Prolonged response of recurrent IDH-wild-type glioblastoma to laser interstitial thermal therapy with pembrolizumab.

Despite the improved understanding of the molecular and genetic heterogeneity of glioblastoma, there is still an unmet need for better therapeutics, as treatment approaches have remained unchanged in recent years. Research into the role of the immune microenvironment has generated enthusiasm for testing immunotherapy (specifically, immune checkpoint inhibitors). However, to date, trials of immunotherapy in glioblastoma have not demonstrated a survival advantage. Combination approaches aimed at optimally inducing response to immune checkpoint inhibitors with radiotherapy are currently being investigated. Herein, the authors describe their experience of the potential benefit and clinical outcomes of using combination pembrolizumab (an immune checkpoint inhibitor) and laser interstitial thermal therapy in a case series of patients with recurrent IDH-wild-type glioblastoma.

Managing Advanced Parkinson Disease.

Advanced Parkinson disease (PD) is associated with treatment-related motor fluctuations and reduced ability to perform activities of daily living. Progression of non-motor symptoms and medication-induced adverse effects complicate focused approach to motor symptom management, frequently accelerating reduced quality of life. It is thus critical for clinicians to consider disease progression versus therapeutic contributions when balancing management decisions. Such an approach requires careful recognition of inflection points resulting from therapeutic decisions and should prompt consideration of reduced pharmacologic burden and increased reliance on non-pharmacologic strategies in advanced disease. The successful approach to advanced PD requires a multidisciplinary effort focused on improving the patient's and family's quality of life, sometimes requiring sacrifice of motor symptom benefit. Here, we emphasize management strategies in advanced PD, focusing on the need to balance the therapeutic approach across advancing motor symptoms, progressive non-motor features, and potential pharmacologic adverse effects.

Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases.

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.

Single-molecule real-time detection of telomerase extension activity.

The ends of eukaryotic chromosomes are capped by telomeres which consist of tandem G-rich DNA repeats stabilized by the shelterin protein complex. Telomeres shorten progressively in most normal cells due to the end replication problem. In more than 85% of cancers however, the telomere length is maintained by telomerase; a reverse transcriptase that adds telomeric TTAGGG repeats using its integral RNA template. The strong association between telomerase activity and malignancy in many cancers suggests that telomerase activity could serve as a diagnostic marker. We demonstrate single-molecule, real-time telomerase extension activity observed digitally as the telomeric repeats are added to a substrate. The human telomerase complex pulled down from mammalian cells displays extension activity dependent on dNTP concentration. In complex with the processivity factor, POT1-TPP1, telomerase adds repeats at an accelerated rate and yields longer products. Our assay provides a unique detection platform that enables the study of telomerase kinetics with single molecule resolution.

Telomeric overhang length determines structural dynamics and accessibility to telomerase and ALT-associated proteins.

The G-rich single-stranded DNA at the 3' end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. Using single-molecule fluorescence spectroscopy, we discovered that lengthening the telomeric overhang also increased the rate of dynamic exchanges between structural conformations. Overhangs with five to seven TTAGGG repeats, compared with four repeats, showed much greater dynamics and accessibility to telomerase binding and activity and loading of the ALT-associated proteins RAD51, WRN, and BLM. Although the eight repeats are highly dynamic, they can fold into two GQs, which limited protein accessibility. In contrast, the telomere-specific protein POT1 is unique in that it binds independently of repeat number. Our results suggest that the telomeric overhang length and dynamics may contribute to the regulation of telomere extension via telomerase action and the ALT mechanism.

Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions.

Single molecule studies of protein-nucleic acid interactions shed light on molecular mechanisms and kinetics involved in protein binding, translocation, and unwinding of DNA and RNA substrates. In this review, we provide an overview of a single molecule fluorescence method, termed "protein induced fluorescence enhancement" (PIFE). Unlike FRET where two dyes are required, PIFE employs a single dye attached to DNA or RNA to which an unlabeled protein is applied. We discuss both ensemble and single molecule studies in which PIFE was utilized.

POT1-TPP1 regulates telomeric overhang structural dynamics.

Human telomeres possess a single-stranded DNA (ssDNA) overhang of TTAGGG repeats, which can self-fold into a G-quadruplex structure. POT1 binds specifically to the telomeric overhang and partners with TPP1 to regulate telomere lengthening and capping, although the mechanism remains elusive. Here, we show that POT1 binds stably to folded telomeric G-quadruplex DNA in a sequential manner, one oligonucleotide/oligosaccharide binding fold at a time. POT1 binds from 3' to 5', thereby unfolding the G-quadruplex in a stepwise manner. In contrast, the POT1-TPP1 complex induces a continuous folding and unfolding of the G-quadruplex. We demonstrate that POT1-TPP1 slides back and forth on telomeric DNA and also on a mutant telomeric DNA to which POT1 cannot bind alone. The sliding motion is specific to POT1-TPP1, as POT1 and ssDNA binding protein gp32 cannot recapitulate this activity. Our results reveal fundamental molecular steps and dynamics involved in telomere structure regulation.

Serial specification of diverse neuroblast identities from a neurogenic placode by Notch and Egfr signaling.

We used the brain insulin-producing cell (IPC) lineage and its identified neuroblast (IPC NB) as a model to understand a novel example of serial specification of NB identities in the Drosophila dorsomedial protocerebral neuroectoderm. The IPC NB was specified from a small, molecularly identified group of cells comprising an invaginated epithelial placode. By progressive delamination of cells, the placode generated a series of NB identities, including the single IPC NB, a number of other canonical Type I NBs, and a single Type II NB that generates large lineages by transient amplification of neural progenitor cells. Loss of Notch function caused all cells of the placode to form as supernumerary IPC NBs, indicating that the placode is initially a fate equivalence group for the IPC NB fate. Loss of Egfr function caused all placodal cells to apoptose, except for the IPC NB, indicating a requirement of Egfr signaling for specification of alternative NB identities. Indeed, both derepressed Egfr activity in yan mutants and ectopic EGF activity produced supernumerary Type II NBs from the placode. Loss of both Notch and Egfr function caused all placode cells to become IPC NBs and survive, indicating that commitment to NB fate nullified the requirement of Egfr activity for placode cell survival. We discuss the surprising parallels between the serial specification of neural fates from this neurogenic placode and the fly retina.

Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity.

Single-molecule FRET has been widely used for monitoring protein-nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0-3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG-I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.

Rural HIV-infected women's access to medical care: ongoing needs in California.

HIV-infected women living in rural areas often have considerably less access to care than their urban and suburban counterparts. In much of the USA, little is known about HIV care among rural populations. This study elucidated barriers to care for rural women in California. Methods included retrospective structured interviews conducted with 64 women living in rural areas and receiving HIV care at 11 California healthcare facilities. Facilities were randomly sampled and all HIV-infected female patients seeking care at those facilities during a specified time period were eligible. The most commonly cited barriers to accessing care included physical health problems that prevented travel to care (32.8%), lack of transportation (31.2%), and lack of ability to navigate the healthcare system (25.0%). Being divorced/separated/widowed (compared to being either married or single) was associated with reporting physical health as a barrier to care (p=0.03); being unemployed (p=0.003) or having to travel 31-90 minutes (p=0.007, compared to less than 31 or greater than 90) were both associated with transportation as a barrier; and speaking English rather than Spanish was associated with reporting "difficulty navigating the system" (p=0.04). Twenty-nine women (45.3%) reported difficulty in traveling to appointments. Overall, 24 (37.5%) women missed an HIV medical appointment in the previous 12-month period, primarily due to their physical health and transportation limitations. Physical health and transportation problems were both the major barriers to accessing health services and the primary reasons for missing HIV care appointments among this population of HIV-infected women living in rural areas. Providing transportation programs and/or mobile clinics, as well as providing support for patients with physical limitations, may be essential to improving access to HIV care in rural areas.

Supplementary eye field activity reflects a decision rule governing smooth pursuit but not the decision.

Animals depend on learned rules to guide their actions. Prefrontal (PFC) and premotor (PMC) cortex of primates have been reported to display rule-related neural activity, but it is unclear how signals encoded here are utilized to enforce the decision to act. The supplementary eye field (SEF) is a candidate for enforcing rule-guided ocular decisions because the activity of neurons here is correlated with the rule in an ocular decision-making task and because this area is anatomically more proximal to movement structures than PFC and PMC and receives inputs from them. However, in the previous work, the rule encoding and ocular outcome were confounded, leaving open the question of whether SEF activity is related to the rule or the behavior. In the present study, we attempted to discriminate between these alternatives by increasing task difficulty and forcing errors, thereby putting the stimulus and the behavior at odds. Single SEF neurons were recorded while monkeys performed the task in which the rule is to pursue a moving target if it intersects a visible square and maintain fixation if it does not. A delay period was imposed to monitor neural activity while the target approached the square. Two complementary populations of go and nogo neurons were found. When task difficulty was increased, the monkeys made more errors, and the neurons took longer to encode the rule. However, in error trials, most neurons continued to reflect the rule rather the monkey's ocular decision in both the delay period and after square intersection (movement period). This was the case for both directionally tuned and nondirectional SEF neurons. The results suggest that SEF neurons encode the ocular decision rule but that the decision itself likely occurs in a different structure that sums rule information from the SEF with information from other areas.

Quantitative determination of polymorphic impurity by X-ray powder diffractometry in an OROS formulation.

An ALZA OROS drug delivery system was evaluated for its potential to increase drug load and reduce side effects when used with RWJ-333369 (S-2-O-carbamoyl-1-o-chlorophenyl-ethanol), a novel neuromodulatory agent initially developed by SK Bio-Pharmaceuticals and licensed by Johnson & Johnson. RWJ-333369 was found to have two crystalline polymorphs (A and B) that are enantiotropically related. Polymorph A, used in the formulation, was thermodynamically stable at room temperature. A partial polymorphic conversion in the solid state was observed at an elevated temperature of 60 degrees C during a two-week stability test. The OROS RWJ-333369 manufacturing process included milling, granulation, compression, sub-coating, membrane coating, drilling, and drying to produce a capsule-shaped OROS tablet. The potential for polymorphic conversion during manufacturing and stability testing was evaluated using Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy (Raman), and X-ray Powder Diffractometry (XRD) to detect impurities; the latter was determined to be preferred method. Pure polymorph A and polymorph B reference materials were used for method development. Mixtures with different ratios of polymorph A and polymorph B were scanned using XRD, and the peak heights and areas were used to generate a calibration curve. OROS RWJ-333369 formulations were spiked with polymorph B reference, and the detection limit was about 2% using the 22 degrees 2theta diffraction angle relative peak area. Samples from different OROS manufacturing process and stability tests were analyzed. The results indicated that polymorph A was not converted to polymorph B during manufacturing process. Polymorph B impurity was, however, detected in stability samples.

Impact of psychiatric disorders on Parkinson's disease : a nationwide follow-up study from Sweden.

OBJECTIVES: To analyze whether hospitalization for a psychiatric disorder predicts Parkinson's disease (PD) in men and women in different age groups after accounting for socioeconomic status and geographical region. METHODS: Data from the MigMed database were used to identify all people in Sweden hospitalized for psychiatric disorder and PD during the study period (1987 to 2001). Standardized incidence ratios (SIRs) with 95% confidence intervals (CI) for PD were calculated among those with and without hospitalization for psychiatric disorder. RESULTS: There were 1876 cases of PD among those with psychiatric disorder during the study period. The risk of developing PD was strongest among those under age 50; the SIR was 11.56 (95% CI 9.15-14.41). The risk was attenuated with increasing age in both men and women. There were similar risk patterns in all subtypes of psychiatric disorders in PD patients. The overall risk of PD among people with psychiatric disorders was higher for women than men. CONCLUSIONS: A psychiatric disorder is an appreciable risk factor for the development of PD, particularly in people under age 50. The association between PD and psychiatric disorders should be taken into account by clinicians and health care providers.