Safety and functional outcome analysis of ventriculoperitoneal shunt placement for hydrocephalus within the critical phase of possible delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

Shunt-dependent hydrocephalus (HC) is a common sequela following aneurysmal subarachnoid hemorrhage (aSAH). However, there is still poor evidence regarding the optimal timing of ventriculoperitoneal shunt (VPS) placement, particularly in the context of early aSAH-associated complications such as delayed cerebral ischemia (DCI). The purpose of this study was to compare the impact of early (< 21 days after aSAH) versus late (≥ 21 days after aSAH) VPS placement on the functional clinical outcome. We retrospectively analyzed data from 82 patients with VPS placement after aSAH enrolled in our institutional database between 2011 and 2021. We compared two groups, early VPS placement (< 21 days after aSAH) versus late VPS placement (≥ 21 days after aSAH) in terms of demographics, SAH grading, radiological parameters, externalized cerebrospinal fluid diversions, DCI, VPS variables, and functional outcome. We identified 53 patients with early and 29 patients with late VPS implantation. Baseline variables, such as the modified Rankin Scale (mRS), the World Federation of Neurological Surgeons Scale, the Glasgow Coma Scale, and Fisher grade were not significantly different between the groups. Postoperatively, the mRS (p = 0.0037), the Glasgow Outcome Scale (p = 0.0037), and the extended Glasgow Outcome Scale (p = 0.0032) showed significantly better functional results in patients with early cerebrospinal fluid diversion. The rate of DCI did not differ significantly between the groups (p = 0.53). There was no difference in the rate of VPS placement associated complications (p = 0.44) or overall mortality (p = 0.39). Early shunt implantation, within 21 days after aSAH and therefore during the timeframe of possible DCI, might not be harmful in patients developing HC after aSAH.

Primary Decompressive Craniectomy After Traumatic Brain Injury: A Literature Review.

Traumatic brain injuries (TBIs) still put a high burden on public health worldwide. Medical and surgical treatment strategies are continuously being studied, but the role and indications of primary decompressive craniectomy (DC) remain controversial. In medically refractory intracranial hypertension after severe traumatic brain injury, secondary decompressive craniectomy is a last resort treatment option to control intracranial pressure (ICP). Randomized controlled studies have been extensively performed on secondary decompressive craniectomy and its role in the management of severe traumatic brain injuries. Indications, prognostic factors, and long-term outcomes in primary decompressive craniectomy during the evacuation of an epidural, subdural, or intracerebral hematoma in the acute phase are still a matter of ongoing research and controversy to this day. Prospective trials have been designed, but the results are yet to be published. In isolated epidural hematoma without underlying brain injury, osteoplastic craniotomy is likely to be sufficient. In acute subdural hematoma (ASDH) with relevant brain swelling and preoperative CT signs such as effaced cisterns, overly proportional midline-shift compared to a relatively small acute subdural hematoma, and accompanying brain contusions as well as pupillary abnormalities, intraventricular hemorrhage, and coagulation disorder, primary decompressive craniectomy is more likely to be of benefit for patients with traumatic brain injury. The role of intracranial pressure monitoring after primary decompressive craniectomy is recommended, but prospective trials are pending. More refined guidelines and hopefully class I evidence will be established with the ongoing trials: randomized evaluation of surgery with craniectomy for patients undergoing evacuation of acute subdural hematoma (RESCUE-ASDH), prospective randomized evaluation of decompressive ipsilateral craniectomy for traumatic acute epidural hematoma (PREDICT-AEDH), and pragmatic explanatory continuum indicator summary (PRECIS).

Long-term hepatitis B virus infection of rhesus macaques requires suppression of host immunity.

Hepatitis B virus has infected a third of the world's population, and 296 million people are living with chronic infection. Chronic infection leads to progressive liver disease, including hepatocellular carcinoma and liver failure, and there remains no reliable curative therapy. These gaps in our understanding are due, in large part, to a paucity of animal models of HBV infection. Here, we show that rhesus macaques regularly clear acute HBV infection, similar to adult humans, but can develop long-term infection if immunosuppressed. Similar to patients, we longitudinally detected HBV DNA, HBV surface antigen, and HBV e antigen in the serum of experimentally infected animals. In addition, we discovered hallmarks of HBV infection in the liver, including RNA transcription, HBV core and HBV surface antigen translation, and covalently closed circular DNA biogenesis. This pre-clinical animal model will serve to accelerate emerging HBV curative therapies into the clinic.

HIV testing behaviour and associated factors in men who have sex with men by level of urbanisation: a cross-sectional study in the Netherlands.

OBJECTIVES: Regular HIV testing in men who have sex with men (MSM) enables timely entry into care and reduces the likelihood of HIV transmission. We aimed to assess HIV-testing behaviour and associated factors in MSM by urbanisation of place of residence. DESIGN: Data were derived from online survey ('Men & Sexuality') in the Netherlands, which was mainly advertised on social media (Facebook and Instagram), dating websites, apps for MSM (Grindr and PlanetRomeo) and gay media. PRIMARY AND SECONDARY OUTCOME MEASURES: HIV testing was defined as recent (<1 year), not recent (≥1 year) or never. Using multinominal regression analyses, factors associated with not recent testing and never testing, compared with recent testing, were assessed among MSM living in highly (>2500 residences/km2) or non-highly (≤2500 residences/km2) urbanised areas. PARTICIPANTS: The study sample included 3815 MSM, currently living in the Netherlands. The mean age was 36 years (SD 14.7), and 67.6% were highly educated. RESULTS: In highly urbanised areas, 11.8% was never and 19.8% was not recently HIV-tested. In non-highly urbanised areas, this was 25.2% and 19.6%. Among MSM living in highly urbanised areas, independently associated with never and not recent testing were younger age, self-identification as bisexual, fewer sex partners, never notified of HIV and no recent condomless anal intercourse. Among MSM living in non-highly urbanised areas, lower perceived HIV severity, higher perceived HIV risk and a lower proportion gay friends were associated with never and not recent testing. Among never tested MSM, those in non-highly urbanised areas preferred self-sampling/self-testing over facility-based testing; those in highly urbanised areas preferred testing at healthcare facilities. CONCLUSIONS: The proportion of never tested MSM was high (25%) in non-highly urbanised areas in the Netherlands. MSM living in non-highly urbanised areas may possibly be reached with targeted approaches to increase HIV testing uptake such as self-testing/self-sampling strategies.

Culture-Independent Genotyping Revealed 3 Strain Clusters in a Potential Neisseria gonorrhoeae Outbreak in Young Heterosexuals (<25 Years), the Netherlands, October 2017 to March 2019.

BACKGROUND: Investigation was undertaken to determine the genetic relatedness of Neisseria gonorrhoeae (NG) isolates of young (<25 years) heterosexuals of a potential outbreak from October 2017 to March 2019 in South-Limburg, the Netherlands. METHODS: Data and residual sample material of routine diagnostics were retrieved for outbreak cases (78/81), young heterosexuals at baseline (January 2016 to September 2017, n = 30), and men who have sex with men (2018, n = 47). Total DNA was isolated, and NG was genotyped using culture-free NG multiantigen sequence typing. Sanger sequence data were used to construct a phylogenetic tree. Cases of outbreak clusters were geographically mapped, and descriptive analyses were performed on patient characteristics, comparing these clusters. RESULTS: Outbreak investigation showed 81 cases of young heterosexuals between October 2017 and March 2019 (4.5 per month) compared with 30 between January 2016 and September 2017 (1.4 per month), which was considered as baseline. Culture-independent genotyping of NG was performed to assess the genetic relatedness, as only 21 outbreak cases were culture confirmed. This revealed 3 independent outbreak clusters G2 (n = 18), G13113 (n = 11), and GNewST (n = 24). None of the clusters were geographically linked or introduced by bridging with men who have sex with men networks. Number of sex partners reported by men and Chlamydia trachomatis coinfection were associated with clusters G2 and GNewST, respectively. CONCLUSIONS: Culture-independent typing proved to be essential to identify the 3 outbreak clusters. However, targeted interventions were difficult because information on sex partners was limited. Therefore, prospective culture-independent typing could be used for early outbreak detection and aid in transmission prevention.

Event-Based Near-Eye Gaze Tracking Beyond 10,000 Hz.

The cameras in modern gaze-tracking systems suffer from fundamental bandwidth and power limitations, constraining data acquisition speed to 300 Hz realistically. This obstructs the use of mobile eye trackers to perform, e.g., low latency predictive rendering, or to study quick and subtle eye motions like microsaccades using head-mounted devices in the wild. Here, we propose a hybrid frame-event-based near-eye gaze tracking system offering update rates beyond 10,000 Hz with an accuracy that matches that of high-end desktop-mounted commercial trackers when evaluated in the same conditions. Our system, previewed in Figure 1, builds on emerging event cameras that simultaneously acquire regularly sampled frames and adaptively sampled events. We develop an online 2D pupil fitting method that updates a parametric model every one or few events. Moreover, we propose a polynomial regressor for estimating the point of gaze from the parametric pupil model in real time. Using the first event-based gaze dataset, we demonstrate that our system achieves accuracies of 0.45°-1.75° for fields of view from 45° to 98°. With this technology, we hope to enable a new generation of ultra-low-latency gaze-contingent rendering and display techniques for virtual and augmented reality.

Men and Women Have an Equal Oropharyngeal and Anorectal Chlamydia trachomatis Bacterial Load: A Comparison of 3 Anatomic Sites.

BACKGROUND: The Chlamydia trachomatis bacterial load could have impact on transmission and sequelae. This is the first study providing comparison of C. trachomatis load at 3 anatomic sites estimated by cycle quantification (Cq) values. METHODS: Data from 7900 C. trachomatis-positive samples were included (2012-2018). Cq value was used as an inversely proportional measure for C. trachomatis load. Multivariable linear regression analyses assessed differences in mean Cq values. RESULTS: Vaginal swabs had the lowest Cq values (31.0) followed by urine (32.5), anorectal swabs (34.0), and oropharyngeal swabs (36.8) (P < .001). Men and women had similar oropharyngeal (36.4 vs 37.3; P = .13) and anorectal (34.2 vs 33.9; P = .19) Cq values. Men (32.2) and women (30.7) aged <25 years had lower urogenital Cq values than men (32.8) and women (31.9) aged ≥25 years (P < .001). HIV-positive patients had higher urogenital Cq values than HIV-negative patients (33.8 vs 32.6; P < .03). CONCLUSIONS: Men and women have a similar C. trachomatis load at extragenital locations arguing for similar transmission potential and clinical relevance. Older patients and HIV-coinfected patients had lower C. trachomatis load, suggesting exposure to previous C. trachomatis infections potentially leading to partial immunity reducing load.

The characteristics of patients frequently tested and repeatedly infected with Chlamydia trachomatis in Southwest Limburg, the Netherlands.

BACKGROUND: Repeat Chlamydia trachomatis (CT) infections are common. To better understand the characteristics of patients frequently infected with CT at our sexually transmitted infection (STI) care services, we assessed the differences between patients repeatedly infected with CT and those who repeatedly tested negative. METHODS: In this cross-sectional analysis of cohort data, we assessed individuals tested for CT at different STI care providers between 2011 and mid-2018 in Southwest Limburg, the Netherlands (n = 17,616). Patients with ≥2 repeat CT infections in the study period were categorized as "patients with repeat CT infections." Multivariable logistic regression analyses were performed for the binary outcome measure: patients with repeat CT infections versus patients who repeatedly tested negative (reference group). Additional analyses were performed for only the STI clinic population. RESULTS: Patients aged < 25 years (OR: 1.83; 95%CI:1.38-2.43), co-infected with HIV (OR: 2.07; 95%CI: 1.02-4.22) or co-infected with Neisseria gonorrhoeae (NG) (OR: 5.04; 95%CI: 3.33-7.63) had more repeat CT infections. In additional analyses among exclusively STI clinic visitors, patients with urogenital symptoms (OR: 2.17; 95%CI: 1.41-3.35), and patients notified for STIs (OR: 4.55; 95%CI: 3.17-6.54) had more frequent repeat CT infections. CONCLUSIONS: Patients aged < 25 years and patients coinfected with HIV or NG had more frequent repeat CT infections, accounting for ~ 20% of the diagnosed CT infections. These patients are likely at the highest risk for transmitting and acquiring CT. Therefore, testing and retesting this group remains important to enhance CT control.

Men and Women Repeatedly Infected With Chlamydia trachomatis Have a Lower Urogenital Bacterial Load.

We assessed whether patients repeatedly infected with Chlamydia trachomatis (CT) have a lower urogenital or anorectal CT load. A CT-positive retest was independently associated with higher vaginal and higher urine Cq values (P<0.01). Partial immunity could play a role in repeat urogenital infections, potentially not in anorectal infections.

Neural Sensors: Learning Pixel Exposures for HDR Imaging and Video Compressive Sensing With Programmable Sensors.

Camera sensors rely on global or rolling shutter functions to expose an image. This fixed function approach severely limits the sensors' ability to capture high-dynamic-range (HDR) scenes and resolve high-speed dynamics. Spatially varying pixel exposures have been introduced as a powerful computational photography approach to optically encode irradiance on a sensor and computationally recover additional information of a scene, but existing approaches rely on heuristic coding schemes and bulky spatial light modulators to optically implement these exposure functions. Here, we introduce neural sensors as a methodology to optimize per-pixel shutter functions jointly with a differentiable image processing method, such as a neural network, in an end-to-end fashion. Moreover, we demonstrate how to leverage emerging programmable and re-configurable sensor-processors to implement the optimized exposure functions directly on the sensor. Our system takes specific limitations of the sensor into account to optimize physically feasible optical codes and we evaluate its performance for snapshot HDR and high-speed compressive imaging both in simulation and experimentally with real scenes.

Chlamydia trachomatis bacterial load, estimated by Cq values, in urogenital samples from men and women visiting the general practice, hospital or STI clinic.

BACKGROUND: The bacterial load of Chlamydia trachomatis (CT) is assumed to play a role in transmission and sequelae. We assessed urogenital CT cycle quantification (Cq) values, as an indicator for CT load, of men and women diagnosed by general practitioners (GPs), hospital physicians and the STI clinic. METHODS: Urogenital CT-positive samples (n = 2,055 vaginal swabs, n = 77 cervical swabs, n = 1,519 urine samples and n = 19 urethral swabs) diagnosed by GPs, hospital physicians and the STI clinic from the Maastricht Medical Microbiology Laboratory were included (2012-2016). The outcome measure 'urogenital Cq values' was used as an inversely proportional measure for CT load. Among all patients, multivariate linear regression analyses were used to assess primary determinants for mean urogenital Cq values, stratified by sex. Additional clinical determinants were assessed among STI clinic patients. RESULTS: In men, mean urogenital Cq values were similar between GPs, hospital physicians and the STI clinic (32.7 and 33.5 vs. 32.7; p>0.05). Women visiting the GP had lower urogenital Cq values than women visiting the STI clinic (30.2 vs. 30.9; p = <0.001). Women visiting the hospital had higher urogenital Cq values than women visiting the STI clinic (32.4 vs. 30.9; p = <0.001). Among STI clinic women, urogenital Cq values were lower in women with concurrent anorectal CT and in rectally untested women compared to anorectal CT-negative women (30.7 and 30.6 vs. 33.9; p = <0.001). CONCLUSION: Men visiting different STI care providers had similar urogenital Cq values, which could be an indicator for similar CT loads. The lower Cq values of women visiting the GP compared to women visiting the STI clinic could be an indicator for higher CT loads and likely higher transmission potential. Notably, urogenital Cq values of STI clinic women were much lower (>3 Cq) when STI clinic women also had anorectal CT. This finding could indicate higher urogenital CT loads and likely higher chances of transmission and sequelae.

Cardiovascular disease after childhood acute lymphoblastic leukaemia: a cohort study.

BACKGROUND AND AIMS: Cardiovascular diseases (CVD) increase late morbidity and mortality in survivors of acute lymphoblastic leukaemia (ALL). We compared the risk of CVD in ALL survivors to siblings, examined time trends, quantified treatment-related risks, and investigated whether risk extends beyond patients treated with anthracyclines and chest radiotherapy. METHODS: The Swiss Childhood Cancer Survivor Study assessed CVD by patient questionnaire in 5-year ALL survivors diagnosed between 1976 and 2005 and their siblings. Participants were asked whether a physician had ever told them that they had hypertension, arrhythmia, heart failure, myocardial infarction, angina pectoris, stroke, thrombosis or valvular problems. We investigated treatment-related risk factors for CVD using multivariable logistic regression, adjusting for demographic and socioeconomic factors, BMI, smoking, diabetes mellitus, alcohol consumption and physical activity. RESULTS: We contacted 707 survivors and 1299 siblings, 511 (72%) and 709 (55%) of whom responded, respectively. Survivors had a higher risk of developing CVD than siblings (odds ratio [OR] 1.9, 95% confidence interval 1.3–2.8), in particular heart failure (OR 13.9, 1.8–107.4). Compared to patients treated 1976–85, the risk of CVD was 1.4 (0.7–2.8) for those treated 1985–1994 and 1.5 (0.6–3.7) for those treated 1995–2005. The overall CVD risks after anthracycline treatment (OR 3.1, 2.0–4.7), haematopoietic stem cell transplantation (OR 8.0, 2.4–26.9) or relapse (OR 4.1, 1.9–8.8) were increased compared to those of siblings, while the CVD risks of survivors treated without anthracycline or chest radiotherapy were similar (OR 1.0; 0.5–2.0). CONCLUSIONS: Despite attempts to reduce cardiotoxicity in childhood cancer treatment, CVD risks in ALL survivors treated more recently do not seem to have declined.

Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner.

Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner.

MPK-1/ERK pathway regulates DNA damage response during development through DAF-16/FOXO.

Ultraviolet (UV) induces distorting lesions to the DNA that can lead to stalling of the RNA polymerase II (RNAP II) and that are removed by transcription-coupled nucleotide excision repair (TC-NER). In humans, mutations in the TC-NER genes CSA and CSB lead to severe postnatal developmental defects in Cockayne syndrome patients. In Caenorhabditis elegans, mutations in the TC-NER genes csa-1 and csb-1, lead to developmental growth arrest upon UV treatment. We conducted a genetic suppressor screen in the nematode to identify mutations that could suppress the developmental defects in csb-1 mutants. We found that mutations in the ERK1/2 MAP kinase mpk-1 alleviate the developmental retardation in TC-NER mutants, while constitutive activation of the RAS-MAPK pathway exacerbates the DNA damage-induced growth arrest. We show that MPK-1 act via insulin/insulin-like signaling pathway and regulates the FOXO transcription factor DAF-16 to mediate the developmental DNA damage response.

Test of cure, retesting and extragenital testing practices for Chlamydia trachomatis and Neisseria gonorrhoeae among general practitioners in different socioeconomic status areas: A retrospective cohort study, 2011-2016.

BACKGROUND: For Chlamydia trachomatis (CT), a test of cure (TOC) within 3-5 weeks is not recommended. International guidelines differ in advising a Neisseria gonorrhoeae (NG) TOC. Retesting CT and NG positives within 3-12 months is recommended in international guidelines. We assessed TOC and retesting practices including extragenital testing in general practitioner (GP) practices located in different socioeconomic status (SES) areas to inform and optimize local test practices. METHODS: Laboratory data of 48 Dutch GP practices between January 2011 and July 2016 were used. Based on a patient's first positive CT or NG test, the proportion of TOC (<3 months) and retests (3-12 months) were calculated. Patient- and GP-related factors were assessed using multivariate logistic regression analyses. RESULTS: For CT (n = 622), 20% had a TOC and 24% had a retest at the GP practice. GP practices in low SES areas were more likely to perform a CT TOC (OR:1.8;95%CI:1.1-3.1). Younger patients (<25 years) were more likely to have a CT TOC (OR:1.6;95%CI:1.0-2.4). For CT (n = 622), 2.4% had a TOC and 6.1% had a retest at another STI care provider. For NG (n = 73), 25% had a TOC and 15% had a retest at the GP practice. For NG (n = 73), 2.7% had a TOC and 12.3% had a retest at another STI care provider. In only 0.3% of the consultations patients were tested on extragenital sites. CONCLUSION: Almost 20% of the patients returned for a CT TOC, especially at GP practices in low SES areas. For NG, 1 out of 4 patients returned for a TOC. Retesting rates were low for both CT (24%) and NG (15%), (re)infections including extragenital infections may be missed. Efforts are required to focus TOC and increase retesting practices of GPs in order to improve CT/NG control.

Vagal and recurrent laryngeal nerves neuromonitoring during thyroidectomy and parathyroidectomy: A prospective study.

OBJECTIVES: The aim of the study was to stimulate the vagal and the recurrent laryngeal nerves during and after thyroidectomy or parathyroidectomy, to record muscle responses, interpret the electrophysiological modifications and identify prognostic factors for postoperative vocal fold mobility. PATIENTS AND METHODS: A prospective study monitored 151 vagal nerves and 144 recurrent laryngeal nerves in 114 patients. Seven patients (14 vagal nerves) underwent continuous monitoring via an automatic periodic stimulation (APS®) electrode. In 15 patients (21 vagal nerves), the stimulation threshold was studied. Muscle response was recorded on direct vagal and/or recurrent laryngeal nerve stimulation by a monopolar electrode or direct repeated stimulation via an electrode on the vagal nerve. In case of signal attenuation on the first operated side, surgery was not extended to the contralateral side. RESULTS: The vagal nerve stimulation checked inferior laryngeal nerve integrity and recurrent status, without risk of false negatives. The vagal nerve stimulation threshold, before and after dissection, that induced a muscle response of at least 100µV ranged from 0.1 to 0.8mA. Similarity between pre- and post-dissection responses to supramaximal stimulation, defined as 1mA, on the one hand, and between post-dissection vagal and laryngeal recurrent nerve responses on the other correlated with normal postoperative vocal cord mobility. Conversely, muscle response attenuation below 100µV and increased latency indicated a risk of vocal fold palsy. CONCLUSION: Vagal nerve stimulation allows suspicion or elimination of lesions on the inferior laryngeal nerve upstream of the stimulation point and detection of non-recurrent inferior laryngeal nerve. Intermittent monitoring assesses nerve function at the moment of stimulation, while continuous monitoring detects the first signs of nerve injury liable to induce postoperative recurrent nerve palsy. When total thyroidectomy is indicated, signal attenuation on the first operated side casts doubt on continuing surgery to the contralateral side in the same step.

Candidate sampling for neuron reconstruction from anisotropic electron microscopy volumes.

The automatic reconstruction of neurons from stacks of electron microscopy sections is an important computer vision problem in neuroscience. Recent advances are based on a two step approach: First, a set of possible 2D neuron candidates is generated for each section independently based on membrane predictions of a local classifier. Second, the candidates of all sections of the stack are fed to a neuron tracker that selects and connects them in 3D to yield a reconstruction. The accuracy of the result is currently limited by the quality of the generated candidates. In this paper, we propose to replace the heuristic set of candidates used in previous methods with samples drawn from a conditional random field (CRF) that is trained to label sections of neural tissue. We show on a stack of Drosophila melanogaster neural tissue that neuron candidates generated with our method produce 30% less reconstruction errors than current candidate generation methods. Two properties of our CRF are crucial for the accuracy and applicability of our method: (1) The CRF models the orientation of membranes to produce more plausible neuron candidates. (2) The interactions in the CRF are restricted to form a bipartite graph, which allows a great sampling speed-up without loss of accuracy.

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing.

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.

Intraoperative laryngeal nerve monitoring during thyroidectomy and parathyroidectomy: A prospective study.

OBJECTIVES: The aim of this study was to stimulate the recurrent laryngeal nerve during thyroidectomy or parathyroidectomy and to record the muscle responses in an attempt to predict postoperative vocal fold mobility. PATIENTS AND METHODS: Intraoperative recurrent laryngeal nerve monitoring during general anaesthesia was performed by using an electrode-bearing endotracheal tube (nerve integrity monitor EMG endotracheal tube [Medtronic Xomed, Jacksonville, Flo, USA]). Two hundred and fifteen recurrent laryngeal nerves from 141 patients undergoing total thyroidectomy (n=74), hemithyroidectomy (n=63), or parathyroidectomy (n=4) were prospectively monitored. In each case, the muscle potential was recorded after stimulation of the recurrent laryngeal nerve by a monopolar probe. RESULTS: The nerve stimulation threshold before and after dissection that induced a muscle response of at least 100 µV ranged from 0.1 to 0.85 mA (mean 0.4 mA). The supramaximal stimulation intensity was defined as 1 mA. The amplitude of muscle response varied considerably from one patient to another, but the similarity of the muscle response at supramaximal intensity between pre- and postdissection and between postdissection at the proximal and distal exposed portions of the nerve was correlated with normal postoperative vocal fold function. Inversely, alteration of the muscle response indicated a considerable risk of recurrent laryngeal nerve palsy, but was not predictive of whether or not this lesion would be permanent. CONCLUSIONS: Recurrent laryngeal nerve monitoring with a system using surface electrodes is a simple, non-invasive technique that is just as sensitive as monitoring by intramuscular electrodes. Monitoring is helpful for initial nerve identification and is useful to determine nerve function during and after surgery, and to adapt the surgical strategy accordingly.

RNAi-based screening identifies the Mms22L-Nfkbil2 complex as a novel regulator of DNA replication in human cells.

Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)-Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L-Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks.