A Diagnosis of Choriocarcinoma in a Parturient Presenting With Intracranial Hemorrhage.

Choriocarcinoma in a viable pregnancy is uncommon. The diagnosis can easily be missed when there is an explanation for the clinical symptoms that the cancer can mimic. We present the case of a primigravid patient whose choriocarcinoma was initially missed as a result of seemingly obvious explanations for her atypical history and disease manifestation. The patient is a Caucasian female at 30 weeks and 5 days of gestation who presented with persistent headaches and new-onset tonic-clonic seizures found on brain magnetic resonance imaging (MRI) to have a left intracranial hematoma, a 5 mm midline shift, and multiple foci of restricted diffusion. Cerebral angiogram demonstrated arteriovenous malformations (AVMs). The fetus was emergently delivered 1 week into hospitalization for non-reassuring fetal heart tracings in the setting of maternal lethargy secondary to continued AVM hemorrhage. The patient's hospital course was complicated by four episodes of intracranial bleeding and edema requiring neurosurgical intervention. Three weeks after hospitalization she was discharged to a rehabilitation center, shortly after which placental biopsy demonstrated choriocarcinoma. MRI after readmission demonstrated extensive metastatic disease and human chorionic gonadotropin (hCG) levels were greater than 225,000 mIU/mL. Despite two additional neurosurgical procedures and extensive chemotherapy the patient died 3 months after initial presentation. Choriocarcinoma is extremely rare in viable pregnancies, but it should be considered when a parturient presents with intracranial bleeding. A high level of suspicion and serial serum hCG levels may lead to early and potentially life-saving multidrug chemotherapy. With a broader differential, earlier hCG measurement, and earlier treatment, our patient may have survived.

The Effect of Adding Subarachnoid Epinephrine to Hyperbaric Bupivacaine and Morphine for Repeat Cesarean Delivery: A Double-Blind Prospective Randomized Control Trial.

BACKGROUND: Spinal anesthesia has become the most common type of anesthetic for cesarean delivery. The major limitation to spinal anesthesia is that the duration of the anesthetic may not be adequate in the event of a prolonged surgery. Some practitioners add epinephrine to hyperbaric bupivacaine to increase the duration, although its effect has not been fully studied. We therefore aimed to evaluate whether adding epinephrine to the spinal medication prolongs the duration of action of the resultant block in women presenting for repeat cesarean delivery. METHODS: Sixty-eight patients were randomized to receive no epinephrine (NE group), epinephrine 100 µg (low-dose [LD] group), or epinephrine 200 µg (high-dose [HD] group) with a standardized spinal mixture (1.5 mL 0.75% hyperbaric bupivacaine with 0.25 mg morphine). Sixty-five patients were included for primary analysis. Our primary outcome was time to intraoperative activation of the epidural catheter or postoperative regression of sensory blockade to T-10 dermatome level as measured by pinprick sensation; motor recovery was a secondary outcome, and graded via a Modified Bromage scale. RESULTS: Block onset time, vital sign changes, and the incidence of hypotension; nausea, and vomiting were similar among groups. Median difference in time to T-10 regression was greatest in the HD group compared to the NE group (median difference [min] [95% confidence interval]: 40 [15-60]; P = .007), followed by the HD group to the LD group (30 [15-45]; P = .007). Comparisons of LD to NE were not significant, but trended to an increase in T-10 regression time (10 [-15 to 30]; P = .76). Median difference in time to knee extension (Bromage 3) was also greatest in the HD group when compared to both the LD and NE group (median difference [min] [95% confidence interval]: 30 [0-60]; P = .034, 60 [0-93]; P = .007). Median difference time to knee extension (min) between the LD and NE group was also significant (37.5 [15-60]; P = .001]. Pain scores during the procedure were higher in the NE group (median [interquartile range] HD: 0 [0-0], LD: 0 [0-0], NE: 0 [0-3]; P = .02) during uterine closure and were otherwise not significantly different from the other groups. CONCLUSIONS: In this single center, prospective, double-blind, randomized control trial, the addition of epinephrine 200 µg to hyperbaric bupivacaine and preservative-free morphine for repeat cesarean delivery prolonged the duration of the sensory blockade. Motor blockade was similarly prolonged and block quality may have been enhanced.

Impediment of Replication Forks by Long Non-coding RNA Provokes Chromosomal Rearrangements by Error-Prone Restart.

Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη) were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

Palindromic amplification of the ERBB2 oncogene in primary HER2-positive breast tumors.

Oncogene amplification confers a growth advantage to tumor cells for clonal expansion. There are several, recurrently amplified oncogenes throughout the human genome. However, it remains unclear whether this recurrent amplification is solely a manifestation of increased fitness resulting from random amplification mechanisms, or if a genomic locus-specific amplification mechanism plays a role. Here we show that the ERBB2 oncogene at 17q12 is susceptible to palindromic gene amplification, a mechanism characterized by the inverted (palindromic) duplication of genomic segments, in HER2-positive breast tumors. We applied two genomic approaches to investigate amplification mechanisms: sequencing of DNA libraries enriched with tumor-derived palindromic DNA (Genome-wide Analysis of Palindrome Formation) and whole genome sequencing (WGS). We observed significant enrichment of palindromic DNA within amplified ERBB2 genomic segments. Palindromic DNA was particularly enriched at amplification peaks and at boundaries between amplified and normal copy-number regions. Thus, palindromic gene amplification shaped the amplified ERBB2 locus. The enrichment of palindromic DNA throughout the amplified segments leads us to propose that the ERBB2 locus is amplified through the mechanism that repeatedly generates palindromic DNA, such as Breakage-Fusion-Bridge cycles. The genomic architecture surrounding ERBB2 in the normal genome, such as segmental duplications, could promote the locus-specific mechanism.

Replication fork integrity and intra-S phase checkpoint suppress gene amplification.

Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs.

Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles.

Breakage-fusion-bridge (BFB) cycle is a series of chromosome breaks and duplications that could lead to the increased copy number of a genomic segment (gene amplification). A critical step of BFB cycles leading to gene amplification is a palindromic fusion of sister chromatids following the rupture of a dicentric chromosome during mitosis. It is currently unknown how sister chromatid fusion is produced from a mitotic break. To delineate the process, we took an integrated genomic, cytogenetic and molecular approach for the recurrent MCL1 amplicon at chromosome 1 in human tumor cells. A newly developed next-generation sequencing-based approach identified a cluster of palindromic fusions within the amplicon at ∼50-kb intervals, indicating a series of breaks and fusions by BFB cycles. The physical location of the amplicon (at the end of a broken chromosome) further indicated BFB cycles as underlying processes. Three palindromic fusions were mediated by the homologies between two nearby inverted Alu repeats, whereas the other two fusions exhibited microhomology-mediated events. Such breakpoint sequences indicate that homology-mediated fold-back capping of broken ends followed by DNA replication is an underlying mechanism of sister chromatid fusion. Our results elucidate nucleotide-level events during BFB cycles and end processing for naturally occurring mitotic breaks.

A common copy-number breakpoint of ERBB2 amplification in breast cancer colocalizes with a complex block of segmental duplications.

INTRODUCTION: Segmental duplications (low-copy repeats) are the recently duplicated genomic segments in the human genome that display nearly identical (> 90%) sequences and account for about 5% of euchromatic regions. In germline, duplicated segments mediate nonallelic homologous recombination and thus cause both non-disease-causing copy-number variants and genomic disorders. To what extent duplicated segments play a role in somatic DNA rearrangements in cancer remains elusive. Duplicated segments often cluster and form genomic blocks enriched with both direct and inverted repeats (complex genomic regions). Such complex regions could be fragile and play a mechanistic role in the amplification of the ERBB2 gene in breast tumors, because repeated sequences are known to initiate gene amplification in model systems. METHODS: We conducted polymerase chain reaction (PCR)-based assays for primary breast tumors and analyzed publically available array-comparative genomic hybridization data to map a common copy-number breakpoint in ERBB2-amplified primary breast tumors. We further used molecular, bioinformatics, and population-genetics approaches to define duplication contents, structural variants, and haplotypes within the common breakpoint. RESULTS: We found a large (> 300-kb) block of duplicated segments that was colocalized with a common-copy number breakpoint for ERBB2 amplification. The breakpoint that potentially initiated ERBB2 amplification localized in a region 1.5 megabases (Mb) on the telomeric side of ERBB2. The region is very complex, with extensive duplications of KRTAP genes, structural variants, and, as a result, a paucity of single-nucleotide polymorphism (SNP) markers. Duplicated segments are varied in size and degree of sequence homology, indicating that duplications have occurred recurrently during genome evolution. CONCLUSIONS: Amplification of the ERBB2 gene in breast tumors is potentially initiated by a complex region that has unusual genomic features and thus requires rigorous, labor-intensive investigation. The haplotypes we provide could be useful to identify the potential association between the complex region and ERBB2 amplification.

Duration of deep hypothermia during aortic surgery and the risk of perioperative blood transfusion.

Deep hypothermia, which is used during thoracic aortic surgery for neuroprotection, is associated with coagulation abnormalities in animal and in vitro models. However, there is a paucity of data regarding the impact of deep hypothermia duration on perioperative bleeding. The objective of the current study was to examine the relationship between the duration of deep hypothermia and perioperative bleeding. A retrospective review of 507 consecutive thoracic aortic surgery patients who had surgery with deep hypothermic circulatory arrest was performed. The degree of bleeding and coagulopathy was estimated using perioperative transfusion. Log linear modeling with Poisson regression was used to analyze the relationship between deep hypothermia duration and perioperative bleeding, while controlling for other preselected variables. There was a significant association between deep hypothermia duration and RBC transfusion (P = 0.001). There was no significant association between deep hypothermia duration and FFP and platelet transfusion (P = 0.18 and P = 0.06). The association between deep hypothermia duration and the amount of bleeding (RBC transfusion) was dependent on total CPB time. In general, for shorter CPB times (approximately 120 to 180 minutes) there was an upward sloping line or positive relationship between deep hypothermia duration and bleeding. However, for cases with longer CPB times (300 to 360 minutes), there was no such relationship. The relationship between deep hypothermia duration and perioperative bleeding is dependent on CPB time. For surgeries with short CPB times (120 to 180 minutes), prolonged deep hypothermia is associated with increased post-operative bleeding, as estimated by RBC transfusion. For cases with longer CPB times (300 to 360 minutes), there appears to be no relationship.

Molecular trajectories leading to the alternative fates of duplicate genes.

Gene duplication generates extra gene copies in which mutations can accumulate without risking the function of pre-existing genes. Such mutations modify duplicates and contribute to evolutionary novelties. However, the vast majority of duplicates appear to be short-lived and experience duplicate silencing within a few million years. Little is known about the molecular mechanisms leading to these alternative fates. Here we delineate differing molecular trajectories of a relatively recent duplication event between humans and chimpanzees by investigating molecular properties of a single duplicate: DNA sequences, gene expression and promoter activities. The inverted duplication of the Glutathione S-transferase Theta 2 (GSTT2) gene had occurred at least 7 million years ago in the common ancestor of African great apes and is preserved in chimpanzees (Pan troglodytes), whereas a deletion polymorphism is prevalent in humans. The alternative fates are associated with expression divergence between these species, and reduced expression in humans is regulated by silencing mutations that have been propagated between duplicates by gene conversion. In contrast, selective constraint preserved duplicate divergence in chimpanzees. The difference in evolutionary processes left a unique DNA footprint in which dying duplicates are significantly more similar to each other (99.4%) than preserved ones. Such molecular trajectories could provide insights for the mechanisms underlying duplicate life and death in extant genomes.

DNA methylation of developmental genes in pediatric medulloblastomas identified by denaturation analysis of methylation differences.

DNA methylation might have a significant role in preventing normal differentiation in pediatric cancers. We used a genomewide method for detecting regions of CpG methylation on the basis of the increased melting temperature of methylated DNA, termed denaturation analysis of methylation differences (DAMD). Using the DAMD assay, we find common regions of cancer-specific methylation changes in primary medulloblastomas in critical developmental regulatory pathways, including Sonic hedgehog (Shh), Wingless (Wnt), retinoic acid receptor (RAR), and bone morphogenetic protein (BMP). One of the commonly methylated loci is the PTCH1-1C promoter, a negative regulator of the Shh pathway that is methylated in both primary patient samples and human medulloblastoma cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) increases the expression of PTCH1 and other methylated loci. Whereas genetic mutations in PTCH1 have previously been shown to lead to medulloblastoma, our study indicates that epigenetic silencing of PTCH1, and other critical developmental loci, by DNA methylation is a fundamental process of pediatric medulloblastoma formation. This finding warrants strong consideration for DNA demethylating agents in future clinical trials for children with this disease.

Ischemic colitis after endovascular aortoiliac aneurysm repair: a 10-year retrospective study.

OBJECTIVE: To examine the incidence, cause, and outcomes of ischemic colitis after endovascular stent graft repair of aortoiliac aneurysms (EVAR). DESIGN: Medical record review. SETTING: University teaching hospital. PATIENTS: Eight hundred nine patients treated during 10 years were included in the study. Preoperative data regarding the size of the aneurysm, hypogastric coil embolization, and inferior mesenteric artery patency were evaluated by means of computed tomographic scans and aortograms. Ischemic colitis was diagnosed by lower endoscopy or pathology reports. MAIN OUTCOME MEASURES: Ischemic colitis after EVAR. RESULTS: Eleven patients (1.4%) developed ischemic colitis. Seven patients' episode occurred less than 30 days from repair (early), whereas 4 occurred 30 days or more from repair (late). Ten of 11 patients had preoperative inferior mesenteric artery occlusion. Microembolization was seen histologically in 2 patients in the early group, both of whom died. A significant increase in ischemic colitis was seen in patients undergoing preoperative unilateral hypogastric coil embolization (P = .02). Three of the patients with late ischemic colitis had comorbidities other than the EVAR to explain the ischemia. CONCLUSIONS: The incidence of ischemic colitis is decreased in patients undergoing EVAR vs open repair. The cause of the ischemia is multifactorial and seems to differ between patients in the early and late groups. Microembolization tends to produce severe ischemic colitis and is usually fatal. There should be a low threshold for performing endoscopy in any patient thought to have ischemic colitis after EVAR.

Linkage disequilibrium between two high-frequency deletion polymorphisms: implications for association studies involving the glutathione-S transferase (GST) genes.

Copy number variations (CNVs) represent a large source of genetic variation in humans and have been increasingly studied for disease association. A deletion polymorphism of the gene encoding the cytosolic detoxification enzyme glutathione S-transferase theta 1 (GSTT1) has been extensively studied for cancer susceptibility (919 studies, from HuGE navigator, http://www.HuGEnavigator.net/). However, clear conclusions have not been reached. Since the GSTT1 gene is located within a genomic region of segmental duplications (SD), there may be a confounding effect from another, yet-uncharacterized CNV at the same locus. Here we describe a previously uncharacterized 38-kilo-base (kb) long deletion polymorphism of GSTT2B located within a 61-kb DNA inverted repeat. GSTT2B is a duplicated copy of GSTT2, the only paralogue of GSTT1 in humans. A newly developed PCR assay revealed that a microhomology-mediated breakpoint appears to be shared among individuals at high frequency. The GSTT2B deletion polymorphism was in strong linkage disequilibrium (LD) (D' = 0.841) with the neighboring GSTT1 deletion polymorphism in the Caucasian population. Alleles harboring a single deletion were significantly overrepresented (p = 2.22 x 10(-16)), suggesting a selection against alleles with both deletions. The deletion alleles are almost certainly the derived ones, because the GSTT2B-GSTT2-GSTT1 genes were strictly retained in chimpanzees. Extremely low GSTT2 mRNA expression was associated with the GSTT2B deletion, suggesting an influence of the deletion on the flanking region and loss of GSTT2 function. Genome-wide LD analysis between deletion polymorphisms further points to the uniqueness of two deletions, because strong LD between deletion polymorphisms might be very rare in humans. These results show a complex genomic organization and unexpected biological functions of CNVs within segmental duplications and emphasize the importance of detailed structural characterization for disease association studies.

The feasibility of a three-dimensional charting interface for general dentistry.

BACKGROUND: Most current paper- and computer-based formats for patient documentation use a two-dimensional dental chart, a design that originated almost 150 years ago in the United States. No studies have investigated the inclusion of a three-dimensional (3-D) charting interface in a general dental record. METHODS: A multidisciplinary research team with expertise in human-computer interaction, dental informatics and computer science conducted a 14-week project to develop and evaluate a proof of concept for a 3-D dental record. Through several iterations of paper- and computer-based prototypes, the project produced a high-fidelity (hi-fi) prototype that was evaluated by two dentists and two dental students. RESULTS: The project implemented a prototypical patient record built around a 3-D model of a patient's maxillofacial structures. Novel features include automatic retrieval of images and radiographs; a flexible view of teeth, soft tissue and bone; access to historical patient data through a timeline; and the ability to focus on a single tooth. CONCLUSIONS: Users tests demonstrated acceptance for the basic design of the prototype, but also identified several challenges in developing intuitive, easy-to-use navigation methods and hi-fi representations in a 3-D record. CLINICAL IMPLICATIONS: Test participants in this project accepted the preliminary design of a 3-D dental record. Significant further research must be conducted before the concept can be applied and evaluated in clinical practice.

Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication.

OBJECTIVE: Mechanisms underlying mucosal transmission of HIV-1 are incompletely understood. We describe the anti-HIV-1 activity of human beta-defensins (hBD), small cationic molecules that provide protection at mucosal surfaces. METHODS AND RESULTS: HIV-1 induced expression of hBD-2 and -3 mRNA (but not that of hBD-1) 4- to 78-fold, respectively, above baseline in normal human oral epithelial cells. HIV-1 failed to infect these cells, even after 5 days of exposure. Recombinant hBD-1 had no antiviral activity, while rhBD-2 and rhBD-3 showed concentration-dependent inhibition of HIV-1 replication without cellular toxicity. Inhibition was greater against CXCR4-tropic than against the CCR5-tropic HIV-1 isolates. hBD-2 and hBD-3 induced an irreversible effect on virion infectivity, with electron microscopy confirming binding of hBDs to viral particles. Finally, hBD-2 and -3 induced downmodulation of the HIV-1 coreceptor CXCR4 (but not CCR5) in peripheral blood mononuclear cells and T lymphocytic cells as shown by confocal microscopy and flow cytometry. CONCLUSIONS: This study shows for the first time that HIV-1 induces beta-defensin expression in human oral epithelial cells and that beta-defensins block HIV-1 replication via a direct interaction with virions and through modulation of the CXCR4 coreceptor. These properties may be exploited as strategies for mucosal protection against HIV-1 transmission.

Role of Baseline pol Genotype in HIV-1 Fitness Evolution.

Viral fitness can be modified upon development of antiretroviral drug resistance, usually by selection of compensatory mutations. In this study, we have used HIV-1 isolates from individuals receiving a protease inhibitor (PI)-based regimen to analyze the impact of basal genetic background on viral fitness evolution. Paired plasma samples and HIV-1 isolates were obtained from 10 PI-naive HIV-infected individuals enrolled in 2 different studies of combination antiretroviral therapy. Genomic regions from pol and env were sequenced. Viral fitness was measured using growth competition experiments followed by heteroduplex tracking analysis. Baseline genotypic analyses of pol showed that 9 of 10 viruses had a different degree of secondary mutations in the protease gene at codons associated with PI resistance (i.e., 10I, 36I, 63P, 71T, and 77I). After 48 weeks of PI-based therapy, a strong correlation was observed between protease genetic divergence and viral fitness difference (r = 0.78, P = 0.03), but not with reverse transcription or Env divergence, suggesting that genotypic changes in the protease gene were driving HIV-1 evolution in these patients. As expected, an inverse correlation was observed between the number of protease and reverse transcription primary mutations and viral fitness (r = -0.65, P < 0.0001). However, our results suggest that the preexistence of secondary mutations in protease genetic background may have implications in HIV-1 fitness evolution and virologic response to antiretroviral therapy.

A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy.

Despite numerous studies on human immunodeficiency virus type 1 (HIV-1) fitness, many key conceptual and technical questions are still unsolved. For example, the proper system to determine virus fitness of HIV-1 is still unknown. In this study, an assay was developed to estimate HIV-1 fitness based on growth competition experiments and TaqMan real-time PCR. This novel technique was compared with several methods (i.e. virus growth kinetics, growth competition/heteroduplex-tracking analysis and single-cycle replication capacity assay) in order to analyse the impact of various genomic regions and overall genetic background on virus fitness. HIV-1 primary isolates and three different sets of recombinant viruses [i.e. recombinant clones carrying protease (PR), reverse transcriptase (RT) or the 3' end of Gag, PR and RT (3'Gag/PR/RT), sequences amplified by PCR from the same primary isolates)] were evaluated. Here, it is demonstrated that, in spite of intrinsic differences, both growth competition/TaqMan and single-cycle replication assays detected a significant reduction in HIV-1 fitness as a consequence of drug-resistant mutations in pol. However, this new assay, based on HIV-1 isolates, may be useful to quantify replicative fitness in viruses from patients treated simultaneously with antiretroviral drugs targeting different genomic regions of HIV-1 (e.g. pol and env).

Role of the human immunodeficiency virus type 1 envelope gene in viral fitness.

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.