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Visual experience gives rise to persistent theta oscillations in the mouse primary visual cortex (V1) that are specific to the familiar stimulus. Our recent work demonstrated the presence of these oscillations in higher visual areas (HVAs), where they are synchronized with V1 in a context-dependent manner. However, it remains unclear where these unique oscillatory dynamics originate. To investigate this, we conducted paired extracellular electrophysiological recordings in two visual thalamic nuclei (dorsal lateral geniculate nucleus [dLGN] and lateral posterior nucleus [LP]), the retrosplenial cortex (RSC), and the hippocampus (HPC). Oscillatory activity was not found in either of the thalamic nuclei, but a sparse ensemble of oscillating neurons was observed in both the RSC and HPC, similar to V1. To infer functional connectivity changes between the brain regions, we performed directed information analysis, which indicated a trend toward decreased connectivity in all V1-paired regions, with a consistent increase in V1 → V1 connections, suggesting that the oscillations appear to initiate independently within V1. Lastly, complete NMDA lesioning of the HPC did not abolish theta oscillations in V1 that emerge with familiarity. Altogether, these results suggest that (1) theta oscillations do not originate in the thalamus; (2) RSC exhibits theta oscillations, which may follow V1 given the temporal delay present; and (3) the HPC had a sparse group of neurons, with theta oscillations matching V1; however, lesioning suggests that these oscillations emerge independent of each other. Overall, our findings pave the way for future studies to determine the mechanisms by which diverse inputs and outputs shape this memory-related oscillatory activity in the brain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Transverse (t)-tubules drive the rapid and synchronous Ca 2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca 2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca 2+ ., Methods: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca 2+ , and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure., Results: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca 2+ release in the cell interior. Systolic Ca 2+ , I Ca-L , sarcoplasmic reticulum Ca 2+ content, and sarcoendoplasmic reticulum Ca 2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca 2+ transient, the rate of Ca 2+ removal, and the peak L-type Ca 2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria., Conclusions: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of I Ca-L , sarcoplasmic reticulum Ca 2+ content, and systolic Ca 2+ . We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy., Competing Interests: None.

Fragile X syndrome (FX) is the most prevalent inheritable form of autism spectrum disorder (ASD), characterized by hypersensitivity, difficulty in habituating to new sensory stimuli, and intellectual disability. Individuals with FX often experience visual perception and learning deficits. Visual experience leads to the emergence of the familiarity-evoked theta band oscillations in the primary visual cortex (V1) and the lateromedial area (LM) of mice. These theta oscillations in V1 and LM are synchronized with each other, providing a mechanism of sensory multi-areal binding. However, how this multi-areal binding and the corresponding theta oscillations are altered in FX is not known. Using iDISCO whole brain clearing with light-sheet microscopy, we quantified immediate early gene Fos expression in V1 and LM, identifying deficits in experience-dependent neural activity in FX mice. We performed simultaneous in vivo recordings with silicon probes in V1 and LM of awake mice and channelrhodopsin-2-assisted circuit mapping (CRACM) in acute brain slices to examine the neural activity and strength of long-range synaptic connections between V1 and LM in both wildtype (WT) and Fmr1 knockout (KO) mice, the model of FX, before and after visual experience. Our findings reveal synchronized familiarity-evoked theta oscillations in V1 and LM, the increased strength of V1→LM functional and synaptic connections, which correlated with the corresponding changes of presynaptic short-term plasticity in WT mice. The LM oscillations were attenuated in FX mice and correlated with impaired functional and synaptic connectivity and short-term plasticity in the feedforward (FF) V1→LM and feedback (FB) LM→V1 pathways. Finally, using 4Pi single-molecule localization microscopy (SMLM) in thick brain tissue, we identified experience-dependent changes in the density and shape of dendritic spines in layer 5 pyramidal cells of WT mice, which correlated with the functional synaptic measurements. Interestingly, there was an increased dendritic spine density and length in naïve FX mice that failed to respond to experience. Our study provides the first comprehensive characterization of the role of visual experience in triggering inter-areal neural synchrony and shaping synaptic connectivity in WT and FX mice.

Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca 2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Visual perceptual experience induces persistent 4-8 Hz oscillations in the mouse primary visual cortex (V1), encoding visual familiarity. Recent studies suggest that higher-order visual areas (HVAs) are functionally specialized and segregated into information streams processing distinct visual features. However, whether visual memories are processed and stored within the distinct streams is not understood. We report here that V1 and lateromedial (LM), but not V1 and anterolateral, become more phase synchronized in 4-8 Hz after the entrainment of visual stimulus that maximally induces responses in LM. Directed information analysis reveals changes in the top-down functional connectivity between V1 and HVAs. Optogenetic inactivation of LM reduces post-stimulus oscillation peaks in V1 and impairs visual discrimination behavior. Our results demonstrate that 4-8 Hz familiarity-evoked oscillations are specific for the distinct visual features and are present in the corresponding HVAs, where they may be used for the inter-areal communication with V1 during memory-related behaviors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Insufficient oxygen supply (hypoxia) during fetal and embryonic development can lead to latent phenotypical changes in the adult cardiovascular system, including altered cardiac function and increased susceptibility to ischemia reperfusion injury. While the cellular mechanisms underlying this phenomenon are largely unknown, several studies have pointed towards metabolic disturbances in the heart of offspring from hypoxic pregnancies. To this end, we investigated mitochondrial function in the offspring of a mouse model of prenatal hypoxia. Pregnant C57 mice were subjected to either normoxia (21%) or hypoxia (14%) during gestational days 6-18. Offspring were reared in normoxia for up to 8 months and mitochondrial biology was assessed with electron microscopy (ultrastructure), spectrophotometry (enzymatic activity of electron transport chain complexes), microrespirometry (oxidative phosphorylation and H 2 0 2 production) and Western Blot (protein expression). Our data showed that male adult offspring from hypoxic pregnancies possessed mitochondria with increased H 2 0 2 production and lower respiratory capacity that was associated with reduced protein expression of complex I, II and IV. In contrast, females from hypoxic pregnancies had a higher respiratory capacity and lower H 2 0 2 production that was associated with increased enzymatic activity of complex IV. From these results, we speculate that early exposure to hypoxia has long term, sex-dependent effects on cardiac metabolic function, which may have implications for cardiovascular health and disease in adulthood., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d 0 -acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Fragile X syndrome (FX), the most common inherited form of autism and intellectual disability, is a condition associated with visual perceptual learning deficits. We recently discovered that perceptual experience can encode visual familiarity via persistent low-frequency oscillations in the mouse primary visual cortex (V1). Here, we combine this paradigm with a multifaceted experimental approach to identify neurophysiological impairments of these oscillations in FX mice. Extracellular recordings reveal shorter durations, lower power, and lower frequencies of peak oscillatory activity in FX mice. Directed information analysis of extracellularly recorded spikes reveals differences in functional connectivity from multiple layers in FX mice after the perceptual experience. Channelrhodopsin-2 assisted circuit mapping (CRACM) reveals increased synaptic strength from L5 pyramidal onto L4 fast-spiking cells after experience in wild-type (WT), but not FX, mice. These results suggest differential encoding of visual stimulus familiarity in FX via persistent oscillations and identify circuit connections that may underlie these changes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Lisano, JK, Smith, JD, Mathias, AB, Christensen, M, Smoak, P, Phillips, KT, Quinn, CJ, and Stewart, LK. Performance and health-related characteristics of physically active men using marijuana. J Strength Cond Res 33(6): 1659-1669, 2019-The influence of chronic marijuana use on the performance and health of physically active individuals has yet to be fully elucidated. The purpose of this study was to explore pulmonary function, aerobic and anaerobic fitness, strength, serum testosterone, cortisol, C-reactive protein (CRP), Δ-9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-Δ-9-tetrahydrocannabinol (THC-COOH), and 11-hydroxy-Δ-9-tetrahydrocannabinol (THC-OH) concentrations in a physically active population either using or not using marijuana. Healthy, physically active males (N = 24) were compared based on their marijuana-use status: marijuana users (MU; n = 12) and nonusers (NU; n = 12). Statistical analysis (p = 0.05) revealed no difference between groups for age, body mass, body mass index, body fat, forced expiratory volume in 1 second percentage, VO2max, anaerobic power output, strength measures, testosterone, or cortisol concentrations. Although not statistically significant, MU showed a trend to fatigue to a greater percentage of absolute power output than NU from the beginning to the end of the Wingate Anaerobic Power Assessment (p = 0.08, effect size = 0.75). C-reactive protein in MU (1.76 ± 2.81 mg·L) and NU (0.86 ± 1.49 mg·L) was not significantly different (p = 0.60) but placed MU at moderate risk and NU at low risk for cardiovascular disease. Anaerobic fatigue was the only performance variable to show a trend for difference between groups. These results suggest that marijuana use in physically active males may not have significant effects on performance; however, it may be linked to elevated concentrations of CRP which place users at a higher risk for cardiovascular disease.

The prevalence of myopia is high and increasing. Approximately 5 billion people around the world are expected to be myopic by the year 2050. Methods to slow the progression of myopia and therefore potentially decrease the associated sight-threatening complications have been the subject of a number of investigations. A workshop, sponsored by the United States Food and Drug Administration (FDA) Center for Devices and Radiological Health, American Academy of Ophthalmology, American Academy of Optometry, American Association for Pediatric Ophthalmology and Strabismus, American Optometric Association, American Society of Cataract and Refractive Surgery, and Contact Lens Association of Ophthalmologists, Inc, convened myopia experts from around the world to discuss principles to consider in the design of clinical trials investigating the effectiveness and safety of myopia control devices. Experts discussed parameters such as study endpoints, duration, enrollment criteria, patient-reported outcomes, recruitment, and retention. The discussions among the experts, FDA, and audience members should help to facilitate the development and evaluation of reasonably safe and effective myopia control devices.

Background: The skeletal muscle toxicity that accompanies the chemotherapy drug doxorubicin (DOX) may lead to cancer patient weakness and fatigue. This myotoxicity involves myogenic regulatory factor (MRF) disruption which alters muscle integrity and regeneration. Endurance exercise enhances MRF expression and thereby may mitigate DOX-induced MRF disruptions. This study examined the effects of endurance training and DOX treatment on myogenic regulatory factor (MRF) expression., Methods: Male rats were exercise trained (EXER) or remained sedentary (SED) for two weeks. EXER and SED then received either DOX (15 mg/kg) or saline (SAL). Soleus, extensor digitorum longus (EDL), and diaphragm were excised 24 hours post injection, and MRF expression was analyzed., Results: Significant Myf5 drug and activity effects were observed in the soleus with EXER+DOX expressing higher Myf5 than SED+DOX. A significant drug effect was detected in soleus MyoD, and a significant activity effect was detected in soleus Mrf4. No main effects or interactions were observed in the EDL, but in the diaphragm, a significant activity effect was observed for Myf5 with EXER+DOX expressing higher levels than SED+DOX., Conclusion: Doxorubicin treatment increased soleus MRFs and exercise boosted MRF response in soleus and diaphragm suggesting that exercise may enhance regenerative signaling with DOX treatment., Level of Evidence: I b, individual randomized controlled trial.

The use of prior exercise training has shown promise in minimizing doxorubicin (DOX)-induced physical impairments. The purpose of this study was to compare changes in thymus mass, thymocyte (T-cell) number, and tissue peroxidation following chronic endurance exercise and DOX treatment in the rat. The thymus mass, number of viable T-cells, and levels of malondialdehyde and 4-hydroxyalkenals (MDA+4-HAE) were compared 3 days post-injection between rats assigned to the following treatment conditions: (a) 10 weeks of endurance training, followed by a saline injection 24 hours after the last training session (TM+SAL); (b) treadmill training as above, followed by a single, bolus 10-mg/kg injection of DOX (TM+10); (c) treadmill training with 12.5 mg/kg of DOX (TM+12.5); (d) sedentary (without exercise) and a saline injection (SED+SAL); (e) sedentary with 10 mg/kg of DOX (SED+10); and (f) sedentary with 12.5 mg/kg (SED+12.5). Thymic mass and T-cell numbers significantly decreased following DOX injections. TM rats exhibited significantly less lipid peroxidation compared with paired-dose SED groups. TM+10 did not significantly differ from SED+SAL in thymic levels of lipid peroxidation. We conclude that chronic endurance exercise decreases levels of lipid peroxidation in the thymus seen with acute DOX treatment., (© The Author(s) 2015.)

Introduction: Chemotherapy treatment with doxorubicin (DOX) can have a negative effect on normal skeletal muscle function. Recent research demonstrates the potential value of exercise in alleviating DOX-induced cardiotoxicity. Yet up to now, little research has been done to examine whether exercise might also be effective in addressing DOX's skeletal muscle adverse effects, especially because posttreatment skeletal muscle dysfunction may cause patient difficulties with completing activities of daily living. The main aim of this study was to examine how resistance training (RT) and treadmill (TM) training play a role in preventing DOX-induced skeletal muscle dysfunction., Methods: Male Sprague-Dawley rats were randomly placed into an RT, TM, or sedentary (SED) group for 10 wk and then received either a bolus injection of DOX (15 mg·kg) or saline as a control. Skeletal muscle function was then assessed ex vivo 5 d after injection., Results: SED animals treated with DOX showed significantly lower maximal twitch force, maximal rate of force production, and maximal rate of force decline versus SED + saline in the soleus (SOL) (Type I muscle). In the extensor digitorum longus (Type II muscle), treatment with DOX resulted in a significantly lower maximal rate of force production and maximal rate of force decline. RT preserved maximal twitch force and maximal rate of force decline in the SOL. TM attenuated DOX-induced fatigue in the SOL but not in the extensor digitorum longus., Conclusion: These findings suggest that RT and TM before DOX could be useful in preserving skeletal muscle function and minimizing fatigue after chemotherapy, but this protection may be dependent on the skeletal muscle type.

1. Doxorubicin (DOX) is a highly effective and commonly used anthracycline antibiotic used to treat cancer patients. The side effects of DOX are manifested in a more delayed manner in children and multidrug resistant proteins (MRPs) may factor into this phenomenon. MRPs are known to extrude DOX and may factor into the degree of cardiac DOX accumulation. 2. The purpose of this study was to examine age-related differences in muscle MRP expression and DOX accumulation. 3. Female Sprague-Dawley rats were randomly selected to receive a 15-mg DOX/kg body weight bolus injection (i.p.) at various ages. 4. Cardiac and extensor digitorum longus DOX accumulation was markedly increased as animals aged from 4 to 24 weeks. In contrast, no differences in soleus accumulation were observed. A significant age-related reduction in MRP-2 and MRP-7 expression was detected in cardiac and extensor digitorum longus tissues with no age differences in MRP-1 expression in any tissues analyzed. MRP-6 was not detected in any tissues. 5. These data suggest that aging is associated with increased DOX accumulation and an age-related decrease in MRP expression may be a factor.

Background: Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA., Methods: High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment., Results: 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells., Conclusions: Rapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Woody stems that have completed some maturation of metaxylem elements should not be capable of further axial extension ("secondary stem lengthening"). However, this mechanism has been claimed by Renninger and Phillips (American Journal of Botany 99: 607-613) to be a feature of the palm Iriartea deltoidea. In response, we describe structural features of palm stems based on extensive known features of their anatomy and development. In addition to the inability of metaxylem vessels to extend after they are mature, fully differentiated fibers of the vascular bundle sheath, which would exist at the time of proposed stem elongation would not be capable of belated extension. "Vessel spirals" claimed by these authors to be capable of stretching to accommodate secondary stem lengthening does not refer to well-established features of the course of vascular bundles. The approach adopted by Renninger and Phillips simply measures stems of different sizes as an implied developmental series. Consequently, results do not take into account changes in the development of the palm stem as it ages. The existence of secondary stem lengthening in the palm Iriartea deltoidea, something never before observed in any tree, cannot occur because it would indeed disrupt mature metaxylem vessels and would also require the secondary extension of mature lignified fibers.

Background: Psychogenic polydipsia is prevalent amongst psychiatric patients, but less common in the general population. Generally, hyponatremia ensues with complications of cerebral edema resulting in confusion, seizures, coma, and death. Rapid correction of serum sodium levels can lead to further complications of osmotic demyelination of neurons, e.g. central pontine myelinolysis., Case Report: We present a case of a 32-year-old male who presented with seizures while being treated at a drug rehabilitation facility. He was discovered to be hyponatremic secondary to suspected psychogenic polydipsia. The patient impressively responded to treatment of fluid restriction and desmopressin and symptoms improved., Conclusions: Among the causes of hyponatremia, psychogenic polydipsia may be more difficult to diagnose especially if an apparent psychiatric condition is not present. Current literature supports cautious correction of hyponatremia to prevent complications. However, rapid corrections may be driven by the physiology of the patient and may not be avoidable. Fortunately, our case illustrates rapid, positive outcomes for the patient.

Concentrations of cadmium in the grain of durum wheat (Triticum turgidum L. var durum) are often above the internationally acceptable limit of 0.2 mg kg(-1). Cultivars that vary in concentrations of cadmium in the grain have been identified but the physiology behind differential accumulation has not been determined. Three pairs of near-isogenic lines (isolines) of durum wheat that vary in aboveground cadmium accumulation (8982-TL 'high' and 'low', W9260-BC 'high' and 'low', and W9261-BG 'high' and 'low') were used to test the hypothesis that the greater amounts of cadmium in shoots of the 'high' isolines are correlated with greater volumes of water transpired. In general, cadmium content was positively correlated with transpiration only in the 'low' isolines. Although shoots of the 'high' isolines of W9260-BC and W9261-BG contained higher concentrations of cadmium than did their corresponding 'low' isolines, they did not transpire larger volumes of water. In addition, isolines of 8982-TL transpired less water than did the other pairs of isolines yet both 'high' and 'low' isolines of 8982-TL contained higher amounts of cadmium than did the other pairs. The difference between 'high' and 'low' isolines appears to be related to the relative contribution of transpiration to cadmium translocation to the shoot. Increased transpiration was associated with increased cadmium content in the 'low' isolines but in the 'high' isolines increased cadmium in the shoot occurred independently of the volume of water transpired.

Advances in recording technologies have given neuroscience researchers access to large amounts of data, in particular, simultaneous, individual recordings of large groups of neurons in different parts of the brain. A variety of quantitative techniques have been utilized to analyze the spiking activities of the neurons to elucidate the functional connectivity of the recorded neurons. In the past, researchers have used correlative measures. More recently, to better capture the dynamic, complex relationships present in the data, neuroscientists have employed causal measures-most of which are variants of Granger causality-with limited success. This paper motivates the directed information, an information and control theoretic concept, as a modality-independent embodiment of Granger's original notion of causality. Key properties include: (a) it is nonzero if and only if one process causally influences another, and (b) its specific value can be interpreted as the strength of a causal relationship. We next describe how the causally conditioned directed information between two processes given knowledge of others provides a network version of causality: it is nonzero if and only if, in the presence of the present and past of other processes, one process causally influences another. This notion is shown to be able to differentiate between true direct causal influences, common inputs, and cascade effects in more two processes. We next describe a procedure to estimate the directed information on neural spike trains using point process generalized linear models, maximum likelihood estimation and information-theoretic model order selection. We demonstrate that on a simulated network of neurons, it (a) correctly identifies all pairwise causal relationships and (b) correctly identifies network causal relationships. This procedure is then used to analyze ensemble spike train recordings in primary motor cortex of an awake monkey while performing target reaching tasks, uncovering causal relationships whose directionality are consistent with predictions made from the wave propagation of simultaneously recorded local field potentials.

Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism, in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF fluorescence confirms formation of EI and provides a direct measurement of the association rate. Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*

The design, synthesis, enzymologic, and protein mass spectrometric characterization of benzodioxocin-3-one and N-acyl-3-amino-3-buten-2-one inhibitors of the cysteine protease cathepsin K are described. The benzodioxocin-3-one ring system is chemically unstable giving rise to a mixture of N-acyl-3-amino-3-buten-2-one and hemiketals. This mixture of N-acyl-3-amino-3-buten-2-one and hemiketals potently inhibits recombinant, human cathepsin K (IC50 = 36 nM) by a time-independent, irreversible mechanism. Formation of a covalent adduct between cathepsin K and inhibitor has been confirmed by mass spectrometry.

Purpose: To further investigate the efficacy and safety of temozolomide plus thalidomide in patients with metastatic melanoma without brain metastases., Patients and Methods: Patients with histologically confirmed advanced-stage metastatic melanoma were enrolled in an open-label, phase II study. The primary end point was response rate. Patients received temozolomide (75 mg/m2/d x 6 weeks with a 2-week rest between cycles) plus concomitant thalidomide (200 mg/d with dose escalation to 400 mg/d for patients < 70 years old, or 100 mg/d with dose escalation to 250 mg/d for patients >/= 70 years old). Treatment was continued until unacceptable toxicity or disease progression occurred., Results: Thirty-eight patients (median age, 62 years) with stage IV (three patients with M1a, eight with M1b, and 26 with M1c) or stage IIIc (one patient) melanoma and a median of four metastatic sites were enrolled, and received a median of two cycles of therapy. Twelve patients (32%) had an objective tumor response, including one with an ongoing complete response of 25+ months' duration and 11 with partial responses. Five patients achieving partial response with a more than 90% reduction of disease were converted to a complete response with surgery. Treatment was generally well tolerated. Median survival was 9.5 months (95% confidence interval, 6.05 to 19.38 months), with a median follow-up among survivors of 24.3 months., Conclusion: The combination of temozolomide plus thalidomide seems to be a promising and well-tolerated oral regimen for metastatic melanoma that merits further study.

Purpose: To establish a safe and tolerated regimen of an oral cytotoxic agent, temozolomide, and a cytostatic agent, thalidomide, in patients with unresectable stage III or IV malignant melanoma., Patients and Methods: Patients with unresectable stage III or IV melanoma without brain metastases were entered successively onto four treatment cohorts: level 1, temozolomide 50 mg/m(2)/d for 6 weeks followed by a 4-week break; levels 2, 3, and 4, temozolomide 75 mg/m(2)/d for 6 weeks followed, respectively, by breaks of 4, 3, and 2 weeks. Thalidomide was started at 200 mg/d, and escalated to a maximum dose of 400 mg/d. Safety was assessed at weeks 2 and 4 and every 4 weeks thereafter; tumor response was evaluated every 8 to 10 weeks., Results: Twelve patients were enrolled, three on each cohort. Therapy was generally well tolerated on all of the treatment schedules. Thalidomide at a dose of 400 mg/d was well tolerated in patients younger than 70, and 200 mg/d was well tolerated in older patients. The most common adverse events were grade 2 or 3 constipation and neuropathy, which were attributed to thalidomide. Five major responses (one complete, four partial) were documented, all at dose levels 2 to 4. Three of the five responding patients were in the over-70 age group. The median duration of response was 6 months (range, 4 to 17+ months), and the median overall survival was 12.3 months (range, 4 to 19+ months)., Conclusion: The combination of temozolomide and thalidomide was well tolerated and had antitumor activity in patients with advanced melanoma, including elderly patients over 70 years old.

Cladistic analyses are presented of matK sequence data as well as a nonmolecular database for an identical set of exemplar species chosen to represent the core genera or groups of genera in Myrtaceae. Eleven robust clades are recognized on the molecular data. Polyphyly of the previously recognized Metrosideros and Leptospermum alliances is confirmed, and several smaller informal taxonomic groupings are recognized from among the members of the former alliance, i.e., the Tristania, Tristaniopsis, Metrosideros, and Lophostemon groups. The nonmolecular analysis provides only limited resolution of relationships. A degree of congruence exists between the two analyses in that two separate fleshy-fruited clades, the Acmena and Myrtoid groups, are identified, as are the Eucalypt and Tristania groups, and Psiloxylon and Heteropyxis are the first lineages to diverge in both analyses. A combined analysis recognized all 11 clades that received strong support from the molecular data. A high level of homoplasy is revealed in many of the nonmolecular characters when they are examined against the combined estimate of phylogeny.

Morphological and ITS (internal transcribed spacer) sequence data for 40 species of the Austral-Pacific genera Camptacra, Kippistia, Minuria, Peripleura, Tetramolopium, and Vittadinia as well as one semiherbaceous species of Olearia were subjected to cladistic analysis, separately and together. Minuria, Peripleura, and Tetramolopium are paraphyletic as currently defined. Tetramolopium vagans from Australia appears to represent an undescribed genus. Both Kippistia suadefolia and Peripleura diffusa show close affinity to Minuria species, and Minuria macrorhiza appears to contain two distinct but closely related species. Vittadinia and the remaining species of Tetramolopium and Peripleura form a strong affinity group. The distribution of indels and the combined analysis each provide evidence that the Hawaiian and Cook Island species of Tetramolopium are descended from New Guinea species. The combined analysis also suggests that the Cook Island species T. mitiaroense is sister to the Hawaiian clade. Olearia arguta groups strongly with Camptacra and shows no close affinity with either of the arborescent species of Olearia used to root these analyses. Marked homoplasy among morphological characters indicates why generic delimitation in the group has been problematic.

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.

Cathepsin K (EC 3.4.22.38), a cysteine protease of the papain superfamily, is predominantly expressed in osteoclasts and has been postulated as a target for the treatment of osteoporosis. Crystallographic and structure--activity studies on a series of acyclic ketone-based inhibitors of cathepsin K have led to the design and identification of two series of cyclic ketone inhibitors. The mode of binding for four of these cyclic and acyclic inhibitors to cathepsin K is discussed and compared. All of the structures are consistent with addition of the active site thiol to the ketone of the inhibitors with the formation of a hemithioketal. Cocrystallization of the C-3 diastereomeric 3-amidotetrahydrofuran-4-one analogue 16 with cathepsin K showed the inhibitor to occupy the unprimed side of the active site with the 3S diastereomer preferred. This C-3 stereochemical preference is in contrast to the X-ray cocrystal structures of the 3-amidopyrrolidin-4-one inhibitors 29 and 33 which show these inhibitors to prefer binding of the 3R diastereomer. The 3-amidopyrrolidin-4-one inhibitors were bound in the active site of the enzyme in two alternate directions. Epimerization issues associated with the labile alpha-amino ketone diastereomeric center contained within these inhibitor classes has proven to limit their utility despite promising pharmacokinetics displayed in both series of compounds.

Sequence data from the noncoding region separating the plastid genes atpbeta and rbcL were gathered for 27 epacrid taxa, representing all previously recognized infrafamilial groups, and four outgroup taxa (Ericaceae), to address several persistent phylogenetic questions in the group. Parsimony analyses were conducted on these data, as well as on a complementary rbcL sequence dataset assembled from the literature and the combined dataset. The atpbeta-rbcL spacer was notable for the high frequency of insertion-deletion mutations (indels); their distributions were coded as binary characters and included as a adjunct matrix in some of the analyses. The phylogenetic patterns derived from the spacer and rbcL data and the combined analyses, both including and excluding the indel data, concur in resolving seven major lineages corresponding to the tribes of Crayn et al. (1998, Aust. J. Bot. 46, 187-200), viz. Prionoteae, Archerieae, Oligarrheneae, Cosmelieae, Richeeae, Epacrideae, and Styphelieae. The relationships of the tribes and within Styphelieae, however, are not convincingly resolved. Minor conflicts in the positions of some taxa between the spacer and the rbcL trees are poorly supported. Among epacrids, the spacer region provided more cladistically informative characters than rbcL and resulted in trees with lower homoplasy. Further, the spacer data, when analyzed alone and when combined with rbcL, resolved several clades that could not be retrieved on rbcL data alone and provided increased support for many other relationships. The evolution of a putative three-base inversion associated with a hairpin secondary structure in the spacer region is discussed in the light of the inferred phylogeny., (Copyright 2000 Academic Press.)

Parsimony analysis of matK and rbcL sequence data, together with a nonmolecular database, yielded a well-resolved phylogeny of Cupressaceae sensu lato. Monophyly of Cupressaceae sensu stricto is well supported, and separate northern and southern hemisphere subclades are resolved, with Tetraclinis within the northern subclade; there is no support for any of the tribes sensu Li. Taxodiaceae comprise five separate lineages. Chamaecyparis nootkatensis falls within Cupressus, clustering with a robust clade of New World species. Libocedrus Florin is paraphyletic and should incorporate Pilgerodendron. Evolution of several characters of wood and leaf anatomy and chemistry is discussed in light of this estimate of the phylogeny; numerous parallelisms are apparent. A new infrafamilial classification is proposed in which seven subfamilies are recognized: Callitroideae Saxton, Athrotaxidoideae Quinn, Cunninghamioideae (Sieb. & Zucc.) Quinn, Cupressoideae Rich. ex Sweet, Sequoioideae (Luerss.) Quinn, Taiwanioideae (Hayata) Quinn, Taxodioideae Endl. ex K. Koch. The rbcL sequence for Taxodium distichum is corrected, and the implications for a previously published estimate of the minimum rate of divergence of the gene since the Miocene are highlighted.

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-delta 4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amide-linked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans had a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-delta 13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.

Cystoid macular edema (CME) may develop in association with a wide variety of ocular conditions. It is the result of cystic accumulation of extracellular intraretinal fluid in the outer plexiform and inner nuclear layers of the retina, as a result of breakdown of the blood-retinal barrier. It is most common following intraocular surgery, and in patients with venous occlusive disease, diabetic retinopathy, and posterior segment inflammatory conditions. A variety of approaches to the treatment of CME have been attempted, with a variable degree of success. These options have included topical and oral steroids, nonsteroidal anti-inflammatory agents, and laser photocoagulation treatment. The exact cause of CME and the effective treatment of this condition have remained elusive.

Nucleotide sequences of the chloroplast gene, rbcL, were obtained for Suriana maritima, Cadellia pentastylis, Guilfoylia monostylis, Stylobasium australe, Quassia amara, Brucea mollis, Cupaniopsis anacardioides, Mangifera indica, Connarus conchocarpus, and Comesperma ericinum. Phylogenetic analyses of these, along with published sequences of representatives of rosid families, support the monophyly of the Surianaceae sensu Cronquist and provide evidence of an affinity with the Polygalaceae and Fabaceae. These conclusions are discussed in the light of available morphological data.