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Background: Lemierre syndrome is characterized by head/neck vein thrombosis and septic embolism usually complicating an acute oropharyngeal bacterial infection in adolescents and young adults. We described the course of Lemierre syndrome in the contemporary era. Methods: In our individual‐level analysis of 712 patients (2000–2017), we included cases described as Lemierre syndrome if these criteria were met: (i) primary site of bacterial infection in the head/neck; (ii) objectively confirmed local thrombotic complications or septic embolism. The study outcomes were new or recurrent venous thromboembolism or peripheral septic lesions, major bleeding, all‐cause death and clinical sequelae. Results: The median age was 21 (Q1–Q3: 17–33) years, and 295 (41%) were female. At diagnosis, acute thrombosis of head/neck veins was detected in 597 (84%) patients, septic embolism in 582 (82%) and both in 468 (80%). After diagnosis and during in‐hospital follow‐up, new venous thromboembolism occurred in 34 (5.2%, 95% CI 3.8–7.2%) patients, new peripheral septic lesions became evident in 76 (11.7%; 9.4–14.3%). The rate of either was lower in patients who received anticoagulation (OR: 0.59; 0.36–0.94), higher in those with initial intracranial involvement (OR: 2.35; 1.45–3.80). Major bleeding occurred in 19 patients (2.9%; 1.9–4.5%), and 26 died (4.0%; 2.7–5.8%). Clinical sequelae were reported in 65 (10.4%, 8.2–13.0%) individuals, often consisting of cranial nerve palsy (n = 24) and orthopaedic limitations (n = 19). Conclusions: Patients with Lemierre syndrome were characterized by a substantial risk of new thromboembolic complications and death. This risk was higher in the presence of initial intracranial involvement. One‐tenth of survivors suffered major clinical sequelae. [ABSTRACT FROM AUTHOR]

Until recently, most patients with sentinel lymph node-positive (SLN+) melanoma underwent a completion lymph node dissection (CLND), as mandated in published trials of adjuvant systemic therapies. Following multicenter selective lymphadenectomy trial-II, most patients with SLN+ melanoma no longer undergo a CLND prior to adjuvant systemic therapy. A retrospective analysis of clinical outcomes in SLN+ melanoma patients treated with adjuvant systemic therapy after July 2017 was performed in 21 international cancer centers. Of 462 patients who received systemic adjuvant therapy, 326 patients received adjuvant anti-PD-1 without prior immediate (IM) CLND, while 60 underwent IM CLND. With median follow-up of 21 months, 24-month relapse-free survival (RFS) was 67% (95% CI 62% to 73%) in the 326 patients. When the patient subgroups who would have been eligible for the two adjuvant anti-PD-1 clinical trials mandating IM CLND were analyzed separately, 24-month RFS rates were 64%, very similar to the RFS rates from those studies. Of these no-CLND patients, those with SLN tumor deposit >1 mm, stage IIIC/D and ulcerated primary had worse RFS. Of the patients who relapsed on adjuvant anti-PD-1, those without IM CLND had a higher rate of relapse in the regional nodal basin than those with IM CLND (46% vs 11%). Therefore, 55% of patients who relapsed without prior CLND underwent surgery including therapeutic lymph node dissection (TLND), with 30% relapsing a second time; there was no difference in subsequent relapse between patients who received observation vs secondary adjuvant therapy. Despite the increased frequency of nodal relapses, adjuvant anti-PD-1 therapy may be as effective in SLN+ pts who forego IM CLND and salvage surgery with TLND at relapse may be a viable option for these patients., Competing Interests: Competing interests: ZE: Advisory boards: Array, Pfizer, OncoSec, Regeneron, Genentech, Novartis, Natera. Research funding: Novartis, Pfizer, Boehringer-Ingelheim. JFT: Advisory boards: BMS Australia, MSD Australia, GSK, Provectus Inc. Travel support: GSK, Provectus Inc and Novartis. TJH: Research funding: Genentech, SkylineDX. EKB: Research funding: SkylineDx, Honorarium: Excite International inc. DEG: Honoraria: BMS, Novartis, Q biotics. GK; Advisory Board: Merck. AVA: Advisory Boards: Amgen, Bristol-Myers Squibb, Novartis, MSD-Merck, Merck-Pfizer, Pierre Fabre, Provectus, Sanofi, Sirius Medical, 4SC Research funding: Amgen, Merck-Pfizer. JV: Speaker: Caste Biosciences. GB: Research funding: Istari Oncology, Delcath, Oncosec Medical, Replimune, Checkmate Pharmaceuticals. ROB: Advisory boards: Amgen, BD/BARD, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Roche and Sanofi Genzyme. Research funding: Bristol-Myers Squibb, SkyLineDx. Speaker honorarium: Roche and Pfizer. Shareholder in SATMEG Ventures AB. NIK: Advisory boards: Bristol-Myers Squibb, AstraZeneca, Regeneron, Array, Immunocore, Merck, Incyte, Jounce Therapeutics,Pfizer,Novartis, Nektar,Castle Biosciences, Instil Bio. Research funding: Bristol-Myers Squibb, Merck, Novartis, Celgene, Replimune, Amgen, Regneron, HUYA, GlaxoSmithKline Stocks: Bellicum Pharmaceuticals, Amarin Corporation, Asensus Surgical., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The complex processes and interactions that regulate aging and determine lifespan are not fully defined for any organism. Here, taking advantage of recent technological advances in studying aging in budding yeast, we discovered a previously unappreciated relationship between the number of copies of the ribosomal RNA gene present in its chromosomal array and replicative lifespan (RLS). Specifically, the chromosomal ribosomal DNA (rDNA) copy number (rDNA CN) positively correlated with RLS and this interaction explained over 70% of variability in RLS among a series of wild-type strains. In strains with low rDNA CN, SIR2 expression was attenuated and extrachromosomal rDNA circle (ERC) accumulation was increased, leading to shorter lifespan. Suppressing ERC formation by deletion of FOB1 eliminated the relationship between rDNA CN and RLS. These data suggest that previously identified rDNA CN regulatory mechanisms limit lifespan. Importantly, the RLSs of reported lifespan-enhancing mutations were significantly impacted by rDNA CN, suggesting that changes in rDNA CN might explain the magnitude of some of those reported effects. We propose that because rDNA CN is modulated by environmental, genetic, and stochastic factors, considering rDNA CN is a prerequisite for accurate interpretation of lifespan data.

Hollow gold nanospheres (HGNs) have been used as the template for seed-mediated growth of multibranched hollow gold nanostars (HNS). The HGNs were synthesized via anerobic reduction of cobalt chloride to cobalt nanoparticles and then formation of a gold shell via galvanic replacement followed by the oxidation of the cobalt core. We obtained control of the inner core size of the HGNs by increasing the size of the sacrificial cobalt core and by varying the ratio of B(OH) 3 /BH 4 using boric acid rather than 48 h aged borohydride. We synthesized the HNS by reducing Au 3+ ions in the presence of Ag + ions using ascorbic acid, creating a spiky morphology that varied with the Au 3+ /Ag + ratio. A broadly tunable localized surface plasmon resonance was achieved through control of both the inner core and the spike length. Amyloid beta (Aβ) was conjugated to the HNS by using a heterobifunctional PEG linker and identified by the vibrational modes associated with the conjugated ring phenylalanine side chain. A bicinchoninic acid assay was used to determine the concentration of Aβ conjugated to HNS as 20 nM, which is below the level of Aβ that negatively affects long-term potentiation. Both the core size and spike length were shown to affect the optical properties of the resulting nanostructures. This HGN templated method introduced a new parameter for enhancing the plasmonic properties of gold nanostars, namely, the addition of a hollow core. Hollow gold nanostars are highly desirable for a wide range of applications, including high sensitivity disease detection and monitoring.

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z 3 EB42, that gives lower expression than Z 3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Background and aimHepatitis E virus (HEV) is a virus of emerging importance to transfusion medicine. Studies from several European countries, including Switzerland, have reported high seroprevalence of hepatitis E as a consequence of endemic infections. Published HEV seroprevalence estimates within developed countries vary considerably; primarily due to improved diagnostic assays. The purpose of this study was to investigate the seroprevalence of anti-HEV IgG in Swiss blood donations. Methods: We used the highly sensitive Wantai HEV IgG EIA and assessed regional distribution patterns. We analysed age- and sex-matched archive plasma dating back 20 years from canton Bern to investigate recent changes in HEV seroprevalence levels. Results: On average, 20.4% (95% confidence intervals: 19.1-21.8) of the 3,609 blood samples collected in 2014-16 were anti-HEV IgG positive; however, distinct differences between geographical regions were observed (range: 12.8-33.6%). Seroprevalence increased with age with 30.7% of males and 34.3% of women being positive donors over > 60 years old. Differences between sexes may be attributed to dissimilarities in the average age of this group. Within the specified region of the Bern canton, overall prevalence has declined over two decades from 30.3% in 1997/98 to 27.0% in 2006 and 22.3% in 2015/6. Conclusions: HEV seroprevalence in Switzerland is high, but has declined over the last decades. The result shows that primarily endemic HEV infections occur and that current blood products may pose a risk to vulnerable transfusion recipients. Nucleic acid screening of all blood products for HEV will begin in November 2018.

Chemotaxis is a specialized form of directed cell migration important for normal development, wound healing, and cancer metastasis. In the social amoeba Dictyostelium discoideum, four signaling pathways act synergistically to maintain directional cell migration. However, it is unknown how these pathways are coordinated in space and time to achieve persistent chemotaxis. Here, we show that the mRNAs and proteins of these four chemotaxis pathways and actin are preferentially enriched at the cell front during dynamic cell migration, which requires the Pumilio-related RNA-binding protein Puf118. Significantly, disruption of the Pumilio-binding sequence in chemotaxis pathway mRNAs, or mislocalization of Puf118 and its target mRNAs to the cell rear perturbs efficient chemotaxis in shallow cAMP gradients, without affecting the abundance of the mRNAs or encoded proteins. Thus, the polarized localization of Puf118-bound mRNAs coordinates the distribution of different chemotaxis pathway proteins in time and space, leading to cell polarization and persistent chemotaxis.

Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by "friendly fire", a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection., Competing Interests: We acknowledge a potential competing interest. T.S.C., O.J., M.H., L.C., J.S., C.K.S., and B.S. are employees of MedImmune L.L.C. L.M. receives funding from MedImmune L.L.C.

The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.

Like many asymmetrically dividing cells, budding yeast segregates mitotic spindle poles nonrandomly between mother and daughter cells. During metaphase, the spindle positioning protein Kar9 accumulates asymmetrically, localizing specifically to astral microtubules emanating from the old spindle pole body (SPB) and driving its segregation to the bud. Here, we show that the SPB component Nud1/centriolin acts through the mitotic exit network (MEN) to specify asymmetric SPB inheritance. In the absence of MEN signaling, Kar9 asymmetry is unstable and its preference for the old SPB is disrupted. Consistent with this, phosphorylation of Kar9 by the MEN kinases Dbf2 and Dbf20 is not required to break Kar9 symmetry but is instead required to maintain stable association of Kar9 with the old SPB throughout metaphase. We propose that MEN signaling links Kar9 regulation to SPB identity through biasing and stabilizing the age-insensitive, cyclin-B-dependent mechanism of symmetry breaking., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A recent analysis of spindle positioning in yeast sheds light on how interactions between microtubules and the cleavage apparatus are modulated through mitosis to promote and maintain proper positioning of the spindle and cleavage plane relative to each other., (2010 Elsevier Ltd. All rights reserved.)

Aims: Whereas cortical EEG effects of benzodiazepines are well characterized, information about benzodiazepine effects in other areas of the central nervous system is sparse. This study investigated the action of midazolam and its active metabolite alpha-hydroxy-midazolam on different parts of the auditory pathway in six healthy volunteers in a randomized, double-blind, three-way cross-over study., Methods: Acoustically evoked short (SLP) and middle (MLP) latency potentials, transitory evoked otoacoustic emissions (TEOAE), and EEG power spectra were analysed after short i. v. injections of placebo, or 0.15 mg kg-1 midazolam, or alpha-hydroxy-midazolam, respectively., Results: All subjects fell asleep during the 4 min infusion of active drug. SLP showed a significant transient increase of Jewett wave V 10 min after injection for midazolam and alpha-hydroxy-midazolam while the latency of wave I was unchanged. Both benzodiazepines induced a marked and long-lasting MLP amplitude decrease for 240 min with slow recovery over the following 360 min. No changes of TEOAE were observed. In agreement with earlier reports, increases in EEG beta activity and decreases in alpha activity were observed after administration of either drug., Conclusions: Systemically administered benzodiazepines modulate the auditory pathway above the level of the cochlea. While SLP changes were closely associated with sedation and high plasma benzodiazepine concentrations, MLP effects persisted for hours after sedation even at low benzodiazepine plasma levels. Evoked potentials may therefore be more sensitive than EEG as a tool to monitor benzodiazepine effects.

Exposure of exponentially growing human promyelocytic of lymphocytic leukemic cells to the putative DNA topoisomerase II inhibitor fostriecin (FST), at a concentration of 1 microM, results in the suppression of their rate of progression through the S and G2 phases of the cell cycle. At concentrations between 5 microM and 0.5 mM, FST triggers endonucleolytic DNA degradation in human promyelocytic leukemia cells, resulting in apoptotic cell death; this effect is not selective for any particular phase of the cell cycle. Little or no apoptotic cell death is observed in lymphocytic leukemic cells at any FST concentration. Because FST, unlike other inhibitors of topoisomerase II, such as teniposide (TN) or amsacrine (m-AMSA), does not stabilize cleavable DNA-topoisomerase complexes, the observed differences between the effects of FST versus TN or m-AMSA on the cell cycle may provide clues regarding the role of such complexes in the kinetic effects of these inhibitors. The present results, therefore, are compared with our earlier data on the effects of TN and m-AMSA on the same cells. The only observed difference is the loss of cell cycle phase-specific triggering of DNA degradation by FST in human promyelocytic leukemia cells, compared to the S phase-specific effects of TN and m-AMSA. Therefore, stabilization of the DNA-topoisomerase cleavable complexes may be essential in the selectivity of cell kill during S phase. However, it appears that the presence of stabilized complexes is not essential to the suppression of cell progression through S or G2 or the induction of apoptotis or necrosis, in general, by topoisomerase II inhibitors.

Two samples of complete cleft cases between five and seven years of age are compared (11 UCLP + 9 BCLP versus 21 UCLP + 12 BCLP). The same early orthopedic procedures were applied to all cases, but surgical management differed for the two groups: "classical" surgery (lip 3 months, palate 2--2 1/2 years) versus delayed conservative surgery (lip 6 months, soft palate 18 months, hard palate 6--8 years). It is evident that early maxillary orthopedic treatment is of little consequence for long-term development unless concomitant surgery complies with growth and functional requirements. In contrast to "classical" surgery, palatal closure in two steps is more favourable to skeletal growth and does not interfere to any relevant degree with speech development.