First-in-human prediction of chronic pain state using intracranial neural biomarkers.

Chronic pain syndromes are often refractory to treatment and cause substantial suffering and disability. Pain severity is often measured through subjective report, while objective biomarkers that may guide diagnosis and treatment are lacking. Also, which brain activity underlies chronic pain on clinically relevant timescales, or how this relates to acute pain, remains unclear. Here four individuals with refractory neuropathic pain were implanted with chronic intracranial electrodes in the anterior cingulate cortex and orbitofrontal cortex (OFC). Participants reported pain metrics coincident with ambulatory, direct neural recordings obtained multiple times daily over months. We successfully predicted intraindividual chronic pain severity scores from neural activity with high sensitivity using machine learning methods. Chronic pain decoding relied on sustained power changes from the OFC, which tended to differ from transient patterns of activity associated with acute, evoked pain states during a task. Thus, intracranial OFC signals can be used to predict spontaneous, chronic pain state in patients.

Don't Ditch the Laptop Just Yet: A Direct Replication of Mueller and Oppenheimer's (2014) Study 1 Plus Mini Meta-Analyses Across Similar Studies.

In this direct replication of Mueller and Oppenheimer's (2014) Study 1, participants watched a lecture while taking notes with a laptop (n = 74) or longhand (n = 68). After a brief distraction and without the opportunity to study, they took a quiz. As in the original study, laptop participants took notes containing more words spoken verbatim by the lecturer and more words overall than did longhand participants. However, laptop participants did not perform better than longhand participants on the quiz. Exploratory meta-analyses of eight similar studies echoed this pattern. In addition, in both the original study and our replication, higher word count was associated with better quiz performance, and higher verbatim overlap was associated with worse quiz performance, but the latter finding was not robust in our replication. Overall, results do not support the idea that longhand note taking improves immediate learning via better encoding of information.

Origin of rebound virus in chronically SIV-infected Rhesus monkeys following treatment discontinuation.

Viral rebound following antiretroviral therapy (ART) discontinuation in HIV-1-infected individuals is believed to originate from a small pool of CD4+ T cells harboring replication-competent provirus. However, the origin and nature of the rebound virus has remained unclear. Recent studies have suggested that rebound virus does not originate directly from individual latent proviruses but rather from recombination events involving multiple proviruses. Here we evaluate the origin of rebound virus in 16 ART-suppressed, chronically SIV-infected rhesus monkeys following ART discontinuation. We sequence viral RNA and viral DNA in these animals prior to ART initiation, during ART suppression, and following viral rebound, and we compare rebound viral RNA after ART discontinuation with near full-length viral DNA from peripheral blood and lymph node mononuclear cells (PBMC and LNMC) during ART suppression. Sequences of initial rebound viruses closely match viral DNA sequences in PBMC and LNMC during ART suppression. Recombinant viruses are rare in the initial rebound virus populations but arise quickly within 2-4 weeks after viral rebound. These data suggest that intact proviral DNA in PBMC and LNMC during ART suppression is likely the direct origin of viral rebound in chronically SIV-infected rhesus monkeys following ART discontinuation.

Discovery of GS-9688 (Selgantolimod) as a Potent and Selective Oral Toll-Like Receptor 8 Agonist for the Treatment of Chronic Hepatitis B.

Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC50 > 50 µM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:(R)-7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.

Blood-brain barrier dysfunction in aging induces hyperactivation of TGFß signaling and chronic yet reversible neural dysfunction.

Aging involves a decline in neural function that contributes to cognitive impairment and disease. However, the mechanisms underlying the transition from a young-and-healthy to aged-and-dysfunctional brain are not well understood. Here, we report breakdown of the vascular blood-brain barrier (BBB) in aging humans and rodents, which begins as early as middle age and progresses to the end of the life span. Gain-of-function and loss-of-function manipulations show that this BBB dysfunction triggers hyperactivation of transforming growth factor-ß (TGFß) signaling in astrocytes, which is necessary and sufficient to cause neural dysfunction and age-related pathology in rodents. Specifically, infusion of the serum protein albumin into the young rodent brain (mimicking BBB leakiness) induced astrocytic TGFß signaling and an aged brain phenotype including aberrant electrocorticographic activity, vulnerability to seizures, and cognitive impairment. Furthermore, conditional genetic knockdown of astrocytic TGFß receptors or pharmacological inhibition of TGFß signaling reversed these symptomatic outcomes in aged mice. Last, we found that this same signaling pathway is activated in aging human subjects with BBB dysfunction. Our study identifies dysfunction in the neurovascular unit as one of the earliest triggers of neurological aging and demonstrates that the aging brain may retain considerable latent capacity, which can be revitalized by therapeutic inhibition of TGFß signaling.

Structure-guided discovery of a novel, potent, and orally bioavailable 3,5-dimethylisoxazole aryl-benzimidazole BET bromodomain inhibitor.

The bromodomain and extra-terminal (BET) family of proteins, consisting of the bromodomains containing protein 2 (BRD2), BRD3, BRD4, and the testis-specific BRDT, are key epigenetic regulators of gene transcription and has emerged as an attractive target for anticancer therapy. Herein, we describe the discovery of a novel potent BET bromodomain inhibitor, using a systematic structure-based approach focused on improving potency, metabolic stability, and permeability. The optimized dimethylisoxazole aryl-benzimidazole inhibitor exhibited high potency towards BRD4 and related BET proteins in biochemical and cell-based assays and inhibited tumor growth in two proof-of-concept preclinical animal models.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

Optical Probes for Neurobiological Sensing and Imaging.

Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells. Over the past decade, significant technological advances in optical probes and imaging methods have enabled entirely new possibilities for studying neural cells and circuits at the chemical level. These optical imaging modalities have shown promise for combining chemical, temporal, and spatial information. This potential makes them ideal candidates to unravel the complex neural interactions at multiple scales in the brain, which could be complemented by traditional electrophysiological methods to obtain a full spatiotemporal picture of neurochemical dynamics. Despite the potential, only a handful of probe candidates have been utilized to provide detailed chemical information in the brain. To date, most live imaging and chemical mapping studies rely on fluorescent molecular indicators to report intracellular calcium (Ca2+) dynamics, which correlates with neuronal activity. Methodological advances for monitoring a full array of chemicals in the brain with improved spatial, temporal, and chemical resolution will thus enable mapping of neurochemical circuits with finer precision. On the basis of numerous studies in this exciting field, we review the current efforts to develop and apply a palette of optical probes and nanosensors for chemical sensing in the brain. There is a strong impetus to further develop technologies capable of probing entire neurobiological units with high spatiotemporal resolution. Thus, we introduce selected applications for ion and neurotransmitter detection to investigate both neurons and non-neuronal brain cells. We focus on families of optical probes because of their ability to sense a wide array of molecules and convey spatial information with minimal damage to tissue. We start with a discussion of currently available molecular probes, highlight recent advances in genetically modified fluorescent probes for ions and small molecules, and end with the latest research in nanosensors for biological imaging. Customizable, nanoscale optical sensors that accurately and dynamically monitor the local environment with high spatiotemporal resolution could lead to not only new insights into the function of all cell types but also a broader understanding of how diverse neural signaling systems act in conjunction with neighboring cells in a spatially relevant manner.

Astrocyte-derived interleukin-33 promotes microglial synapse engulfment and neural circuit development.

Neuronal synapse formation and remodeling are essential to central nervous system (CNS) development and are dysfunctional in neurodevelopmental diseases. Innate immune signals regulate tissue remodeling in the periphery, but how this affects CNS synapses is largely unknown. Here, we show that the interleukin-1 family cytokine interleukin-33 (IL-33) is produced by developing astrocytes and is developmentally required for normal synapse numbers and neural circuit function in the spinal cord and thalamus. We find that IL-33 signals primarily to microglia under physiologic conditions, that it promotes microglial synapse engulfment, and that it can drive microglial-dependent synapse depletion in vivo. These data reveal a cytokine-mediated mechanism required to maintain synapse homeostasis during CNS development.

Discovery of a 2'-fluoro-2'-C-methyl C-nucleotide HCV polymerase inhibitor and a phosphoramidate prodrug with favorable properties.

A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.

Diastereoselective synthesis of (±)-heliotropamide by a one-pot, four-component reaction.

The first synthesis of heliotropamide is reported. The preparation of this 2-oxopyrrolidine (γ-lactam) natural product relied on a diastereoselective one-pot, four-component reaction (4CR) for the assembly of the core structure. On the basis of chemical shift correlation and NOESY experiments, the previously unknown alkene geometry of heliotropamide is assigned as E.

C. elegans germ cells switch between distinct modes of double-strand break repair during meiotic prophase progression.

Chromosome inheritance during sexual reproduction relies on deliberate induction of double-strand DNA breaks (DSBs) and repair of a subset of these breaks as interhomolog crossovers (COs). Here we provide a direct demonstration, based on our analysis of rad-50 mutants, that the meiotic program in Caenorhabditis elegans involves both acquisition and loss of a specialized mode of double-strand break repair (DSBR). In premeiotic germ cells, RAD-50 is not required to load strand-exchange protein RAD-51 at sites of spontaneous or ionizing radiation (IR)-induced DSBs. A specialized meiotic DSBR mode is engaged at the onset of meiotic prophase, coincident with assembly of meiotic chromosome axis structures. This meiotic DSBR mode is characterized both by dependence on RAD-50 for rapid accumulation of RAD-51 at DSB sites and by competence for converting DSBs into interhomolog COs. At the mid-pachytene to late pachytene transition, germ cells undergo an abrupt release from the meiotic DSBR mode, characterized by reversion to RAD-50-independent loading of RAD-51 and loss of competence to convert DSBs into interhomolog COs. This transition in DSBR mode is dependent on MAP kinase-triggered prophase progression and coincides temporally with a major remodeling of chromosome architecture. We propose that at least two developmentally programmed switches in DSBR mode, likely conferred by changes in chromosome architecture, operate in the C. elegans germ line to allow formation of meiotic crossovers without jeopardizing genomic integrity. Our data further suggest that meiotic cohesin component REC-8 may play a role in limiting the activity of SPO-11 in generating meiotic DSBs and that RAD-50 may function in counteracting this inhibition.

Induction of apoptosis by monastrol, an inhibitor of the mitotic kinesin Eg5, is independent of the spindle checkpoint.

Spindle poisons such as paclitaxel are widely used as cancer therapeutics. By interfering with microtubule dynamics, paclitaxel induces mitotic arrest and apoptosis. Targeting the kinesin Eg5, which is required for the formation of a bipolar spindle, is a promising therapeutic alternative to drugs that interfere with microtubule dynamics. Recent data suggest that the spindle checkpoint can determine the response of tumor cells to microtubule poisons. The relationship between checkpoint function and Eg5 inhibition, however, has not yet been fully investigated. Here, we used time-lapse video microscopy and biochemical analysis to study the effect of spindle checkpoint abrogation on the response of HeLa cells to monastrol, a selective Eg5 inhibitor. In HeLa cells, monastrol activated the spindle checkpoint, leading to mitotic arrest and apoptosis. Small interfering RNA-mediated depletion of the spindle checkpoint proteins BubR1 or Mad2 significantly shortened drug-induced arrest, causing premature mitotic exit without cell division. Time-lapse microscopy as well as analysis of caspase activation shows that these checkpoint-deficient cells initiate apoptosis after mitotic exit in response to monastrol. Checkpoint-deficient cells treated with paclitaxel, on the other hand, yielded a higher frequency of cells with >4N DNA content and a decreased incidence of apoptotic events, particularly in Mad2-depleted cells. These results indicate that the immediate fate of postmitotic cells is influenced by both the nature of the checkpoint defect and the type of drug used. Furthermore, these results show that inactivation of the kinesin Eg5 can induce apoptosis in tumor cells in the absence of critical spindle checkpoint components.

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay.

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.

Middle turbinate resection: impact on outcomes in endoscopic sinus surgery.

There is considerable controversy concerning the advisability and efficacy of middle turbinate resection in endoscopic sinus surgery. Some have reported that it causes an increase in the incidence of frontal sinusitis, while others have found no such association. Surprisingly, in a 35-year review of turbinate surgery literature, Clement and White did not find a single prospective study of turbinate surgery. In this article, we briefly report our prospective study of middle turbinate resection during endoscopic sinus surgery. Our findings lead us to believe that middle turbinate resection has no deleterious effects on the results of endoscopic sinus surgery.

Fibrin glue prevents complications of septal surgery: findings in a series of 100 patients.

Septal surgery is a common type of otolaryngology--head and neck surgery, and it is often performed in combination with other procedures. Complications of septal surgery include bleeding, hematoma, infection, abscess formation, and perforation. The most common methods of preventing these complications are the use of nasal packing, septal splints, and quilting sutures as a means of approximating the septal flaps. In this article, we describe our study of an alternate method: fibrin glue. We used fibrin glue as the sole method of approximating flaps on 100 consecutive septal surgery patients. Our results indicate that the use of fibrin glue is effective, rapid, comfortable, and inexpensive.

Transnasal endoscopic closure of cerebrospinal fluid leaks.

OBJECTIVES/HYPOTHESIS: To demonstrate an effective method for transnasal endoscopic closure of anterior skull base fistulas that does not involve lumbar drains or fat packing. STUDY DESIGN: Retrospective. METHODS: We reviewed the charts of 36 patients who had 37 anterior skull base defects that were repaired endoscopically between 1993 and 2001. RESULTS: Thirty-three defects were successfully closed on the first attempt. Three were successfully closed on the second attempt. One large defect was repaired by neurosurgery after a failed endoscopic attempt. Our results are similar to those of other published series. CONCLUSION: Our method is effective and does not require adjuvant procedures or prolonged hospital stays.

Correlation of presentation and pathologic condition in primary ciliary dyskinesia.

OBJECTIVES: To better characterize primary ciliary dyskinesia (PCD) and improve the diagnosis of this uncommon disorder. STUDY DESIGN AND SETTING: We retrospectively reviewed the records of 118 patients with ciliary biopsy or brushing specimens examined at Yale University School of Medicine from 1991 to 2001. RESULTS: Sinonasal, middle ear, and pulmonary infections were more common in patients with PCD-positive biopsy results than in those with negative results. In addition, PCD caused by random ciliary orientation presented similarly to PCD caused by other ultrastructural defects. CONCLUSIONS: Patients who present with cough alone are highly unlikely to have PCD (chi(2 ) test, 24.85; P<.001). In contrast, patients who present with multiple manifestations are highly likely to have PCD (chi(2) test, 22.2; P<.001). This information may assist the clinician in the diagnosis of PCD.