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Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single-molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches, we show that S. cerevisiae Pif1 can use its ATP-dependent 5' to 3' translocase activity to chemomechanically push a single human RPA (hRPA) heterotrimer directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity, Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein. These results highlight the two basic requirements for any processive DNA helicase: transient DNA base pair melting (supplied by hRPA) and ATP-dependent directional ssDNA translocation (supplied by Pif1) and that these functions can be unlinked by using two separate proteins.

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain., Competing Interests: The authors declare no conflict of interest.

Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB "arm" domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

The interferon-inducible, double-stranded (ds) RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays, and small-angle X-ray scattering (SAXS) to dissect the thermodynamic basis for the specificity of the dsRBD termed "p20" for various RNAs and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA-DNA hybrid segments and a central dsRNA segment, which was either unbulged ("perfect") or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (K(d) ~ 30 nM) and weaker binding to the bulged duplex (K(d) ~ 2-5 μM). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change in perfect dsRNA but largely straightens bulged dsRNA. Together, these observations support the dsRBD's ability to tightly bind to only A-form RNA and suggest that in a noninfected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs with complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.

The protein kinase PKR is activated by RNA to phosphorylate eIF-2α, inhibiting translation initiation. Long dsRNA activates PKR via interactions with the dsRNA-binding domain (dsRBD). Weakly structured RNA also activates PKR and does so in a 5'-triphosphate (ppp)-dependent fashion, however relatively little is known about this pathway. We used a mutant T7 RNA polymerase to incorporate all four triphosphate-containing nucleotides into the first position of a largely single-stranded RNA and found absence of selectivity, in that all four transcripts activate PKR. Recognition of 5'-triphosphate, but not the nucleobase at the 5'-most position, makes this RNA-mediated innate immune response sensitive to a broad array of viruses. PKR was neither activated in the presence of γ-GTP nor recognized NTPs other than ATP in activation competition and ITC binding assays. This indicates that the binding site for ATP is selective, which contrasts with the site for the 5' end of ppp-ssRNA. Activation experiments reveal that short dsRNAs compete with 5'-triphosphate RNAs and heparin for activation, and likewise gel-shift assays reveal that activating 5'-triphosphate RNAs and heparin compete with short dsRNAs for binding to PKR's dsRBD. The dsRBD thus plays a critical role in the activation of PKR by ppp-ssRNA and even heparin. At the same time, cross-linking experiments indicate that ppp-ssRNA interacts with PKR outside of the dsRBD as well. Overall, 5'-triphosphate-containing, weakly structured RNAs activate PKR via interactions with both the dsRBD and a distinct triphosphate binding site that lacks 5'-nucleobase specificity, allowing the innate immune response to provide broad-spectrum protection from pathogens.

The malachite green aptamer binds two closely related ligands, malachite green (MG) and tetramethylrosamine (TMR), with nearly equal affinity. The MG ligand consists of three phenyl rings emanating from a central carbon, while TMR has two of the three rings connected by an ether linkage. The binding pockets for MG and TMR in the aptamer, known from high-resolution structures, differ only in the conformation of a few nucleotides. Herein, we applied isothermal titration calorimetry (ITC) to compare the thermodynamics of binding of MG and TMR to the aptamer. Binding heat capacities were obtained from ITC titrations over the temperature range of 15-60 °C. Two temperature regimes were found for MG binding: one from 15 to 45 °C where MG bound with a large negative heat capacity and an apparent stoichiometry (n) of ~0.4 and another from 50 to 60 °C where MG bound with a positive heat capacity and an n of ~1.1. The binding of TMR, on the other hand, revealed only one temperature regime for binding, with a more modest negative heat capacity and an n of ~1.2. The large difference in heat capacity between the two ligands suggests that significantly more conformational rearrangement occurs upon the binding of MG than that of TMR, which is consistent with differences in solvent accessible surface area calculated for available ligand-bound structures. Lastly, we note that the binding stoichiometry of MG was improved not only by an increase in the temperature but also by a decrease in the concentration of Mg(2+) or an increase in the time between ITC injections. These studies suggest that binding of a dynamical ligand to a functional RNA requires the RNA itself to have significant dynamics.

Noncoding RNAs serve myriad functions in the cell, but their biophysical properties are not well understood. Calorimetry offers direct and label-free means for characterizing the ligand-binding and thermostability properties of these RNA. We apply two main types of calorimetry--isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)--to the characterization of these functional RNA molecules. ITC can describe ligand binding in terms of stoichiometry, affinity, and heat (enthalpy), while DSC can provide RNA stability in terms of heat capacity, melting temperature, and folding enthalpy. Here, we offer detailed experimental protocols for studying such RNA systems with commercially available conventional and high-throughput ITC and DSC instruments.

Biological RNAs, like their DNA counterparts, contain helical stretches, which have standard Watson-Crick base pairs in the anti conformation. Most functional RNAs also adopt geometries with far greater complexity such as bulges, loops, and multihelical junctions. Occasionally, nucleobases in these regions populate the syn conformation wherein the base resides close to or over the ribose sugar, which leads to a more compact state. The importance of the syn conformation to RNA function is largely unknown. In this study, we analyze 51 RNAs with tertiary structure, including aptamers, riboswitches, ribozymes, and ribosomal RNAs, for number, location, and properties of syn nucleobases. These RNAs represent the set of nonoverlapping, moderate- to high-resolution structures available at present. We find that syn nucleobases are much more common among purines than pyrimidines, and that they favor C2'-endo-like conformations especially among those nucleobases in the intermediate syn conformation. Strikingly, most syn nucleobases participate in tertiary stacking and base-pairing interactions: Inspection of RNA structures revealed that the majority of the syn nucleobases are in regions assigned to function, with many syn nucleobases interacting directly with a ligand or ribozyme active site. These observations suggest that judicious placement of conformationally restricted nucleotides biased into the syn conformation could enhance RNA folding and catalysis. Such changes could also be useful for locking RNAs into functionally competent folds for use in X-ray crystallography and NMR.

Traumatic brain injury features deficits are often ameliorated by dopamine (DA) agonists. We have previously shown deficits in striatal DA neurotransmission using fast scan cyclic voltammetry after controlled cortical impact (CCI) injury that are reversed after daily treatment with the DA uptake inhibitor methylphenidate (MPH). The goal of this study was to determine how a single dose of MPH (5 mg/kg) induces changes in basal DA and metabolite levels and with electrically evoked overflow (EO) DA in the striatum of CCI rats. MPH-induced changes in EO DA after a 2-week daily pre-treatment regime with MPH was also assessed. There were no baseline differences in basal DA or metabolite levels. MPH injection significantly increased basal [DA] output in dialysates for control but not injured rats. Also, MPH injection increased striatal peak EO [DA] to a lesser degree in CCI (176% of baseline) versus control rats (233% of baseline). However, daily pre-treatment with MPH resulted in CCI rats having a comparable increase in EO [DA] after MPH injection when compared with controls. The findings further support the concept that daily MPH therapy restores striatal DA neurotransmission after CCI.

The therapeutic benefits of dopamine (DA) agonists after traumatic brain injury (TBI) imply a role for DA systems in mediating functional deficits post-TBI. We investigated how experimental TBI affects striatal dopamine systems using fast scan cyclic voltammetry (FSCV), western blot, and d-amphetamine-induced rotational behavior. Adult male Sprague-Dawley rats were injured by a controlled cortical impact (CCI) delivered unilaterally to the parietal cortex, or were naïve controls. Amphetamine-induced rotational behavior was assessed 10 days post-CCI. Fourteen days post-CCI, animals were anesthetized and underwent FSCV with bilateral striatal carbon fiber microelectrode placement and stimulating electrode placement in the medial forebrain bundle (MFB). Evoked DA overflow was assessed in the striatum as the MFB was electrically stimulated at 60 Hz for 10 s. In 23% of injured animals, but no naïve animals, rotation was observed with amphetamine administration. Compared with naïves, striatal evoked DA overflow was lower for injured animals in the striatum ipsilateral to injury (p < 0.05). Injured animals exhibited a decrease in V(max) (52% of naïve, p < 0.05) for DA clearance in the hemisphere ipsilateral to injury compared with naïves. Dopamine transporter (DAT) expression was proportionally decreased in the striatum ipsilateral to injury compared with naïve animals (60% of naïve, p < 0.05), despite no injury-related changes in vesicular monoamine transporter or D2 receptor expression (DRD2) in this region. Collectively, these data appear to confirm that the clinical efficacy of dopamine agonists in the treatment of TBI may be related to disruptions in the activity of subcortical dopamine systems.

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions. HIV relies on the RRE RNA interaction with Rev protein for nuclear export of viral mRNAs. The structure of the high-affinity Rev binding site in RRE in two conformations suggests a mechanism for initial Rev binding and oligomerization onto RRE. [ABSTRACT FROM AUTHOR]

Myriad DNA-binding proteins undergo dynamic assembly, translocation, and conformational changes while on DNA or alter the physical configuration of the DNA substrate to control its metabolism. It is now possible to directly observe these activities—often central to the protein function—thanks to the advent of single-molecule fluorescence- and force-based techniques. In particular, the integration of fluorescence detection and force manipulation has unlocked multidimensional measurements of protein–DNA interactions and yielded unprecedented mechanistic insights into the biomolecular processes that orchestrate cellular life. In this review, we first introduce the different experimental geometries developed for single-molecule correlative force and fluorescence microscopy, with a focus on optical tweezers as the manipulation technique. We then describe the utility of these integrative platforms for imaging protein dynamics on DNA and chromatin, as well as their unique capabilities in generating complex DNA configurations and uncovering force-dependent protein behaviors. Finally, we give a perspective on the future directions of this emerging research field. [ABSTRACT FROM AUTHOR]

Neuronal damage resulting from traumatic brain injury (TBI) causes disruption of neuronal projections and neurotransmission that contribute to behavioral deficits. Cellular generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is an early event following TBI. ROS often damage DNA, lipids, proteins, and carbohydrates while RNS attack proteins. The products of lipid peroxidation 4-hydroxynonenal (4-HNE) and protein nitration 3-nitrotyrosine (3-NT) are often used as indicators of oxidative and nitrosative damages, respectively. Increasing evidence has shown that striatum is vulnerable to damage from TBI with a disturbed dopamine neurotransmission. TBI results in neurodegeneration, oxidative stress, neuroinflammation, neuronal apoptosis, and autophagy in the striatum and contribute to motor or behavioral deficits. Pomalidomide (Pom) is a Food and Drug Administration (FDA)-approved immunomodulatory drug clinically used in treating multiple myeloma. We previously showed that Pom reduces neuroinflammation and neuronal death induced by TBI in rat cerebral cortex. Here, we further compared the effects of Pom in cortex and striatum focusing on neurodegeneration, oxidative and nitrosative damages, as well as neuroinflammation following TBI. Sprague–Dawley rats subjected to a controlled cortical impact were used as the animal model of TBI. Systemic administration of Pom (0.5 mg/kg, intravenous [i.v.]) at 5 h post-injury alleviated motor behavioral deficits, contusion volume at 24 h after TBI. Pom alleviated TBI-induced neurodegeneration stained by Fluoro-Jade C in both cortex and striatum. Notably, Pom treatment reduces oxidative and nitrosative damages in cortex and striatum and is more efficacious in striatum (93% reduction in 4-HNE-positive and 84% reduction in 3-NT-positive neurons) than in cerebral cortex (42% reduction in 4-HNE-positive and 55% reduction in 3-NT-positive neurons). In addition, Pom attenuated microgliosis, astrogliosis, and elevations of proinflammatory cytokines in cortical and striatal tissue. We conclude that Pom may contribute to improved motor behavioral outcomes after TBI through targeting oxidative/nitrosative damages and neuroinflammation. [ABSTRACT FROM AUTHOR]

Purpose: Vault (vt) RNAs are noncoding (nc) RNAs transcribed by RNA polymerase III (RNA Pol III) with 5ʹ-triphosphate (5ʹ-PPP) termini that play significant roles and are recognized by innate immune sensors, including retinoic acid-inducible protein 1 (RIG-I). In addition, vtRNAs adopt secondary structures that can be targets of interferon-inducible protein kinase R (PKR) and the oligoadenylate synthetase (OAS)/RNase L system, both of which are important for activating antiviral defenses. However, changes in the expression of vtRNAs have been associated with pathological processes that activate proinflammatory pathways, which influence cellular events such as differentiation, aging, autophagy, apoptosis, and drug resistance in cancer cells. Results: In this review, we summarized the biology of vtRNAs and focused on their interactions with the innate immune system. These findings provide insights into the diverse roles of vtRNAs and their correlation with various cellular processes to improve our understanding of their biological functions. [ABSTRACT FROM AUTHOR]

A liquid-phase radioimmunoassay was used to quantitate sperm-associated IgG in a population of patients with unexplained infertility. Plasma IgG antisperm antibody activity was identified in a subpopulation of these patients. Using the human sperm/hamster egg assay, a unique functional test of human sperm penetrating capacity, we identified a subset of these positive patients whose plasma significantly and reproducibly inhibited penetration. This study implies that antigen-antibody reactions may prevent sperm responses that are critical to sperm-egg fusion.

In an attempt to maximize the success of in vitro fertilization (IVF) and embryo transfer (ET) as a treatment for human infertility, we have examined the relationship of follicular size and number to the rates of oocyte recovery, fertilization, cleavage, and ET in clomiphene citrate-stimulated cycles. The recovery of oocytes from follicles less than 20 mm in diameter was significantly reduced over that from larger follicles, and those oocytes that were obtained from smaller follicles showed a significantly lower rate of fertilization and cleavage. In addition, the overall chance that a patient would undergo ET was greater in a cycle in which more than one follicle 20 mm or larger was developing than in a cycle in which a single large follicle was developing. This latter observation suggests that attempts at laparoscopic oocyte retrieval should be confined to cycles in which more than one accessible large follicle is developing, thereby maximizing the success rate while minimizing the risk and expense for the patient.

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.

We compared fertility potential measurements by the zona-free hamster egg bioassay with the in vitro fertilization of human eggs. Sperm from 24 husbands participating in an in vitro fertilization-embryo transfer program were used in 27 inseminations of the wives' eggs and in simultaneous inseminations of hamster eggs. In seven cases, a positive fertility assessment was obtained in the absence of fertilization of the wives' eggs attributable to egg immaturity or in one case to equipment failure. In the remaining 20 cases, at least one egg was fertilized, for a level of 73%. In 90% of the husbands who fertilized their wives' eggs, a positive fertility assessment was obtained. Two husbands gave false-negative assessments. To account for these, alternative culture conditions were examined; sperm exposure to longer preincubations or at higher concentrations usually improved assessments. These results indicate that fertility assessment with this bioassay is highly correlated with the fertilization of human eggs in vitro.

During a 6-month period, a combination of gamete intrafallopian transfer (GIFT) and in vitro fertilization (IVF) was offered to all couples beginning an IVF treatment cycle in whom the wife had anatomically normal fallopian tubes. It was recommended to these couples that sufficient oocytes be reserved for insemination in vitro to determine whether the husband's spermatozoa could fertilize the wife's oocytes. During this interval, 16 couples underwent the combined IVF-GIFT procedure. All of the IVF-GIFT couples had at least two oocytes inseminated in vitro and at least two oocytes for GIFT. Of the 16 IVF-GIFT couples, only 1 (6.25%) achieved a clinical pregnancy. More important, 50% (8/16) of the IVF-GIFT couples had no oocytes fertilized in vitro. With the information concerning lack of fertilization in vitro, appropriate recommendations concerning future fertility management can be made. If the same couples had undergone the GIFT procedure alone, without additional oocytes fertilized in vitro, this information would not have been obtained.

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, b ut not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.

Human sperm become capable of penetrating zona-free hamster eggs after preincubation in an appropriate culture medium. This observation has led to the development of an assay for characterizing the fertilizing capacity of human spermatozoa. In the present study, the incorporation of sperm by zona-free hamster eggs was quantitated, and several parmeters that contribute to penetration were evaluated. The importance of the motile sperm concentration was established; no penetration was seen at concentrations lower than 6 x 10(5) motile cells/ml, whereas above this level the mean number of incorporated sperm per egg was linearly related to concentration. Freeze-thawed sperm, although capable of penetrating zona-free hamster eggs, did so with lesser frequency than did fresh sperm at equal concentrations of motile sperm. Kinetic experiments indicated that eggs were maximally penetrated after 5 hours of exposure to capacitated sperm and that the cessation in sperm incorporation seen at this time resulted from egg-related changes that occurred during aging in vitro. A protocol for evaluating "fertilizing capacity" of human sperm samples was outlined incorporating the findings from the present study. Using these conditions, reproducible penetration levels were obtained when several ejaculates obtained from the same donor over a 3-month interval were tested at similar motile sperm concentrations.

Human sperm fertility potential, expressed as a quality index (QI), was evaluated objectively from considerations of sperm velocity, percentage of motile forms, sperm density, and ejaculate volume. Turbidimetry was applied in the quantitation of sperm velocity. High QIs (700) were characteristic of semen samples that were capable of penetrating cervical mucus in capillary tube penetration tests, while low QIs (80) were associated with specimens that did not penetrate mucus. The rate of decline in QI as a function of time postejaculation was determined for samples stored at 37 degrees C and at ambient temperature, providing correction factors for the comparative evaluation of semen samples analyzed at different times. The penetrability of cervical mucus was determined by capillary tube penetration testing. Subsequent chemical analysis of sperm-penetrable and impenetrable samples indicated that the concentrations of mucus nondialyzable solids (NDS), mucins, and soluble proteins were significantly higher in impenetrable specimens. These characteristics were also observed in impenetrable mucus obtained from patients with cervical factor infertility. These studies indicate that it is now feasible to employ mucus [NDS] in the diagnosis of cervical factor infertility and turbidimetry in the evaluation of human sperm quality.

The interaction of sperm with the zona-free hamster egg was studied. Hamster sperm were capacitated in Tyrodes media, containing 50% heat-inactivated serum and used to inseminate zona-free eggs in BWW containing 10% serum. Capacitated sperm began fusing with eggs within the first hour of insemination and by 3 h penetration had ceased as indicated by the absence of further changes in the mean number of sperm incorporated per egg. Penetration by capacitated hamster sperm was linearly related to the log of the motile sperm concentration at concentrations above 10(4) cells/ml. The viability of sperm and eggs in culture was limited in these studies to approximately 3-5 h. The existence of a block to unlimited sperm penetration of the zona-free egg was sought in reinsemination experiments. A relatively low sperm concentration was used to initiate egg responses, followed, at timed intervals, by reinsemination with a high challenge concentration of sperm. Subsequent polyspermy levels reflected the presence or absence of the egg's block to polyspermy response. In order to circumvent the problems arising from the rapid aging of hamster sperm in culture, mouse sperm were employed, a convenience afforded by the lack of species specificity in this egg. Reinseminated eggs incorporated additional sperm during the challenge; therefore, the hamster egg is not capable of preventing unlimited sperm penetration. The implications of these findings to the use of the zona-free hamster egg test in fertility evaluation is discussed.

A turbidimetric method has been developed for determining rapidly the fraction of sperm in human ejaculates which show the most vigorous motility. The method is based on the fact that sperm cells so endowed will be the first to swim upward into clear medium from a concentrated cell suspension at the bottom of an optical cuvette. This results in a time-dependent increase in turbidity in the medium which is recorded spectrophotometrically as an increase in absorbance. The determination requires 10 minutes and yields both the fraction of rapidly moving sperm, FRM, and their average velocity, VRM. Examination of 25 samples yielded FRM values of 10% or lower, whereas values for VRM averaged about 100 microns/second. These vigorously motile cells may be the best candidates for fertilization, and samples with a high fraction of such cells should have high fertilizing capacity. It is suggested that this simple turbidimetric test be used in evaluation of human semen as a possible indicator of fertilizing capacity.

A critical aspect of human in vitro fertilization (IVF) treatment programs is the timing of human chorionic gonadotropin (hCG) administration to induce final oocyte maturation. Patients were treated with clomiphene citrate (Serophene, Serono Laboratories, Inc., Randolph, MA), 100 mg, and human menopausal gonadotropin (Pergonal, Serono Laboratories, Inc.) 1 ampule daily from cycle days 4 through 8. The baseline estradiol (E2) level was determined by averaging the E2 values from cycle days 3 and 4. The first day of E2 rise was defined as the day that the E2 level exceeded 150% of the baseline. In general, 5000 IU of hCG (Profasi-HP, Serono Laboratories, Inc.) was administered on the evening of the sixth day of E2 rise. Significantly fewer oocytes were recovered per patient when the hCG was administered on the seventh day of E2 rise, compared with the fifth or sixth day. The recovered oocytes in the seventh-day E2 group had a significantly lower fertilization rate, compared with the other groups. The use of E2-rise days proved to be a simple, successful technique for the timing of hCG administration in an IVF treatment program.

Objective: Feedback facilitates learning by guiding and modifying behaviors through an action-outcome contingency. As the majority of existing studies have focused on the immediate presentation of feedback, the impact of delayed feedback on learning is understudied. Prior work demonstrated that learning from immediate and delayed feedback employed distinct brain regions in healthy individuals, and compared to healthy individuals, individuals with traumatic brain injury (TBI) are impaired in learning from immediate feedback. The goal of the current investigation was to assess the effects of delayed vs immediate feedback on learning in individuals with TBI and examine brain networks associated with delayed and immediate feedback processing., Setting: Nonprofit research organization., Participants: Twenty-eight individuals with moderate-to-severe TBI., Design: Participants completed a paired-associate word learning task while undergoing magnetic resonance imaging. During the task, feedback was presented either immediately, after a delay, or not at all (control condition)., Main Measures: Learning performance accuracy, confidence ratings, post-task questionnaire, and blood oxygen level-dependent signal., Results: Behavioral data showed that delayed feedback resulted in better learning performance than immediate feedback and no feedback. In addition, participants reported higher confidence in their performance during delayed feedback trials. During delayed vs immediate feedback processing, greater activation was observed in the superior parietal and angular gyrus. Activation in these areas has been previously associated with successful retrieval and greater memory confidence., Conclusion: The observed results might be explained by delayed feedback processing circumventing the striatal dopaminergic regions responsible for learning from immediate feedback that are impaired in TBI. In addition, delayed feedback evokes less of an affective reaction than immediate feedback, which likely benefited memory performance. Indeed, compared to delayed feedback, positive or negative immediate feedback was more likely to be rated as rewarding or punishing, respectively. The findings have significant implications for TBI rehabilitation and suggest that delaying feedback during rehabilitation might recruit brain regions that lead to better functional outcomes., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Cyclic AMP (cAMP) is a ubiquitous second messenger synthesized by most living organisms. In bacteria, it plays highly diverse roles in metabolism, host colonization, motility, and many other processes important for optimal fitness. The main route of cAMP perception is through transcription factors from the diverse and versatile CRP–FNR protein superfamily. Since the discovery of the very first CRP protein CAP in Escherichia coli more than four decades ago, its homologs have been characterized in both closely related and distant bacterial species. The cAMP-mediated gene activation for carbon catabolism by a CRP protein in the absence of glucose seems to be restricted to E. coli and its close relatives. In other phyla, the regulatory targets are more diverse. In addition to cAMP, cGMP has recently been identified as a ligand of certain CRP proteins. In a CRP dimer, each of the two cyclic nucleotide molecules makes contacts with both protein subunits and effectuates a conformational change that favors DNA binding. Here, we summarize the current knowledge on structural and physiological aspects of E. coli CAP compared with other cAMP- and cGMP-activated transcription factors, and point to emerging trends in metabolic regulation related to lysine modification and membrane association of CRP proteins. [ABSTRACT FROM AUTHOR]

Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd—a DNA translocase of a marginal NER phenotype1–3. Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo. We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD–RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.Integrated structure–function studies show that transcription-coupled DNA repair (TCR)—rather than global genomic repair—is responsible for most chromosomal repair events in bacteria, and that TCR mainly occurs independently of the Mfd translocase. [ABSTRACT FROM AUTHOR]

There is increasing preclinical and clinical data supporting a potential association between Traumatic Brain Injury (TBI) and Parkinson's disease (PD). It has been suggested that the glutamate-induced excitotoxicity underlying TBI secondary neuronal degeneration (SND) might be associated with further development of PD. Interestingly, an accumulation of extracellular glutamate and olfactory dysfunction are both sharing pathological conditions in TBI and PD. The possible involvement of glutamate excitotoxicity in olfactory dysfunction has been recently described, however, the role of olfactory bulbs (OB) glutamate excitotoxicity as a possible mechanism involved in the association between TBI and PD-related neurodegeneration has not been investigated yet. We examined the number of nigral dopaminergic neurons (TH +), nigral α-synuclein expression, the striatal dopamine transporter (DAT) expression, and motor performance after bilateral OB N-Methyl-D-Aspartate (NMDA)-induced excitotoxic lesions in rodents. Bulbar NMDA administration induced a decrease in the number of correct choices in the discrimination tests one week after lesions (p < 0.01) and a significant decrease in the number of nigral DAergic neurons (p < 0.01) associated with an increase in α-synuclein expression (p < 0.01). No significant striatal changes in DAT expression or motor alterations were observed. Our results show an association between TBI-induced SND and PD-related neurodegeneration suggesting that the OB excitotoxicity occurring in TBI SND may be a filling gap mechanism underlying the link between TBI and PD-like pathology. [ABSTRACT FROM AUTHOR]

Outcome processing, the ability to learn from feedback, is an important component of adaptive behavior and rehabilitation. Evidence from healthy adults implicates the striatum and dopamine in outcome processing. Animal research shows that damage to dopaminergic pathways in the brain can lead to a disruption of dopamine tone and transmission. Such evidence thus suggests that persons with TBI experience deficits in outcome processing. However, no research has directly investigated outcome processing and associated neural mechanisms in TBI. Here, we examine outcome processing in individuals with TBI during learning. Given that TBI negatively impacts striatal and dopaminergic systems, we hypothesize that individuals with TBI exhibit deficits in learning from outcomes. To test this hypothesis, individuals with moderate-to-severe TBI and healthy adults were presented with a declarative paired-associate word learning task. Outcomes indicating performance accuracy were presented immediately during task performance and in the form of either monetary or performance-based feedback. Two types of feedback provided the opportunity to test whether extrinsic and intrinsic motivational aspects of outcome presentation play a role during learning and outcome processing. Our results show that individuals with TBI exhibited impaired learning from feedback compared to healthy participants. Additionally, individuals with TBI exhibited increased activation in the striatum during outcome processing. The results of this study suggest that outcome processing and learning from immediate outcomes is impaired in individuals with TBI and might be related to inefficient use of neural resources during task performance as reflected by increased activation of the striatum. [ABSTRACT FROM AUTHOR]

The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer's disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by β- and γ-secretases in the 'amyloidogenic' proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the 'non-amyloidogenic' pathway which, through the action of the α-secretase a disintegrin and metalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting β–secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the β-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression/activity of α-, β- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. Given the small size and blood-brain-barrier permeability of the drug, further research into its mechanism of action with respect to APP proteolysis may lead to the development of therapies for slowing the progression of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases. UvrD is a model helicase from the non-hexameric Superfamily 1. Here, the authors use optical tweezers to measure directly the stepwise translocation of UvrD along a DNA hairpin, and propose a mechanism in which UvrD moves one base pair at a time, but sequesters the nascent single strands, releasing them after a variable number of ATP hydrolysis cycles. [ABSTRACT FROM AUTHOR]

Sperm motility and concentration are commonly evaluated parameters in semen analysis. Those parameters are assessed objectively with commercial instrumentation such as computer‐assisted sperm analysis systems (CASA) and hemocytometer. In CASA systems, sperm motility is assessed in the horizontal plane imposed by the stage of the microscope. Thus, there is lack of measurement of the vertical velocity of sperm. The female reproductive tract is a tridimensional space which the sperm traverse to reach the ovum, and there is a need for instruments measuring parameters more relevant to this real‐world situation. In this report we describe the design, construction and use of an open‐source hardware (OSH) device for evaluation of the vertical velocity of sperm, called UPSPERM. This device was also used to measure sperm concentration, and agreement with hemocytometer was evaluated. Bland–Altman analysis shows good agreement between these two methods of sperm counting. As a first application of UPSPERM, we evaluated the changes in boar sperm motility at distinct pH values between 7.0 and 8.0. The UPSPERM results showed that the vertical velocity of sperm was highest at pH 7.6 and 7.8. We propose that our UPSPERM offers a reliable and affordable option for obtaining measurements of vertical velocity and sperm concentration. [ABSTRACT FROM AUTHOR]

Irc3 is a superfamily II DNA helicase required for the maintenance of mitochondrial DNA stability in Saccharomyces cerevisiae. Here, we show that recombinant Irc3 is a monomeric protein and that it can form a binary complex with forked DNA. The catalytically active enzyme is a monomer as no positive cooperativity of ATP hydrolysis or DNA unwinding can be detected. Interestingly, we find that Irc3 prefers to unwind the nascent lagging strand at a replication fork. Using DNase I footprinting, we demonstrate that Irc3 captures DNA substrates by establishing a strong contact at the DNA branching point. Additional protections on the lagging strand template suggest a 3′‐to‐5′ polarity for Irc3 movement. [ABSTRACT FROM AUTHOR]

Objective: Traumatic brain injury (TBI) and rapid eye movement sleep behavioural disorder (RBD) are risk factors for Parkinson's disease (PD). Dopaminergic abnormalities are often seen after TBI, but patients usually lack parkinsonian features. We test whether TBI, PD and RBD have distinct striatal dopamine abnormalities using dopamine transporter (DaT) imaging.Methods: 123I-ioflupane single-photon emission CT scans were used in a cross-sectional study to measure DaT levels in moderate/severe TBI, healthy controls, patients with early PD and RBD. Caudate and putamen DaT, putamen to caudate ratios and left-right symmetry of DaT were compared.Results: 108 participants (43 TBI, 26 PD, 8 RBD, 31 controls) were assessed. Patients with early PD scored significantly higher on the Unified Parkinson's Disease Rating Scale motor subscale than other groups. Patients with TBI and PD had reduced DaT levels in the caudate (12.2% and 18.7%, respectively) and putamen (9.0% and 42.6%, respectively) compared with controls. Patients with RBD had reduced DaT levels in the putamen (12.8%) but not in the caudate compared with controls. Patients with PD and TBI showed distinct patterns of DaT reduction, with patients with PD showing a lower putamen to caudate ratio. DaT asymmetry was greater in the PD group than other groups.Conclusions: The results show that patients with early PD and TBI have distinct patterns of striatal dopamine abnormalities. Patients with early PD and moderate/severe TBI showed similar reductions in caudate DaT binding, but patients with PD showed a greater reduction in putamen DaT and a lower putamen to caudate ratio. The results suggest that parkinsonian motor signs are absent in these patients with TBI because of relatively intact putaminal dopamine levels. [ABSTRACT FROM AUTHOR]

Study Question: Is there is an association between follicle size and the quality of oocytes retrieved from them as judged by ability to achieve the blastocyst stage, blastocyst grades and blastocyst ploidy?Summary Answer: Although follicle size is a valuable predictor of oocyte maturity and is a significant predictor of the ability of a fertilized oocyte to become a quality blastocyst, the ploidy of each quality blastocyst is not related to the size of the follicle from which its oocyte was retrieved.What Is Known Already: It is unclear whether the oocytes within larger follicles are the best oocytes of the cohort. Although there have been studies examining follicle size in relation to embryo quality, there has been no study relating the incidence of euploidy in embryos to follicle size.Study Design, Size, Duration: The purpose of this study was to examine follicle sizes and the oocytes from those follicles (and the embryos that result from those oocytes) to see if there is an association between follicle size and the quality of oocytes as judged by ability to achieve the blastocyst stage, blastocyst grades and blastocyst ploidy. Follicle sizes for oocytes were assessed both as diameters (mm) and as Z values (expressed as their size relative to the mean and standard deviation of that donor's follicular cohort). Comparisons were made using cumulative histograms, rolling averages and receiver operator characteristic (ROC) curves and its AUC.Participants/materials, Setting, Methods: Twenty-two oocyte donors (ages: 24.5 ± 3.5 years) whose recipients would use ICSI for insemination were enrolled in this study. Follicles were aspirated one-at-a-time to be certain that the aspirated oocyte was from the same follicle measured. The follicle measurement (size) was noted in the embryology records. Oocytes were cultured individually throughout their time in the embryology laboratory so that follicle sizes could be uniquely associated with each oocyte. Oocytes and embryos were analyzed according to the size of the follicle from which the oocyte was retrieved.Main Results and the Role Of Chance: Three hundred seventeen oocytes (96.1%) had an associated follicle size. Of the oocytes with follicle sizes, 255 (80.4%) had a polar body (MII), and 60 (18.9%) were immature: 31 (9.8%) with a visible germinal vesicle (GV stage) and 29 (9.1%) with neither a polar body nor a visible germinal vesicle (MI). The incidence of MII oocytes was significantly associated with larger follicle size using either mm (ROC's AUC = 0.87; P < 0.0001) or Z values (ROC's AUC = 0.86; P < 0.0001). Among MII oocytes there was no association with follicle size for the appearance of 228 oocytes with two pronuclei (2 PN). Among 2 PN's, the development of 94 quality blastocysts that underwent trophectoderm biopsy (TE Bx) exhibited a significant association with larger follicles using either mm (ROC's AUC = 0.59; P = 0.01) or Z values (ROC's AUC = 0.57; P = 0.01). The use of follicle diameter as a feature to distinguish between fertilized oocytes that would ultimately become blastocysts versus those that would not become blastocysts resulted in an enrichment for blastocyst formation from 20 to 40%. Of the 94 quality blastocysts, 51 were determined by next generation sequencing (NGS) to be euploid.Although oocyte maturity and the incidence of blastocyst formation were associated with follicle size, the incidence of euploidy among biopsied blastocysts was not. Follicles measured by two different methods (mm or Z values) led to predominantly the same conclusions.Limitations, Reasons For Caution: This study investigated the relationship between follicle size and measures of oocyte/embryo quality when donors were treated similarly. Therefore, this study does not investigate the effects of triggering and retrieving oocytes when the follicle cohorts are of different sizes or lead follicles are of different sizes. Although no association was found between follicle size and euploid blastocysts, the fact that blastocyst ploidy is not entirely dependent upon oocyte ploidy (e.g. aneuploidies derived from mitotic errors or from the fertilizing sperm) makes it difficult to infer the relationship between follicle diameter and oocyte ploidy.Wider Implications Of the Findings: It is confirmed that follicle diameter is predictive of oocyte maturity. However, once oocyte maturity is known, the diameter of the follicle from which the oocyte was retrieved is not instructive. Embryos generated through fertilization and development of the mature oocytes from any observed follicle diameter were equally likely to become euploid blastocysts.Study Funding/competing Interest(s): This study was funded by ReproART: Georgian American Center for Reproductive Medicine. None of the authors declare any actual conflicts of interest. D.H.M. received compensation from ReproART, Biogenetics Corporation and the Sperm and Embryo Bank of New York and honoraria and travel funding from Ferring Pharmaceuticals and from Granata Bio. S.M. received compensation from Cooper Genomics and an honorarium and travel funding from Ferring Pharmaceuticals. L.C. is the founder of LTD Ovamedi, the organization that represents Cooper Genomics in Georgia, and received travel funding from the European Society for Human Reproduction and Embryology.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

This chapter encapsulates a short introduction to positron emission tomography (PET) imaging and the information gained by using this technology to detect changes of the dopamine 3 receptor (D3R) at the molecular level in vivo. We will discuss available D3R radiotracers, emphasizing [11C]PHNO. The focus, however, will be on PET findings in conditions including substance abuse, obesity, traumatic brain injury, schizophrenia, Parkinson's disease, and aging. Finally, there is a discussion about progress in producing next-generation selective D3R radiotracers., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Positive effects of methylphenidate (MPH) on attention and cognitive processing speed have been reported in studies of patients with moderate to severe traumatic brain injury (TBI). Studies which have acquired functional brain imaging before and while using MPH have also found alteration of brain activation while performing a cognitive task; in some studies, this alteration of activation in selective brain regions was also related to improved performance on cognitive tests administered outside of the scanning environment. Enhanced cognitive performance has been reported after single doses of MPH and after daily treatment over durations of up to and exceeding 1 month. Preclinical research and both positron emission tomography and single photon emission tomography of humans have shown that MPH increases extracellular dopamine and norepinephrine; the dose effects of MPH have an inverted U -shaped function where high doses may cause insomnia, nervousness, and increased heart rate among other symptoms and impair cognitive performance, whereas too low a dose fails to improve cognitive performance. In the past 5 years, small clinical trials, and experimental pilot studies have found therapeutic effects of single and repeated low doses of MPH in patients with mild TBI who reported cognitive dysfunction. This literature also suggests that MPH may interact with concurrent cognitive interventions to enhance their effects. This focused review will critically evaluate the recent literature on MPH effects on cognitive dysfunction after mild to moderate TBI. To elucidate the neural mechanisms of MPH effects, this review will also include recent imaging research, preclinical, and experimental human studies. [ABSTRACT FROM AUTHOR]

Cognitive impairment is common following traumatic brain injury. Dopaminergic drugs can enhance cognition after traumatic brain injury, but individual responses are highly variable. This may be due to variability in dopaminergic damage between patients. We investigate whether measuring dopamine transporter levels using 123I-ioflupane single-photon emission computed tomography (SPECT) predicts response to methylphenidate, a stimulant with dopaminergic effects. Forty patients with moderate-severe traumatic brain injury and cognitive impairments completed a randomized, double-blind, placebo-controlled, crossover study. 123I-ioflupane SPECT, MRI and neuropsychological testing were performed. Patients received 0.3 mg/kg of methylphenidate or placebo twice a day in 2-week blocks. Subjects received neuropsychological assessment after each block and completed daily home cognitive testing during the trial. The primary outcome measure was change in choice reaction time produced by methylphenidate and its relationship to stratification of patients into groups with normal and low dopamine transporter binding in the caudate. Overall, traumatic brain injury patients showed slow information processing speed. Patients with low caudate dopamine transporter binding showed improvement in response times with methylphenidate compared to placebo [median change = -16 ms; 95% confidence interval (CI): -28 to -3 ms; P = 0.02]. This represents a 27% improvement in the slowing produced by traumatic brain injury. Patients with normal dopamine transporter binding did not improve. Daily home-based choice reaction time results supported this: the low dopamine transporter group improved (median change -19 ms; 95% CI: -23 to -7 ms; P = 0.002) with no change in the normal dopamine transporter group (P = 0.50). The low dopamine transporter group also improved on self-reported and caregiver apathy assessments (P = 0.03 and P = 0.02, respectively). Both groups reported improvements in fatigue (P = 0.03 and P = 0.007). The cognitive effects of methylphenidate after traumatic brain injury were only seen in patients with low caudate dopamine transporter levels. This shows that identifying patients with a hypodopaminergic state after traumatic brain injury can help stratify the choice of cognitive enhancing therapy. [ABSTRACT FROM AUTHOR]

Rationale: The sensorimotor cortex and the striatum are interconnected by the corticostriatal pathway, suggesting that cortical injury alters the striatal function, which may be modulated by dopamine. Objectives: We studied whether the activation of dopamine D1 receptors (D1Rs) modulates the γ-aminobutyric acid (GABA) and glutamate levels in the striatum of recovered rats at 192 h after cortical injury. Methods: The D1R agonist SKF-38393 (0, 2, 3, or 4 mg/kg) was administered at 24, 48, 96, and 192 h post-injury, and then rats were decapitated to determine GABA and glutamate levels and the levels of D1R mRNA on both sides of the striatum. Results: GABAergic imbalance in the striatum contralateral to the injury site was normalized by the administration of the D1R agonist, but this treatment did not produce a significant effect on glutamate levels, suggesting that glutamate was metabolized into GABA. The administration of SKF-38393 (2 mg/kg) decreased the levels of D1R mRNA in the striatum contralateral to the injury, and this effect was blocked by the coadministration of the D1R antagonist SCH-23390 (2 mg/kg). In the striatum ipsilateral to the injury, the D1R agonist increased the D1R mRNA levels, an effect that was blocked by SCH-23390. Conclusion: The reversal of the GABAergic imbalance in the striatum contralateral to the cortical injury can be modulated by extrastriatal D1R activation, and the D1R agonist-induced increases in the D1R mRNA levels in the striatum ipsilateral to the injury suggest that the striatum may be necessary to achieve functional recovery. [ABSTRACT FROM AUTHOR]

Purpose: To assess whether traumatic brain injury (TBI) increases the risks of subsequent problem gambling.Methods: We conducted a matched case-control analysis of adults in Ontario, Canada. The study included those who self-reported their gambling activities in the Canadian Community Health Survey 2007-2008. Using Problem Gambling Severity Index, we defined cases as those who were problem gamblers and controls who were recreational gamblers. Cases were matched to controls 1:2 using propensity scores based on demographics, prior mental health, and self-reported behaviours. The main predictor was prior TBI defined as requiring emergency care and identified using ICD-10 codes from administrative health databases. We estimated the likelihood of prior TBI in problem gamblers compared to controls using conditional logistic regression.Results: Of 30,652 survey participants, 16,002 (53%) reported gambling activity of whom 14,910 (49%) were recreational gamblers and 4% (n = 1092) were problem gamblers. A total of 1469 respondents (5%) had a prior TBI. Propensity score matching yielded 2038 matched pairs with 1019 cases matched to 2037 controls. Case-control analysis showed a significant association between prior TBI and subsequent problem gambling (odds ratio 1.27, 95% confidence interval 1.07-1.51, P = 0.007). The increased risk was mostly apparent in men aged 35 to 64 years who reported alcohol use or smoking. The relative risk of problem gambling in those with two or more TBIs equated to an odds ratio of 2.04 (95% confidence interval 1.05-3.99).Conclusions: We found that a prior TBI was associated with an increased subsequent risk of problem gambling. Our findings support more awareness, screening, and treating problem gambling risks among TBI patients. [ABSTRACT FROM AUTHOR]

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix a6 and helix a13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly. [ABSTRACT FROM AUTHOR]

Resveratrol is a polyphenolic compound produced by plants which makes its way into the human diet through plant-based foods. It has been shown to provide many health benefits, helping to ward of age-related diseases and promoting cardiovascular health. Additionally, resveratrol is a potent activator of the Notch signaling pathway. While resveratrol receives the most attention as a polyphenolic nutraceutical, other compounds with similar structures may be more potent regulators of specific cellular processes. Here, we compare resveratrol, apigenin, chrysin, genistein, luteolin, myricetin, piceatannol, pterostilbene, and quercetin for their ability to regulate Notch signaling. In addition, we compare the ability of these polyphenolic compounds to regulate endothelial cell viability, proliferation, and migration. Out of these compounds we found that resveratrol is the best activator of Notch signaling, however, other similar compounds are also capable of stimulating Notch. We also discovered that several of these polyphenols were able to inhibit endothelial cell proliferation. Finally, we found that many of these polyphenols are potent inhibitors of endothelial migration during wound healing assays. These findings provide the first side-by-side comparison of the regulation of Notch signaling, and endothelial cell proliferation and migration, by nine polyphenolic compounds. [ABSTRACT FROM AUTHOR]