A multidisciplinary pediatric oncofertility team improves fertility preservation and counseling across 7 years.

BACKGROUND: Oncofertility is a developing field of increasing importance, particularly in pediatric oncology, where most patients are likely to survive long-term and have not yet had the opportunity to have children. AIMS: We performed a quality improvement initiative to increase our rates of fertility preservation counseling and referral through the implementation of a pediatric oncofertility team, and we report outcomes 7 years following implementation of our initiative. METHODS AND RESULTS: We compare our baseline oncofertility survey to 44 post-intervention survey respondents and electronic medical record documentation for 149 patients treated in 2019. Ninety-five percent of post-intervention survey respondents recalled fertility counseling (baseline 70%, p = .004) and 89.3% were appropriately referred for fertility preservation (baseline 50%, p = .017). Counseling was documented in 60.4% of charts; 81% of patients analyzed by chart review were appropriately referred for fertility preservation. Fertility preservation outcomes differed by sex assigned at birth. CONCLUSION: Creation of an oncofertility team produced improvements in fertility counseling and fertility preservation referral across an extended period of time.

Double-strand breaks: When DNA repair events accidentally meet.

The cellular response to alkylation damage is complex, involving multiple DNA repair pathways and checkpoint proteins, depending on the DNA lesion, the cell type, and the cellular proliferation state. The repair of and response to O-alkylation damage, primarily O6-methylguaine DNA adducts (O6-mG), is the purview of O6-methylguanine-DNA methyltransferase (MGMT). Alternatively, this lesion, if left un-repaired, induces replication-dependent formation of the O6-mG:T mis-pair and recognition of this mis-pair by the post-replication mismatch DNA repair pathway (MMR). Two models have been suggested to account for MMR and O6-mG DNA lesion dependent formation of DNA double-strand breaks (DSBs) and the resulting cytotoxicity - futile cycling and direct DNA damage signaling. While there have been hints at crosstalk between the MMR and base excision repair (BER) pathways, clear mechanistic evidence for such pathway coordination in the formation of DSBs has remained elusive. However, using a novel protein capture approach, Fuchs and colleagues have demonstrated that DSBs result from an encounter between MMR-induced gaps initiated at alkylation induced O6-mG:C sites and BER-induced nicks at nearby N-alkylation adducts in the opposite strand. The accidental encounter between these two repair events is causal in the formation of DSBs and the resulting cellular response, documenting a third model to account for O6-mG induced cell death in non-replicating cells. This graphical review highlights the details of this Repair Accident model, as compared to current models, and we discuss potential strategies to improve clinical use of alkylating agents such as temozolomide, that can be inferred from the Repair Accident model.

The Trini Sing-Song: Sociophonetic variation in Trinidadian English prosody and differences to other varieties.

The current study provides a phonetic perspective on the questions of whether a high degree of variability in pitch may be considered a characteristic, endonormative feature of Trinidadian English (TrinE) at the level of speech production and contribute to what is popularly described as 'sing-song' prosody. Based on read and spontaneous data from 111 speakers, we analyze pitch level, range, and dynamism in TrinE in comparison to Southern Standard British (BrE) and Educated Indian English (IndE) and investigate sociophonetic variation in TrinE prosody with a view to these global F0 parameters. Our findings suggest that a large pitch range could potentially be considered an endonormative feature of TrinE that distinguishes it from other varieties (BrE and IndE), at least in spontaneous speech. More importantly, however, it is shown that a high degree of pitch variation in terms of range and dynamism is not as much characteristic of TrinE as a whole as it is of female Trinidadian speakers. An important finding of this study is that pitch variation patterns are not homogenous in TrinE, but systematically sociolinguistically conditioned across gender, age, and ethnic groups, and rural and urban speakers. The findings thus reveal that there is a considerable degree of systematic local differentiation in TrinE prosody. On a more general level, the findings may be taken to indicate that endonormative tendencies and sociolinguistic differentiation in TrinE prosody are interlinked.

Novel Approach to Ultrasound-Guided Thoracostomy.

OBJECTIVES: Thoracostomy is often a required treatment in patients with thoracic trauma; however, performing a thoracostomy using traditional techniques can have complications. Ultrasound can be a beneficial tool for identifying the correct thoracostomy insertion site. We designed a randomized prospective study to assess if ultrasound guidance can improve thoracostomy site identification over traditional techniques. METHODS: Emergency medicine residents were randomly assigned to use palpation or ultrasound to identify a safe insertion site for thoracostomy placement. The target population comprised of hemodynamically stable trauma patients who received an extended focused assessment with sonography for trauma (EFAST) and a chest computed tomography (CT) exam. The resident placed a radiopaque marker on the skin of the patient where a safe intercostal space was believed to be located, either by palpation or ultrasound. Clinical ultrasound faculty reviewed the CT to confirm marker placement relative to the diaphragm. A Fischer's exact test was used to analyze the groups. RESULTS: One hundred and forty-seven patients were enrolled in the study, 75 in the ultrasound group and 72 in the landmark group. This resulted in the placement of 271 total thoracostomy site markers, 142 by ultrasound and 129 by palpation and landmarks. The ultrasound group correctly identified thoracostomy insertion sites above the diaphragm in 97.2% (138/142) of patients, while the palpation group identified a safe insertion site in 88.4% (114/129) of patients (P = .0073). CONCLUSION: This study found that emergency medicine residents are more likely to identify a safe tube thoracostomy insertion site in trauma patients by using ultrasound, as compared to using landmarks and palpation.

Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide.

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.

Enrichment of plasmid pool from E. coli for mass sequencing to detect untargeted mutagenic events.

Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a source of genetic diversity. Identifying such untargeted mutations in vivo is challenging due to the bulk of DNA that does not undergo TLS. Here, we present a protocol to enrich a plasmid pool that underwent Pol V-mediated TLS in Escherichia coli for mass sequencing. The concept of this protocol could be applied into any species. For complete details on the use and execution of this protocol, please refer to Isogawa et al. (2018).

Adolescent risk substance use behavior, posttraumatic stress, depression, and resilience: Innovative considerations for disaster recovery.

Natural and technological disasters cause long-term psychological trauma and increase substance use in adults. It is unclear whether these problems also occur in children and whether trauma influences long-term psychological outcomes due to developmental stages at the time of trauma. One community of interest is located in southeastern Louisiana, where, as children, many locals were exposed to Hurricane Katrina in 2005 and the Deepwater Horizon Oil Spill in 2010. We hypothesized individuals exposed to these disasters in early childhood would exhibit higher rates of anxiety, depression, and alcohol use as adolescents than the general population. To test this, we developed a questionnaire with a focus on severity of disaster exposure, indicators of psychological resilience, and current levels of anxiety, depression, and alcohol use. This survey was administered to over 1000 adolescents in local high schools throughout southeastern Louisiana. Structural equation modeling was performed to test correlations and moderation effects. We found disaster exposure was positively associated with trauma-like symptoms and substance use and psychological resilience was negatively related to these outcomes. These findings demonstrate childhood disaster exposure has the potential to cause chronic psychological distress and predispose individuals to substance use later in life. They also suggest resilience may be protective for disaster survivors. Future studies should expand these concepts to other age groups and types of disasters. Whether resilience-focused psychotherapy may be beneficial in these populations is also a relevant topic for exploration.

A Comprehensive View of Translesion Synthesis in Escherichia coli.

The lesion bypass pathway, translesion synthesis (TLS), exists in essentially all organisms and is considered a pathway for postreplicative gap repair and, at the same time, for lesion tolerance. As with the saying "a trip is not over until you get back home," studying TLS only at the site of the lesion is not enough to understand the whole process of TLS. Recently, a genetic study uncovered that polymerase V (Pol V), a poorly expressed Escherichia coli TLS polymerase, is not only involved in the TLS step per se but also participates in the gap-filling reaction over several hundred nucleotides. The same study revealed that in contrast, Pol IV, another highly expressed TLS polymerase, essentially stays away from the gap-filling reaction. These observations imply fundamentally different ways these polymerases are recruited to DNA in cells. While access of Pol IV appears to be governed by mass action, efficient recruitment of Pol V involves a chaperone-like action of the RecA filament. We present a model of Pol V activation: the 3' tip of the RecA filament initially stabilizes Pol V to allow stable complex formation with a sliding ß-clamp, followed by the capture of the terminal RecA monomer by Pol V, thus forming a functional Pol V complex. This activation process likely determines higher accessibility of Pol V than of Pol IV to normal DNA. Finally, we discuss the biological significance of TLS polymerases during gap-filling reactions: error-prone gap-filling synthesis may contribute as a driving force for genetic diversity, adaptive mutation, and evolution.

Chromatin Pull-Down Methodology Based on DNA Triple Helix Formation.

Identification of the protein complexes associated with defined DNA sequence elements is essential to understand the numerous transactions in which DNA is involved, such as replication, repair, transcription, and chromatin dynamics. Here we describe two protocols, IDAP (Isolation of DNA Associated Proteins) and CoIFI (Chromatin-of-Interest Fragment Isolation), that allow for isolating DNA/protein complexes (i.e., nucleoprotein elements) by means of a DNA capture tool based on DNA triple helix (triplex) formation. Typically, IDAP is used to capture proteins that bind to a given DNA element of interest (e.g., a specific DNA sequence, an unusual DNA structure, a DNA lesion) that can be introduced at will into plasmids. The plasmids are immobilized by means of a triplex-forming probe on magnetic beads and incubated in nuclear extracts; by using in parallel a control plasmid (that lacks the DNA element of interest), proteins that preferentially bind to the DNA element of interest are captured and identified by mass spectrometry. Similarly, CoIFI also uses a triplex-forming probe to capture a specific chromatin fragment from a cultured cell line that has been engineered to contain multiple copies of the DNA element of interest.

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes.

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

Severe Cytokine Release Syndrome after Haploidentical Peripheral Blood Stem Cell Transplantation.

Inflammatory cytokines released by activated lymphocytes and innate cells in the context of cellular therapy can cause fever, vasodilatation, and end-organ damage, collectively known as cytokine release syndrome (CRS). CRS can occur after allogeneic blood or marrow transplantation, but is especially prevalent after HLA-haploidentical (haplo) peripheral blood transplantation (PBT). We reviewed charts of all patients who underwent haplo-PBT between October 1, 2013, and September 1, 2017 and graded CRS in these patients. A total of 146 consecutive patients who underwent related haplo-PBT were analyzed. CRS occurred in 130 patients (89%), with most cases of mild severity (grade 0 to 2). Severe CRS (grade 3 to 5) occurred in 25 patients (17%). In this group with severe CRS, 13 patients had encephalopathy, 12 required hemodialysis, and 11 were intubated. Death from the immediate complications of CRS occurred in 6 patients (24% of the severe CRS group and 4% of the entire haplo-PBT cohort). The cumulative probability of nonrelapse mortality (NRM) was 38% at 6 months for the patients with severe CRS and 8% (121 of 146) in patients without severe CRS. In conclusion, CRS occurs in nearly 90% of haplo-PBTs. Older haplo-PBT recipients (odds ratio [OR], 2.4; 95% confidence interval [CI], .83 to 6.75; P = .11) and those with a history of radiation therapy (OR, 3.85; 95% CI, 1.32 to 11.24; P = .01) are at increased risk of developing severe CRS. Although most recipients of haplo-PBT develop CRS, <20% experience severe complications. The development of severe CRS is associated with a significantly increased risk of NRM.

PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.

Chronological Switch from Translesion Synthesis to Homology-Dependent Gap Repair In Vivo.

Cells are constantly exposed to endogenous and exogenous chemical and physical agents that damage their genome by forming DNA lesions. These lesions interfere with the normal functions of DNA such as transcription and replication, and need to be either repaired or tolerated. DNA lesions are accurately removed via various repair pathways. In contrast, tolerance mechanisms do not remove lesions but only allow replication to proceed despite the presence of unrepaired lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS), which is an error-prone strategy and an accurate strategy based on homologous recombination (homology-dependent gap repair [HDGR]). Thus, the mutation frequency reflects the relative extent to which the two tolerance pathways operate in vivo. In the present paper, we review the present understanding of the mechanisms of TLS and HDGR and propose a novel and comprehensive view of the way both strategies interact and are regulated in vivo.

Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Post-replicative Gap Repair.

In vivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events in vivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events at high resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and downstream of a site-specific DNA lesion. Pol V-mediated untargeted mutations are thus spread over several hundred nucleotides, strongly eliciting genetic instability on either side of a given lesion. Consequently, post-replicative gap repair may be a source of untargeted mutations critical for gene diversification in adaptation and evolution.

Glutamatergic neurons of the paraventricular nucleus are critical contributors to the development of neurogenic hypertension.

KEY POINTS: Recurrent periods of over-excitation in the paraventricular nucleus (PVN) of the hypothalamus could contribute to chronic over-activation of this nucleus and thus enhanced sympathetic drive. Stimulation of the PVN glutamatergic population utilizing channelrhodopsin-2 leads to an immediate frequency-dependent increase in baseline blood pressure. Partial lesions of glutamatergic neurons of the PVN (39.3%) result in an attenuated rise in blood pressure following Deoxycorticosterone acetate (DOCA)-salt treatment and reduced index of sympathetic activity. These data suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in blood pressure during hypertension. ABSTRACT: Neuro-cardiovascular dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a key hub for blood pressure (BP) control, producing or relaying the increased sympathetic tone in hypertension. We hypothesize that increased central sympathetic drive is caused by chronic over-excitation of glutamatergic PVN neurons. We tested how stimulation or lesioning of excitatory PVN neurons in conscious mice affects BP, baroreflex and sympathetic activity. Glutamatergic PVN neurons were unilaterally transduced with channelrhodopsin-2 using an adeno-associated virus (CamKII-ChR2-eYFP-AAV2) in wildtype mice (n = 7) to assess the impact of acute stimulation of excitatory PVN neurons selectively on resting BP in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency-dependent (2, 10 and 20 Hz) increase in BP from baseline by ∼9 mmHg at 20 Hz stimulation (P < 0.001). Additionally, in vGlut2-cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre-dependent caspase (AAV2-flex-taCASP3-TEVp). Resting BP and urinary noradrenaline (norepinephrine) levels were then recorded in conscious mice before and after DOCA-salt hypertension. Partial lesions of glutamatergic neurons of the PVN (39.3%, P < 0.05) resulted in an attenuated rise in BP following DOCA-salt treatment (P < 0.05 at 7 day time point, n = 8). Noradrenaline levels as an index of sympathetic activity between the lesion and wildtype groups showed a significant reduction after DOCA-salt treatment in the lesioned animals (P < 0.05). These experiments suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in BP.

Nicotine and the renin-angiotensin system.

Cigarette smoking is the single most important risk factor for the development of cardiovascular and pulmonary diseases (CVPD). Although cigarette smoking has been in constant decline since the 1950s, the introduction of e-cigarettes or electronic nicotine delivery systems 10 yr ago has attracted former smokers as well as a new generation of consumers. Nicotine is a highly addictive substance, and it is currently unclear whether e-cigarettes are "safer" than regular cigarettes or whether they have the potential to reverse the health benefits, notably on the cardiopulmonary system, acquired with the decline of tobacco smoking. Of great concern, nicotine inhalation devices are becoming popular among young adults and youths, emphasizing the need for awareness and further study of the potential cardiopulmonary risks of nicotine and associated products. This review focuses on the interaction between nicotine and the renin-angiotensin system (RAS), one of the most important regulatory systems on autonomic, cardiovascular, and pulmonary functions in both health and disease. The literature presented in this review strongly suggests that nicotine alters the homeostasis of the RAS by upregulating the detrimental angiotensin-converting enzyme (ACE)/angiotensin (ANG)-II/ANG II type 1 receptor axis and downregulating the compensatory ACE2/ANG-(1-7)/Mas receptor axis, contributing to the development of CVPD.

Prazosin induced lysosomal tubulation interferes with cytokinesis and the endocytic sorting of the tumour antigen CD98hc.

The quinazoline based drug prazosin (PRZ) is a potent inducer of apoptosis in human cancer cells. We recently reported that PRZ enters cells via endocytosis and induces tubulation of the endolysosomal system. In a proteomics approach aimed at identifying potential membrane proteins with binding affinity to quinazolines, we detected the oncoprotein CD98hc. We confirmed shuttling of CD98hc towards lysosomes and upregulation of CD98hc expression in PRZ treated cells. Gene knockout (KO) experiments revealed that endocytosis of PRZ still occurs in the absence of CD98hc - suggesting that PRZ does not enter the cell via CD98hc but misroutes the protein towards tubular lysosomes. Lysosomal tubulation interfered with completion of cytokinesis and provoked endoreplication. CD98hc KO cells showed reduced endoreplication capacity and lower sensitivity towards PRZ induced apoptosis than wild type cells. Thus, loss of CD98hc does not affect endocytosis of PRZ and lysosomal tubulation, but the ability for endoreplication and survival of cells. Furthermore, we found that glutamine, lysomototropic agents - namely chloroquine and NH4Cl - as well as inhibition of v-ATPase, interfere with the intracellular transport of CD98hc. In summary, our study further emphasizes lysosomes as target organelles to inhibit proliferation and to induce cell death in cancer. Most importantly, we demonstrate for the first time that the intracellular trafficking of CD98hc can be modulated by small molecules. Since CD98hc is considered as a potential drug target in several types of human malignancies, our study possesses translational significance suggesting, that old drugs are able to act on a novel target.

Characterization of the endolysosomal system in human chordoma cell lines: is there a role of lysosomes in chemoresistance of this rare bone tumor?

Chordoma is a rare tumor of the bone derived from remnants of the notochord with pronounced chemoresistance. A common feature of the notochord and chordoma cells is distinct vacuolization. Recently, the notochord vacuole was described as a lysosome-related organelle. Since lysosomes are considered as mediators of drug resistance in cancer, we were interested whether they may also play a role in chemoresistance of chordoma. We characterized the lysosomal compartment in chordoma cell lines by cytochemistry, electron microscopy (ELMI) and mutational analysis of genes essential for the physiology of lysosomes. Furthermore, we tested for the first time the cytotoxicity of chloroquine, which targets lysosomes, on chordoma. Cytochemical stainings clearly demonstrated a huge mass of lysosomes in chordoma cell lines with perinuclear accumulation. Also vacuoles in chordoma cells were positive for the lysosomal marker LAMP1 but showed no acidic pH. Genetic analysis detected no apparent mutation associated with known lysosomal pathologies suggesting that vacuolization and the huge lysosomal mass of chordoma cell lines is rather a relict of the notochord than a result of transformation. ELMI investigation of chordoma cells confirmed the presence of large vacuoles, lysosomes and autophagosomes with heterogeneous ultrastructure embedded in glycogen. Interestingly, chordoma cells seem to mobilize cellular glycogen stores via autophagy. Our first preclinical data suggested no therapeutically benefit of chloroquine for chordoma. Even though, chordoma cells are crammed with lysosomes which are according to their discoverer de Duve "cellular suicide bags". Destabilizing these "suicide bags" might be a promising strategy for the treatment of chordoma.

Endoplasmic reticulum stress in bipolar disorder? - BiP and CHOP gene expression- and XBP1 splicing analysis in peripheral blood.

BACKGROUND: Endoplasmic Reticulum stress activates the Unfolded Protein Response, which is partially impaired in Bipolar Disorder (BD) according to previous in-vitro studies. Thus, BiP and CHOP gene expression and XBP1 splicing were analyzed in peripheral blood of study participants with BD and controls. METHODS: RNA was isolated from fasting blood of study participants with BD (n = 81) and controls (n = 54) and reverse transcribed into cDNA. BiP and CHOP gene expression was analyzed with quantitative RT-PCR. Atypical splicing of XBP1 mRNA was measured by semi-quantitative RT-PCR, gel-electrophoresis and densitometry. ANCOVAs with the covariates age, BMI, sex, lithium and anticonvulsants intake were used with SPSS. Bonferroni correction was used to correct for multiple testing (adjusted p = 0.0083). RESULTS: BiP gene expression was significantly higher in BD than in controls (F(1/128) = 10.076, p = 0.002, Partial η2 = 0.073). Total XBP1 (F(1/126) = 9.550, p = 0.002, Partial η2 = 0.070) and unspliced XBP1 (F(1/128)= 8.803, p= 0.004, Patial η2 = 0.065) were significantly decreased in BD. Spliced XBP1 (F(1/126) = 5.848, p = 0.017, Partial η2 = 0.044) and the ratio spliced XBP1/ unspliced XBP1 did not differ between BD and controls (F(1/126) = 0.599, p = 0.441, Partial η2 = 0.005). Gene expression did not differ between euthymia, depression and mania. DISCUSSION: BiP gene expression was significantly higher in BD compared to controls. Total and unspliced XBP1 were significantly lower in BD than in the control group. Thus, both genes may be considered as putative trait markers. Nevertheless, XBP1 splicing itself did not differ between both groups.

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation.

The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA (Isolation of DNA Associated Proteins: IDAP). We describe a robust and versatile procedure to pull-down chromatinized DNA sequences-of-interest by formation of a triple helix between a sequence tag present in the DNA and a complementary triple helix forming oligonucleotide (TFO) coupled to a desthiobiotin residue. Following optimization to insure efficient recovery of native plasmids via TFO probe in vitro, the procedure is shown to work under various experimental situations. For instance, it allows capture proteins associated to plasmids hosted in E. coli, and is also successfully applied to recovering nucleosomes in vitro opening many possibilities to study post translational modifications of histones in a genuine nucleosome context. Incubation in human nuclear extracts of a plasmid carrying a NF-κB model promoter is shown to pull-down a specific transcription factor. Finally, isolation of a specific locus from human genomic chromatin has been successfully achieved (Chromatin-of-Interest Fragment Isolation: CoIFI). In conclusion, the methodology can be implemented for capturing proteins that specifically bind to any sequence-of-interest, DNA adduct or secondary structure provided a short sequence tag for triple helix formation is located nearby.