A novel targeted NGS panel identifies numerous homologous recombination deficiency (HRD)-associated gene mutations in addition to known BRCA mutations.

Deleterious mutations in the BRCA1 and BRCA2 genes have significant therapeutic relevance in clinical settings regarding personalized therapy approaches. BRCA1 and BRCA2 play a pivotal role in homologous recombination (HR) and thus are sensitive for PARP inhibitors (PARPi). Beyond the narrow scope of evaluating only the BRCA mutation status, PARPi can be beneficial for HR deficient (HRD) patients, who harbor mutations in other HR-associated genes. In the present retrospective study, a novel targeted HRD gene panel was validated and implemented for use with FFPE tissue. Samples of patients with ovarian, breast, pancreatic and prostate cancer were included. Variants were robustly detected with various DNA input amounts and the use of test samples showed complete concordance between previously known mutations and HRD panel results. From all the 90 samples included in this cohort, TP53 was the most frequently altered gene (73%). Deleterious BRCA1/2 mutations were found in 20 (22%) of all samples. New pathogenic or likely pathogenic mutations in additional HR-associated genes were identified in 22 (24%) patients. Taken together, the present study proves the feasibility of a new HRD gene panel with reliable panel performance and offers the possibility to easily screen for resistance mutations acquired over treatment time.Mutations in HR-associated genes, besides BRCA1/2, might represent promising potential targets for synthetic lethality approaches. Thus, a substantial number of patients may benefit from expanding the scope of therapeutic agents like PARPi.

An assessment of learning rates in habitual prospective memory.

Most laboratory research in the field of prospective memory has focused on newly formed (episodic) intentions that are carried out in the experimental context once or only a small number of times. However, many naturalistic prospective memories are carried out many times and these types of (habitual) intentions have been studied much less in the laboratory. In the current study, our aim was to extend prior work examining habitual intentions in laboratory prospective memory paradigms. Participants formed a typical prospective memory intention and then completed an ongoing task in which the intention could be executed up to 63 times. We examined changes in performance across trials in three traditionally important prospective memory metrics: cue detection, task interference, and cue interference. Across repeated performance of the prospective memory task, we observed an increase in cue detection, elimination of task interference, and elimination of cue interference. These results provide key insights into the operation of learning mechanisms in prospective memory paradigms and promote theory development by showing that many of the resource-demanding processes that are theorized to be necessary for successful prospective memory play much less of a role when intentions are repeatedly completed.

Comparative analysis of nuclear and mitochondrial DNA from tissue and liquid biopsies of colorectal cancer patients.

The current standard for molecular profiling of colorectal cancer (CRC) is using resected or biopsied tissue specimens. However, they are limited regarding sampling frequency, representation of tumor heterogeneity, and sampling can expose patients to adverse side effects. The analysis of cell-free DNA (cfDNA) from blood plasma, which is part of a liquid biopsy, is minimally invasive and in principle enables detection of all tumor-specific mutations. Here, we analyzed cfDNA originating from nucleus and mitochondria and investigated their characteristics and mutation status in a cohort of 18 CRC patients and 10 healthy controls using targeted next-generation sequencing (NGS) and digital PCR. Longitudinal analyses of nuclear cfDNA level and size during chemotherapy revealed a decreasing cfDNA content and a shift from short to long fragments, indicating an appropriate therapy response, while shortened cfDNAs and increased cfDNA content corresponded with tumor recurrence. Comparative NGS analysis of nuclear tissue and plasma DNA demonstrated a good patient-level concordance and cfDNA revealed additional variants in three of the cases. Analysis of mitochondrial cfDNA surprisingly revealed a higher plasma copy number in healthy subjects than in CRC patients. These results highlight the potential clinical utility of liquid biopsies in routine diagnostics and surveillance of CRC patients as complementation to tissue biopsies or as an attractive alternative in cases where tissue biopsies are risky or the quantity/quality does not allow testing.

Wait a second . . . Boundary conditions on delayed responding theories of prospective memory.

Research suggests that forcing participants to withhold responding for as brief as 600 ms eliminates one of the most reliable findings in prospective memory (PM): the cue focality effect. This result undermines the conventional view that controlled attentional monitoring processes support PM, and instead suggests that cue detection results from increased response thresholds that allow more time for PM information to accumulate. Given the significance of such findings, it is critical to examine the generalizability of the delay mechanism. Experiments 1-4 examined boundary conditions of the delay theory of PM, whereas Experiment 5 more directly tested contrasting theoretical predictions from monitoring theory (e.g., multiprocess framework) and delay theory. Using the same (Experiment 1) or conceptually similar (Experiment 2) delay procedure and identical cues (nonfocal "tor" intention) from the original study failed to show any influence of delay on performance. Using a different nonfocal intention (first letter "S") similarly did not influence performance (Experiment 3), and the difference between focal and nonfocal cue detection was never completely eliminated even with delays as long as 2,500 ms (Experiment 4). Experiment 5 did find the anticipated reduction in the focality effect with increased delays with a larger sample (n = 249). However, the focality effect was not moderated by attention control ability despite the fact that participants with impoverished attention control should benefit most from the delay procedure. These results suggest that any theory of PM that considers only a delay mechanism may not fully capture the dynamic attention processes that support cue detection. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

From the Ocean to the Lab-Assessing Iron Limitation in Cyanobacteria: An Interface Paper.

Iron is an essential, yet scarce, nutrient in marine environments. Phytoplankton, and especially cyanobacteria, have developed a wide range of mechanisms to acquire iron and maintain their iron-rich photosynthetic machinery. Iron limitation studies often utilize either oceanographic methods to understand large scale processes, or laboratory-based, molecular experiments to identify underlying molecular mechanisms on a cellular level. Here, we aim to highlight the benefits of both approaches to encourage interdisciplinary understanding of the effects of iron limitation on cyanobacteria with a focus on avoiding pitfalls in the initial phases of collaboration. In particular, we discuss the use of trace metal clean methods in combination with sterile techniques, and the challenges faced when a new collaboration is set up to combine interdisciplinary techniques. Methods necessary for producing reliable data, such as High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS), Flow Injection Analysis Chemiluminescence (FIA-CL), and 77K fluorescence emission spectroscopy are discussed and evaluated and a technical manual, including the preparation of the artificial seawater medium Aquil, cleaning procedures, and a sampling scheme for an iron limitation experiment is included. This paper provides a reference point for researchers to implement different techniques into interdisciplinary iron studies that span cyanobacteria physiology, molecular biology, and biogeochemistry.

Neural mechanisms of perceptive and affective processing of body stimuli in anorexia nervosa - are there developmental effects?

Different components of body image processing seem to be reflected by different neural mechanisms. A core symptom of Anorexia nervosa (AN) is a disturbance of body image with correlates found on a neural level. The present study focuses on the neural processing of visual body stimuli of different weight categories in adolescent and adult AN patients. Thirty-three adolescents aged 12-18 years (15 AN patients, 18 control participants) and 36 adult women (19 AN patients, 17 control participants) underwent functional magnetic resonance imaging (fMRI) while performing a perceptive and an affective body image task involving photographic stimuli of women belonging to different BMI categories. Differential effects on activation, depending on the BMI of the women shown in the pictures, were found in frontal brain regions, the thalamus, the caudate and the fusiform gyrus. Group effects differentiating between AN patients and control participants were seen mainly in the caudate and insula. No significant developmental effect was seen. During a perceptive task, diminished activation of regions involved in perceptive and evaluative functions as well as emotional reasoning was seen in AN. During an affective task there was a tendency towards activation differences reflecting reduced ability of size estimation and impaired integration of visual and body perception with emotions.

Experience with One-Stage Repair of Urethral Strictures Using the Augmented Anastomotic Repair Technique.

INTRODUCTION: We report the results of augmented anastomotic repair (AAR) in the treatment of anterior urethral strictures. MATERIAL AND METHODS: In this retrospective study, we evaluated 71 consecutive patients who had undergone AAR between June 2004 and June 2013. Medical records were reviewed to identify early postoperative complications based on the Clavien-Dindo classification (CDC). Self-developed standardized questionnaires sent to the patients and referring urologists were used to collect data on late complications (>90) days. Stricture recurrence (SR) was defined as any postoperative endoscopic or open surgical intervention on the urethra. The influence of patient demographics, stricture characteristics, and operative procedure performed on the occurrence of SR was analyzed. RESULTS: Early postoperative complications were rare events (11.3%) with only one severe CDC complication. Late complications were reported in 46.5% cases. At a median follow-up of 17 months (range 3-114 months), however, 64 patients had no evidence of SR and required no further intervention, giving an overall success rate of 90.1%. Seven patients with SR had a higher body mass index, were older, and had been operated on by less experienced surgeon(s). Most SRs occurred within the first year after surgery. CONCLUSIONS: AAR was an effective and safe operative technique that allowed one-stage repair in our patients with anterior urethral strictures who needed resection of the scarred urethra and otherwise were not suitable for primary anastomosis or simple substitution urethroplasty.

AbiEi Binds Cooperatively to the Type IV abiE Toxin-Antitoxin Operator Via a Positively-Charged Surface and Causes DNA Bending and Negative Autoregulation.

Bacteria resist phage infection using multiple strategies, including CRISPR-Cas and abortive infection (Abi) systems. Abi systems provide population-level protection from phage predation, via "altruistic" cell suicide. It has recently been shown that some Abi systems function via a toxin-antitoxin mechanism, such as the widespread AbiE family. The Streptococcus agalactiae AbiE system consists of a bicistronic operon encoding the AbiEi antitoxin and AbiEii toxin, which function as a Type IV toxin-antitoxin system. Here we examine the AbiEi antitoxin, which belongs to a large family of transcriptional regulators with a conserved N-terminal winged helix-turn-helix domain. This winged helix-turn-helix is essential for transcriptional repression of the abiE operon. The function of the AbiEi C-terminal domain is poorly characterized, but it contributes to transcriptional repression and is sufficient for toxin neutralization. We demonstrate that a conserved charged surface on one face of the C-terminal domain assists sequence-specific DNA binding and negative autoregulation, without influencing antitoxicity. Furthermore, AbiEi binds cooperatively to two inverted repeats within the abiE promoter and bends the DNA by 72°. These findings demonstrate that the mechanism of DNA binding by the widespread family of AbiEi antitoxins and transcriptional regulators can contribute to negative autoregulation.

Robot-assisted laparoscopic Y-V plasty in 12 patients with refractory bladder neck contracture.

We present preliminary results of a case series on refractory bladder neck contracture (BNC) treated with robot-assisted laparoscopic Y-V plasty (RAYV). Between 01/2013 and 02/2016, 12 consecutive adult male patients underwent RAYV in our hospital. BNC developed after transurethral procedures (n = 9), simple prostatectomy (n = 2) and HIFU therapy of the prostate (n = 1). Each patient had had multiple unsuccessful previous endoscopic treatments. All RAYV procedures were performed using a transperitoneal six-port approach (four-arm robotic setting). There were no intraoperative or major postoperative complications. During a median follow-up of 23.2 months two cases of refractory BNC were observed. In both cases a postoperative International Prostate Symptom Score (IPSS) of 20 and 25 was reported, respectively. In contrast, amongst the patients without evidence of refractory BNC the median IPSS was 6.5 reflecting an only mildly impaired voiding function in most cases, thus, suggesting a treatment success in 83.3% of patients. To the best of our knowledge, this is the first report on RAYV for refractory BNC. In our series RAYV was feasible in all patients, and only two cases of refractory BNC were reported during a median follow-up of almost 2 years. At the same time, no intraoperative or major postoperative complications were observed. More clinical data with a longer follow-up are needed in this promising field to reveal the actual efficacy and relevance of RAYV.

Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002.

BACKGROUND: Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrick-compatible integration modules is desirable to further simplifying chromosomal integrations. RESULTS: We designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the Synechococcus chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for Synechococcus significantly. CONCLUSIONS: The optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in Synechococcus. We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing Synechococcus as the prominent chassis for future synthetic biology applications.

Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation.

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30-100 nt enriched for thymine-stretches in their 3' ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3' cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.

Minocycline exacerbates apoptotic neurodegeneration induced by the NMDA receptor antagonist MK-801 in the early postnatal mouse brain.

NMDA receptor (NMDAR) antagonists induce in perinatal rodent cortical apoptosis and protracted schizophrenia-like alterations ameliorated by antipsychotic treatment. The broad-spectrum antibiotic minocycline elicits antipsychotic and neuroprotective effects. Here we tested, if minocycline protects also against apoptosis triggered by the NMDAR antagonist MK-801 at postnatal day 7. Surprisingly, minocycline induced widespread cortical apoptosis and exacerbated MK-801-triggered cell death. In some areas such as the subiculum, the pro-apoptotic effect of minocycline was even more pronounced than that elicited by MK-801. These data reveal among antipsychotics unique pro-apoptotic properties of minocycline, raising concerns regarding consequences for brain development and the use in children.

Role of the nitric oxide donor sodium nitroprusside in the antidepressant effect of ketamine in mice.

Ketamine may represent an efficient alternative antidepressant with rapid therapeutic onset; however, the clinical use of ketamine is hampered by psychosis-like side-effects. Recent studies suggest that the nitric oxide (NO) donor sodium nitroprusside (SNP) prevents psychosis-like abnormalities triggered by ketamine or another NMDA receptor (NMDAR) antagonist, phencyclidine (PCP) in rats. SNP was shown to elicit antipsychotic effects also in humans. Considering the tight interrelation between NMDAR activation and neuronal NO synthesis, we evaluated the effect of pre-treatment with SNP on the antidepressant action of ketamine. We found that SNP (0.5-1mg/kg, i.p.) did not alter the antidepressant effect of ketamine (30 mg/kg) in the Porsolt Forced Swim Test (FST) in mice. Additionally, SNP by itself produced no effect in the FST or in the openfield. This suggests indirectly a differential involvement of the nitrinergic system in the antidepressant vs. psychotomimetic effect of ketamine, although an influence of species-specific differences cannot be excluded in this interpretation.

Touchscreen-paradigm for mice reveals cross-species evidence for an antagonistic relationship of cognitive flexibility and stability.

The abilities to either flexibly adjust behavior according to changing demands (cognitive flexibility) or to maintain it in the face of potential distractors (cognitive stability) are critical for adaptive behavior in many situations. Recently, a novel human paradigm has found individual differences of cognitive flexibility and stability to be related to common prefrontal networks. The aims of the present study were, first, to translate this paradigm from humans to mice and, second, to test conceptual predictions of a computational model of prefrontal working memory mechanisms, the Dual State Theory, which assumes an antagonistic relation between cognitive flexibility and stability. Mice were trained in a touchscreen-paradigm to discriminate visual cues. The task involved "ongoing" and cued "switch" trials. In addition distractor cues were interspersed to test the ability to resist distraction, and an ambiguous condition assessed the spontaneous switching between two possible responses without explicit cues. While response times did not differ substantially between conditions, error rates (ER) increased from the "ongoing" baseline condition to the most complex condition, where subjects were required to switch between two responses in the presence of a distracting cue. Importantly, subjects switching more often spontaneously were found to be more distractible by task irrelevant cues, but also more flexible in situations, where switching was required. These results support a dichotomy of cognitive flexibility and stability as predicted by the Dual State Theory. Furthermore, they replicate critical aspects of the human paradigm, which indicates the translational potential of the testing procedure and supports the use of touchscreen procedures in preclinical animal research.

Enteral or parenteral nimodipine treatment: a comparative pharmacokinetic study.

UNLABELLED: BACKGROUND AND STUDY AIMS/OBJECT: Oral nimodipine is recommended to reduce poor outcome related to aneurysmal subarachnoid hemorrhage (SAH). In addition, animal experiments and clinical trails revealed a beneficial effect of enteral and parenteral nimodipine for the regeneration of cranial nerves following skull base, laryngeal, and maxillofacial surgery. Despite these findings there is a lack of pharmacokinetic data in the literature, especially concerning its distribution in nerve tissue. PATIENTS/MATERIAL AND METHODS: Samples were taken from a consecutive series of 57 patients suffering from skull base lesions and treated with nimodipine prophylaxis from the day before surgery until the seventh postoperative day. Both groups received standard dosages for enteral (n = 25) and parenteral (n = 32) nimodipine . Nimodipine levels were measured in serum, cerebrospinal fluid (CSF), and tissue samples, including vestibular nerves. RESULTS: Nimodipine levels were significantly higher following parenteral as compared with enteral administration for intraoperative serum (p < 0.001), intraoperative CSF (p < 0.001), tumor tissues (p = 0.01), and postoperative serum (p < 0.001). In addition, nimodipine was significantly more frequently detected in nerve tissue following parenteral administration (Fisher's exact test, p = 0.015). CONCLUSIONS: From a pharmacokinetic point of view, parenteral nimodipine medication leads to higher levels in serum and CSF. Furthermore, traces are more frequently found in nerve tissue following parenteral as compared with enteral nimodipine administration, at least in the early course.

Therapeutic benefits of expressive writing in an electronic format.

Expressive writing is recognized as having therapeutic benefits including fewer stress-related visits to the doctor, improved immune system functioning, reduced blood pressure, improved functional status, fewer days in the hospital, feelings of greater psychological well-being, reduced depressive symptoms, fewer posttraumatic intrusion and avoidance symptoms, improved memory and sleep, and faster healing after surgery. Combine this with current popularity of social networking, and there is a formula for improved patient outcomes.

[Evaluation of group homes for the mentally ill in the county of Copenhagen--1980-1995. A study of a time-limited stay in the group home assessed by the development of the social situation and duration of hospitalization before and after]. / Evaluering af Københavns Amts bofaellesskaber for psykisk syge--1980-1995. En undersøgelse af et tidsbegraenset ophold i bofaellesskab vurderet ud fra udviklingen i social situation samt en opgørelse af sengedagsforbrug før og efter opholdet.

INTRODUCTION: We have assessed the diagnostic and social characteristics of residents, who were living in two small group homes during the period, 1 March 1980-1 August 1994. Almost all the 74 residents had a chronic psychotic disease. They had been living in the group homes for at least three months, on average 19 months. MATERIALS AND METHODS: The investigation was made from case notes and hospital records. The social characteristics were obtained from the first stay in a psychiatric ward, from two years before the patient moved into the group home to the time of moving out and 2, 5, and 10 years thereafter. RESULTS AND DISCUSSION: Almost all the patients ended up with clear symptoms of schizophrenia and were receiving a social pension. The average duration of hospitalisation for this group was reduced from one third to one seventh of the time from 2 years before to 10 years after they had left the group homes.