting BRCA1 or FANCD2 (Fanconi anemia [FA] proteins) or BRG1, a mSWI/SNF subunit, caused HME cells to undergo spontaneous epithelial-to-mesenchymal transition (EMT) and aberrant differentiation. This also occurred when wild-type HMEs were exposed to chemicals that generate DNA interstrand crosslinks (repaired by FA proteins), but not in response to double-strand breaks. Suppressed expression of ΔNP63 also occurred in each of these settings, an effect that links DNA damage to the aberrant differentiation outcome. Taken together with somatic breast cancer genome data, these results point to a breakdown in a BRCA/FA-mSWI/SNF-ΔNP63-mediated DNA repair and differentiation maintenance process in mammary epithelial cells that may contribute to sporadic breast cancer development.

BRCA1/Trp53 heterozygosity and replication stress drive esophageal cancer development in a mouse model.

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.

Infrared thermography of in situ natural freezing and mechanism of winter-thermonasty in Rhododendron maximum.

Evergreen leaves of Rhododendron species inhabiting temperate/montane climates are typically exposed to both high radiation and freezing temperatures during winter when photosynthetic biochemistry is severely inhibited. Cold-induced "thermonasty," that is, lamina rolling and petiole curling, can reduce the amount of leaf area exposed to solar radiation and has been associated with photoprotection in overwintering rhododendrons. The present study was conducted on natural, mature plantings of a cold-hardy and large-leaved thermonastic North American species (Rhododendron maximum) during winter freezes. Infrared thermography was used to determine initial sites of ice formation, patterns of ice propagation, and dynamics of the freezing process in leaves to understand the temporal and mechanistic relationship between freezing and thermonasty. Results indicated that ice formation in whole plants is initiated in the stem, predominantly in the upper portions, and propagates in both directions from the original site. Ice formation in leaves initially occurred in the vascular tissue of the midrib and then propagated into other portions of the vascular system/venation. Ice was never observed to initiate or propagate into palisade, spongy mesophyll, or epidermal tissues. These observations, together with the leaf- and petiole-histology, and a simulation of the rolling effect of dehydrated leaves using a cellulose-based, paper-bilayer system, suggest that thermonasty occurs due to anisotropic contraction of cell wall cellulose fibers of adaxial versus abaxial surface as the cells lose water to ice present in vascular tissues.

BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair.

The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.

RAP80 and BRCA1 PARsylation protect chromosome integrity by preventing retention of BRCA1-B/C complexes in DNA repair foci.

BRCA1 promotes error-free, homologous recombination-mediated repair (HRR) of DNA double-stranded breaks (DSBs). When excessive and uncontrolled, BRCA1 HRR activity promotes illegitimate recombination and genome disorder. We and others have observed that the BRCA1-associated protein RAP80 recruits BRCA1 to postdamage nuclear foci, and these chromatin structures then restrict the amplitude of BRCA1-driven HRR. What remains unclear is how this process is regulated. Here we report that both BRCA1 poly-ADP ribosylation (PARsylation) and the presence of BRCA1-bound RAP80 are critical for the normal interaction of BRCA1 with some of its partners (e.g., CtIP and BACH1) that are also known components of the aforementioned focal structures. Surprisingly, the simultaneous loss of RAP80 and failure therein of BRCA1 PARsylation results in the dysregulated accumulation in these foci of BRCA1 complexes. This in turn is associated with the intracellular development of a state of hyper-recombination and gross chromosomal disorder. Thus, physiological RAP80-BRCA1 complex formation and BRCA1 PARsylation contribute to the kinetics by which BRCA1 HRR-sustaining complexes normally concentrate in nuclear foci. These events likely contribute to aneuploidy suppression.

Visualising the effect of freezing on the vascular system of wheat in three dimensions by in-block imaging of dye-infiltrated plants.

Infrared thermography has shown after roots of grasses freeze, ice spreads into the crown and then acropetally into leaves initially through vascular bundles. Leaves freeze singly with the oldest leaves freezing first and the youngest freezing later. Visualising the vascular system in its native 3-dimensional state will help in the understanding of this freezing process. A 2 cm section of the crown that had been infiltrated with aniline blue was embedded in paraffin and sectioned with a microtome. A photograph of the surface of the tissue in the paraffin block was taken after the microtome blade removed each 20 µm section. Two hundred to 300 images were imported into Adobe After Effects and a 3D volume of the region infiltrated by aniline blue dye was constructed. The reconstruction revealed that roots fed into what is functionally a region inside the crown that could act as a reservoir from which all the leaves are able to draw water. When a single root was fed dye solution, the entire region filled with dye and the vascular bundles of every leaf took up the dye; this indicated that the vascular system of roots was not paired with individual leaves. Fluorescence microscopy suggested the edge of the reservoir might be composed of phenolic compounds. When plants were frozen, the edges of the reservoir became leaky and dye solution spread into the mesophyll outside the reservoir. The significance of this change with regard to freezing tolerance is not known at this time. Thermal cameras that allow visualisation of water freezing in plants have shown that in crops like wheat, oats and barley, ice forms first at the bottom of the plant and then moves upwards into leaves through water conducting channels. Leaves freeze one at a time with the oldest leaves freezing first and then younger ones further up the stem freeze later. To better understand why plants freeze like this, we reconstructed a 3-dimensional view of the water conducting channels. After placing the roots of a wheat plant in a blue dye and allowing it to pull the dye upwards into leaves, we took a part of the stem just above the roots and embedded it in paraffin. We used a microtome to slice a thin layer of the paraffin containing the plant and then photographed the surface after each layer was removed. After taking about 300 images, we used Adobe After Effects software to re-construct the plant with the water conducting system in three dimensions. The 3D reconstruction showed that roots fed into a roughly spherical area at the bottom of the stem that could act as a kind of tank or reservoir from which the leaves pull up water. When we put just one root in dye, the entire reservoir filled up and the water conducting channels in every leaf took up the dye. This indicates that the water channels in roots were not directly connected to specific leaves as we had thought. When plants were frozen, the dye leaked out of the reservoir and spread into cells outside. Research is continuing to understand the significance of this change during freezing. It is possible that information about this effect can be used to help breeders develop more winter-hardy crop plants.

PP2A-mediated regulation of Ras signaling in G2 is essential for stable quiescence and normal G1 length.

Quiescence (G0) allows cycling cells to reversibly cease proliferation. A decision to enter quiescence is suspected of occurring early in G1, before the restriction point (R). Surprisingly, we have identified G2 as an interval during which inhibition of the protein phosphatase PP2A results in failure to exhibit stable quiescence. This effect is accompanied by shortening of the ensuing G1. The PP2A subcomplex required for stable G0 contains the B56γ B subunit. After PP2A inhibition in G2, aberrant overexpression of cyclin E occurs during mitosis and is responsible for overriding quiescence. Strikingly, suppression of Ras signaling re-establishes normal cyclin E levels during M and restores G0. These data point to PP2A-B56γ-driven Ras signaling modulation in G2 as essential for suppressing aberrant cyclin E expression during mitosis and thereby achieving normal G0 control. Thus, G2 is an interval during which the length and growth factor dependence of the next G1 interval are established.

Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage.

BRCA1 is a breast and ovarian tumor suppressor. Given its numerous incompletely understood functions and the possibility that more exist, we performed complementary systematic screens in search of new BRCA1 protein-interacting partners. New BRCA1 functions and/or a better understanding of existing ones were sought. Among the new interacting proteins identified, genetic interactions were detected between BRCA1 and four of the interactors: TONSL, SETX, TCEANC, and TCEA2. Genetic interactions were also detected between BRCA1 and certain interactors of TONSL, including both members of the FACT complex. From these results, a new BRCA1 function in the response to transcription-associated DNA damage was detected. Specifically, new roles for BRCA1 in the restart of transcription after UV damage and in preventing or repairing damage caused by stabilized R loops were identified. These roles are likely carried out together with some of the newly identified interactors. This new function may be important in BRCA1 tumor suppression, since the expression of several interactors, including some of the above-noted transcription proteins, is repeatedly aberrant in both breast and ovarian cancers.

Sequential Protocol Biopsies Post-Liver Transplant From Donors With Moderate Macrosteatosis: What Happens to the Fat?

The number of steatotic deceased donor livers encountered has continued to rise as a result of the obesity epidemic. Little is known about the histological characteristics of moderately macrosteatotic livers over time in the recipient following liver transplantation (LT). All recipients undergoing LT at Mayo Clinic Florida with donor livers with moderate macrosteatosis (30%-60%) from 2000-2017 were identified (n = 96). Routine protocol liver biopsies were performed 1-week and 6-months following LT. All liver donor and protocol biopsies were read by an experienced liver pathologist. Of the 96 moderate macrosteatosis LTs, 70 recipients had post-LT protocol liver biopsies available and comprised the study cohort. Median donor allograft macrosteatosis at the time of transplant was 33% (IQR, 30%-40%) compared with 0% (IQR, 0%-2%) at 1-week (P < 0.001) and 0% (IQR, 0%-0%) at 6-months (P < 0.001) following LT. Biopsies at 1-week post-LT displayed pericentral necrosis in 57.1% of recipients and lipopeliosis in 34.3% of recipients. In the 6-month post-LT biopsies, cholestasis was seen in 3 (4.3%) of the recipients, whereas grade 2 fibrosis was seen in 6 recipients (8.6%). Graft survival at 5 years in the present cohort was 74.0%. Moderate macrosteatosis (30%-60%) in the donor allograft demonstrates complete reversal on liver biopsies performed as early as 7 days following LT and remains absent at 6-months following LT. Both pericentral necrosis and lipopeliosis are common features on day 7 biopsies. Despite these encouraging findings, the perioperative risks of using these livers (postreperfusion cardiac arrest and primary nonfunction) should not be understated. Long-term graft survival is acceptable in patients who are able to overcome the immediate perioperative risk of using moderately steatotic donor livers.

Cell wall modification by the xyloglucan endotransglucosylase/hydrolase XTH19 influences freezing tolerance after cold and sub-zero acclimation.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation.

Microsphere stem blockage as a screen for nitrogen-fixation drought tolerance in soybean.

Symbiotic nitrogen-fixation of soybean (Glycine max [Merr.] L) commonly decreases in response to soil drying in advance of other plant processes. While a few soybean lines express nitrogen-fixation drought tolerance, breeding for genetic variation is hampered by laborious phenotyping procedures. The objective of this research was to explore the potential of an initial screen for nitrogen-fixation drought-tolerant genotypes based on a possible relationship with xylem-vessel diameter. The hypothesis was that nitrogen-fixation drought tolerance might result from fewer, large-diameter xylem vessels in the stem that are vulnerable to disrupted flow as water deficit develops. The disrupted flow could cause nitrogen products to accumulate in nodules resulting in negative feedback on nitrogen-fixation rate. The proposed screen involved exposing de-rooted shoots to a suspension containing microspheres (45-53 µm diameter) and recording the decrease in transpiration rate as a result of microsphere xylem-blockage. Two soybean populations were tested. One population was progeny derived from mating of two parents with high and low nitrogen-fixation drought sensitivity. A high correlation (R2  = 0.68; P < 0.001) was found in this population between decreasing transpiration rate resulting from the microsphere treatment and increasing sensitivity of nitrogen-fixation to soil drying. The second tested population consisted of 16 genotypes, most of which had been previously identified in germplasm screens as expressing nitrogen-fixation drought tolerance. Nearly half of the lines in this second population were identified in the screen as showing minimum blockage of transpiration when exposed to the microspheres. Overall, these results showed the potential of using the microsphere screen to identify candidate genotypes expressing nitrogen-fixation drought tolerance.

BRCA1-IRIS promotes human tumor progression through PTEN blockade and HIF-1α activation.

BRCA1 is an established breast and ovarian tumor suppressor gene that encodes multiple protein products whose individual contributions to human cancer suppression are poorly understood. BRCA1-IRIS (also known as "IRIS"), an alternatively spliced BRCA1 product and a chromatin-bound replication and transcription regulator, is overexpressed in various primary human cancers, including breast cancer, lung cancer, acute myeloid leukemia, and certain other carcinomas. Its naturally occurring overexpression can promote the metastasis of patient-derived xenograft (PDX) cells and other human cancer cells in mouse models. The IRIS-driven metastatic mechanism results from IRIS-dependent suppression of phosphatase and tensin homolog (PTEN) transcription, which in turn perturbs the PI3K/AKT/GSK-3ß pathway leading to prolyl hydroxylase-independent HIF-1α stabilization and activation in a normoxic environment. Thus, despite the tumor-suppressing genetic origin of IRIS, its properties more closely resemble those of an oncoprotein that, when spontaneously overexpressed, can, paradoxically, drive human tumor progression.

Physiological modulation of endogenous BRCA1 p220 abundance suppresses DNA damage during the cell cycle.

Endogenous BRCA1 p220 expression peaks in S and G2 when it is activated, and the protein participates in certain key DNA damage responses. In contrast, its expression is markedly reduced in G0/G1. While variations in transcription represent a significant part of p220 expression control, there is at least one other relevant process. We found that a microRNA, miR-545, that is expressed throughout the cell cycle down-modulates endogenous p220 mRNA and protein abundance directly in both G0/G1 and S/G2. When miR-545 function was inhibited by a specific antagomir, endogenous p220 expression increased in G0/G1, and aberrant p220-associated DNA damage responses and de novo DNA strand breaks accumulated. Analogous results were observed upon inhibition of miR-545 function in S/G2. Both sets of antagomir effects were mimicked by infecting cells with a p220 cDNA-encoding adenoviral vector. Thus, strand breaks were a product of p220 overexpression, and their prevention by miR-545 depends on its modulation of p220 expression. Breaks were also dependent on aberrant, overexpressed p220-driven recruitment of RAD51 to either spontaneously arising or mutagen-based DNA damage sites. Hence, when its level is not physiologically maintained, endogenous p220 aberrantly directs at least one DNA repair protein, RAD51, to damage sites, where their action contributes to the development of de novo DNA damage. Thus, like its loss, a surfeit of endogenous p220 function represents a threat to genome integrity.

Older Patients With Severe Traumatic Brain Injury: National Variability in Palliative Care.

BACKGROUND: Older patients with traumatic brain injury (TBI) have higher mortality and morbidity than their younger counterparts. Palliative care (PC) is recommended for all patients with a serious or life-limiting illness. However, its adoption for trauma patients has been variable across the nation. The goal of this study was to assess PC utilization and intensity of care in older patients with severe TBI. We hypothesized that PC is underutilized despite its positive effects. MATERIALS AND METHODS: The National Inpatient Sample database (2009-2013) was queried for patients aged ≥55 y with International Classification of Diseases, Ninth Revision codes for TBI with loss of consciousness ≥24 h. Outcome measures included PC rate, in-hospital mortality, discharge disposition, length of stay (LOS), and intensity of care represented by craniotomy and or craniectomy, ventilator use, tracheostomy, and percutaneous endoscopic gastrostomy. RESULTS: Of 5733 patients, 78% died in hospital with a median LOS of 1 d, and 85% of the survivors were discharged to facilities. The overall PC rate was 35%. Almost 40% of deaths received PC, with nearly half within 48 h of admission. PC was used in 26% who had neurosurgical procedures, compared with 35% who were nonoperatively managed (P = 0.003). PC was associated with less intensity of care in the entire population. For survivors, those with PC had significantly shorter LOS, compared with those without PC. CONCLUSIONS: Despite high mortality, only one-third of older patients with severe TBI received PC. PC was associated with decreased use of life support and lower intensity of care. Significant efforts need to be made to bridge this quality gap and improve PC in this high-risk population.

Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress.

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.

The impact of postreperfusion syndrome during liver transplantation using livers with significant macrosteatosis.

The impact of postreperfusion syndrome (PRS) during liver transplantation (LT) using donor livers with significant macrosteatosis is largely unknown. Clinical outcomes of all patients undergoing LT with donor livers with moderate macrosteatosis (30%-60%) (N = 96) between 2000 and 2017 were compared to propensity score matched cohorts of patients undergoing LT with donor livers with mild macrosteatosis (10%-29%) (N = 96) and no steatosis (N = 96). Cardiac arrest at the time of reperfusion was seen in eight (8.3%) of the patients in the moderate macrosteatosis group compared to one (1.0%) of the patients in the mild macrosteatosis group (P = .02) and zero (0%) of the patients in the no steatosis group (P = .004). Patients in the moderate macrosteatosis group had a higher rate of PRS (37.5% vs 18.8%; P = .004), early allograft dysfunction (EAD) (76.4% vs 25.8%; P < .001), renal dysfunction requiring continuous renal replacement therapy following transplant (18.8% vs 8.3%; P = .03) and return to the OR within 30 days (24.0% vs 7.3%; P = .002), than the no steatosis group. Both long-term patient (P = .30 and P = .08) and graft survival (P = .15 and P = .12) were not statistically when comparing the moderate macrosteatosis group to the mild macrosteatosis and no steatosis groups. Recipients of LT using livers with moderate macrosteatosis are at a significant increased risk of PRS. If patients are able to overcome the initial increased perioperative risk of using these donor livers, long-term graft survival does not appear to be different than matched recipients receiving grafts with no steatosis.