A small molecule that inhibits the evolution of antibiotic resistance.

Antibiotic resistance rapidly develops against almost all available therapeutics. Therefore, searching for new antibiotics to overcome the problem of antibiotic resistance alone is insufficient. Given that antibiotic resistance can be driven by mutagenesis, an avenue for preventing it is the inhibition of mutagenic processes. We previously showed that the DNA translocase Mfd is mutagenic and accelerates antibiotic resistance development. Here, we present our discovery of a small molecule that inhibits Mfd-dependent mutagenesis, ARM-1 (anti-resistance molecule 1). We found ARM-1 using a high-throughput, small molecule, in vivo screen. Using biochemical assays, we characterized the mechanism by which ARM-1 inhibits Mfd. Critically, we found that ARM-1 reduces mutagenesis and significantly delays antibiotic resistance development across highly divergent bacterial pathogens. These results demonstrate that the mutagenic proteins accelerating evolution can be directly inhibited. Furthermore, our findings suggest that Mfd inhibition, alongside antibiotics, is a potentially effective approach for prevention of antibiotic resistance development during treatment of infections.

Chemical-genetics refines transcription factor regulatory circuits.

Transcriptional dysregulation is a key step in oncogenesis, but our understanding of transcriptional control has relied on genetic approaches that are slow and allow for compensation. Chemical-genetic approaches have shortened the time frame for the analysis of transcription factors from days or weeks to minutes. These studies show that while DNA-binding proteins bind to thousands of sites, they are directly required to regulate only a small cadre of genes. Moreover, these transcriptional control networks are far more distinct, with much less overlap and interconnectivity than predicted from DNA binding. The identified direct targets can then be used to dissect the mechanism of action of these factors, which could identify ways to therapeutically manipulate these oncogenic transcriptional control networks.

The in vivo measurement of replication fork velocity and pausing by lag-time analysis.

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell. To capture these dynamics, we develop lag-time analysis, a method for measuring in vivo dynamics. Using this approach, we provide quantitative locus-specific measurements of fork velocity, in units of kilobases per second, as well as replisome pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In this work, we quantitatively characterize known phenomena, detect brief, locus-specific pauses at ribosomal DNA loci in wild-type cells, and observe temporal fork velocity oscillations in three highly-divergent bacterial species.

O-GlcNAcylation suppresses TRAP1 activity and promotes mitochondrial respiration.

The molecular chaperone TNF-receptor-associated protein-1 (TRAP1) controls mitochondrial respiration through regulation of Krebs cycle and electron transport chain activity. Post-translational modification (PTM) of TRAP1 regulates its activity, thereby controlling global metabolic flux. O-GlcNAcylation is one PTM that is known to impact mitochondrial metabolism, however the major effectors of this regulatory PTM remain inadequately resolved. Here we demonstrate that TRAP1-O-GlcNAcylation decreases TRAP1 ATPase activity, leading to increased mitochondrial metabolism. O-GlcNAcylation of TRAP1 occurs following mitochondrial import and provides critical regulatory feedback, as the impact of O-GlcNAcylation on mitochondrial metabolism shows TRAP1-dependence. Mechanistically, loss of TRAP1-O-GlcNAcylation decreased TRAP1 binding to ATP, and interaction with its client protein succinate dehydrogenase (SDHB). Taken together, TRAP1-O-GlcNAcylation serves to regulate mitochondrial metabolism by the reversible attenuation of TRAP1 chaperone activity.

SARM1 knockout does not rescue neuromuscular phenotypes in a Charcot-Marie-Tooth disease Type 1A mouse model.

Charcot-Marie-Tooth disease Type 1A (CMT1A) is caused by duplication of the PMP22 gene and is the most common inherited peripheral neuropathy. Although CMT1A is a dysmyelinating peripheral neuropathy, secondary axon degeneration has been suggested to drive functional deficits in patients. Given that SARM1 knockout is a potent inhibitor of the programmed axon degeneration pathway, we asked whether SARM1 knockout rescues neuromuscular phenotypes in CMT1A model (C3-PMP) mice. CMT1A mice were bred with SARM1 knockout mice to generate CMT1A/SARM1-/- mice. A series of behavioral assays were employed to evaluate motor and sensorimotor function. Electrophysiological and histological studies of the tibial branch of the sciatic nerve were performed. Additionally, gastrocnemius and soleus muscle morphology were evaluated histologically. Although clear behavioral and electrophysiological deficits were observed in CMT1A model mice, genetic deletion of SARM1 conferred no significant improvement. Nerve morphometry revealed predominantly myelin deficits in CMT1A model mice and SARM1 knockout yielded no improvement in all nerve morphometry measures. Similarly, muscle morphometry deficits in CMT1A model mice were not improved by SARM1 knockout. Our findings demonstrate that programmed axon degeneration pathway inhibition does not provide therapeutic benefit in C3-PMP CMT1A model mice. Our results indicate that the clinical phenotypes observed in CMT1A mice are likely caused primarily by prolonged dysmyelination, motivate further investigation into mechanisms of dysmyelination in these mice and necessitate the development of improved CMT1A rodent models that recapitulate the secondary axon degeneration observed in patients.

Case report: A case of classic hairy cell leukemia with CNS involvement treated with vemurafenib.

Hairy cell leukemia (HCL) is a rare mature B-cell lymphoproliferative disorder and most often presents as classic hairy cell leukemia. This entity is characterized by an indolent course and the presence of the BRAF V600E mutation. We report the case of an 80-year-old man with a history of classical hairy cell leukemia who presented with fatigue, dizziness, shortness of breath, blurring of vision, and headache. His initial diagnosis was 9 years prior, and he received treatments with cladribine, pentostatin, and rituximab. The workup showed an elevated white blood cell count with atypical lymphocytes, anemia, and thrombocytopenia. A peripheral blood smear confirmed HCL relapse, and a magnetic resonance imaging (MRI) of the brain showed diffuse, nonenhancing masses in the supratentorial and infratentorial regions of the brain. He was initiated on treatment with vemurafenib, with improvements in his white blood cell count and a recovery of his platelet count and hemoglobin. A repeat MRI of the brain after 3 months showed complete resolution of the lesions. Vemurafenib was discontinued after 6 months, with bone marrow biopsy showing no evidence of residual hairy cell leukemia. There have only been limited reports of HCL involvement in the central nervous system in the literature. Due to the rarity of the condition, it is not clear which treatments can be effective for intracranial disease control. Our report shows the successful use of vemurafenib, resulting in complete remission of relapsed HCL with CNS involvement.

GADD45A is a protective modifier of neurogenic skeletal muscle atrophy.

Neurogenic muscle atrophy is the loss of skeletal muscle mass and function that occurs with nerve injury and in denervating diseases, such as amyotrophic lateral sclerosis. Aside from prompt restoration of innervation and exercise where feasible, there are currently no effective strategies for maintaining skeletal muscle mass in the setting of denervation. We conducted a longitudinal analysis of gene expression changes occurring in atrophying skeletal muscle and identified growth arrest and DNA damage-inducible A (Gadd45a) as a gene that shows one of the earliest and most sustained increases in expression in skeletal muscle after denervation. We evaluated the role of this induction using genetic mouse models and found that mice lacking GADD45A showed accelerated and exacerbated neurogenic muscle atrophy, as well as loss of fiber type identity. Our genetic analyses demonstrate that, rather than directly contributing to muscle atrophy as proposed in earlier studies, GADD45A induction likely represents a protective negative feedback response to denervation. Establishing the downstream effectors that mediate this protective effect and the pathways they participate in may yield new opportunities to modify the course of muscle atrophy.

New evidence for secondary axonal degeneration in demyelinating neuropathies.

Development of peripheral nervous system (PNS) myelin involves a coordinated series of events between growing axons and the Schwann cell (SC) progenitors that will eventually ensheath them. Myelin sheaths have evolved out of necessity to maintain rapid impulse propagation while accounting for body space constraints. However, myelinating SCs perform additional critical functions that are required to preserve axonal integrity including mitigating energy consumption by establishing the nodal architecture, regulating axon caliber by organizing axonal cytoskeleton networks, providing trophic and potentially metabolic support, possibly supplying genetic translation materials and protecting axons from toxic insults. The intermediate steps between the loss of these functions and the initiation of axon degeneration are unknown but the importance of these processes provides insightful clues. Prevalent demyelinating diseases of the PNS include the inherited neuropathies Charcot-Marie-Tooth Disease, Type 1 (CMT1) and Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) and the inflammatory diseases Acute Inflammatory Demyelinating Polyneuropathy (AIDP) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). Secondary axon degeneration is a common feature of demyelinating neuropathies and this process is often correlated with clinical deficits and long-lasting disability in patients. There is abundant electrophysiological and histological evidence for secondary axon degeneration in patients and rodent models of PNS demyelinating diseases. Fully understanding the involvement of secondary axon degeneration in these diseases is essential for expanding our knowledge of disease pathogenesis and prognosis, which will be essential for developing novel therapeutic strategies.

Cognitive-affective strategies and cortisol stress reactivity in children and adolescents: Normative development and effects of early life stress.

This study examined cognitive-affective strategies as predictors of hypothalamic-pituitary-adrenal (HPA) axis responses to a social-evaluative stressor in adolescence as compared to late childhood as a function of early life experiences. Participants included 159 children (9-10 years) and adolescents (15-16 years) divided into two groups based on early care experiences: non-adopted youth raised in their birth families (n = 81) and post-institutionalized youth internationally adopted from orphanage care (n = 78). Youth completed a version of the Trier Social Stress Test modified for use with children and reported on their trait emotion regulation and coping strategies. Children reported more use of suppression and disengagement than adolescents, while adolescents reported more engagement coping strategies. Non-adopted and post-institutionalized youth did not differ in reported strategies. Cognitive reappraisal predicted higher cortisol reactivity in non-adopted children and adolescents, and was not associated with reactivity in the post-institutionalized group. This study has implications for efforts aimed at promoting self-regulation and adaptive stress responses during the transition to adolescence for both typically developing children and children who experienced adverse early care.

The astrocytic transporter SLC7A10 (Asc-1) mediates glycinergic inhibition of spinal cord motor neurons.

SLC7A10 (Asc-1) is a sodium-independent amino acid transporter known to facilitate transport of a number of amino acids including glycine, L-serine, L-alanine, and L-cysteine, as well as their D-enantiomers. It has been described as a neuronal transporter with a primary role related to modulation of excitatory glutamatergic neurotransmission. We find that SLC7A10 is substantially enriched in a subset of astrocytes of the caudal brain and spinal cord in a distribution corresponding with high densities of glycinergic inhibitory synapses. Accordingly, we find that spinal cord glycine levels are significantly reduced in Slc7a10-null mice and spontaneous glycinergic postsynaptic currents in motor neurons show substantially diminished amplitudes, demonstrating an essential role for SLC7A10 in glycinergic inhibitory function in the central nervous system. These observations establish the etiology of sustained myoclonus (sudden involuntary muscle movements) and early postnatal lethality characteristic of Slc7a10-null mice, and implicate SLC7A10 as a candidate gene and auto-antibody target in human hyperekplexia and stiff person syndrome, respectively.

Early Life Stress: Effects on the Regulation of Anxiety Expression in Children and Adolescents.

This study examined children's (N = 79; 9-10 years) and adolescents' (N = 82; 15-16 years) ability to regulate their emotion expressions of anxiety as they completed a modified version of the Trier Social Stress Test for Children (TSST-C). Approximately half in each age group were internationally adopted from institutional care (N = 79) and half were non-adopted, age-matched peers (N = 82). Institutional care was viewed as a form of early life stress. Coders who were reliable and blind to group status watched videos of the session to assess anxiety expressions using the Child and Adolescent Stress and Emotion Scale developed for this study. Children exhibited more expressions of anxiety than adolescents, and youth adopted from institutions showed more expressions of anxiety than their non-adopted counterparts. The role of early life stress on observed anxiety expressions remained significant after controlling for differences in age, physiological stress responses measured through salivary cortisol reactivity, and self-reports of stress during the TSST-C. This suggests possible deficits in the regulation of expressive behavior for youth with early life stress histories, which cannot be explained by experiencing the task as more stressful.

Early social deprivation and the social buffering of cortisol stress responses in late childhood: An experimental study.

The goal of the present study was to investigate the role of early social deprivation in shaping the effectiveness of parent support to alleviate hypothalamic-pituitary-adrenal (HPA)-axis-stress responses of children (ages 8.9-11, M = 9.83 years, SD = .55). The sample was equally divided between children who had been adopted internationally from orphanage care by age 5 (n = 40) and an age- and gender-matched group of nonadopted (NA) children (n = 40). On average, internationally adopted children were invited to the laboratory 7.6 years postadoption (SD = 1.45). We experimentally manipulated the provision of parent support during the 5-min speech preparation period before a modified Trier Social Stress Test (TSST) and examined its effect on levels of salivary cortisol secreted in response to this laboratory stressor. All participants were randomly assigned to receive support from their parent or a stranger. Analyses revealed a significant interaction of support condition and group such that parent support significantly dampened the cortisol-stress response in NA children compared with support from a stranger, whereas the cortisol response curves of postinstitutionalized (PI) children did not differ between the parent- and stranger-support conditions. Cortisol reactivity for PI children in both conditions was lower than that of NA children in the stranger-support condition. Social deprivation during the first few years of life may shape neurobehavioral development in ways that reduce selective responses to caregivers versus strangers.

The impact of early neglect on defensive and appetitive physiology during the pubertal transition: a study of startle and postauricular reflexes.

This study tested the effect of early neglect on defensive and appetitive physiology during puberty. Emotion-modulated reflexes, eye-blink startle (defensive) and postauricular (appetitive), were measured in 12-to-13-year-old internationally adopted youth (from foster care or from institutional settings) and compared to non-adopted US born controls. Startle Reflex: adopted youth displayed lower overall startle amplitude across all valences and startle potentiation to negative images was negatively related to severity of pre-adoption neglect. Postauricular reflex (PAR): adopted youth showed larger PAR magnitude across all valences. Puberty: adopted youth showed diminished PAR potentiation to positive images and startle potentiation during mid/late puberty versus the opposite pattern in not-adopted. Early neglect was associated with blunted fast defensive reflexes and heightened fast appetitive reflexes. After puberty, early neglected youth showed physiological hyporeactivity to threatening and appetitive stimuli versus heightened reactivity in not adopted youth. Behavioral correlates in this sample and possible neurodevelopmental mechanisms of psychophysiological differences are discussed.

Parent support is less effective in buffering cortisol stress reactivity for adolescents compared to children.

The goal of the present study was to investigate developmental differences in the effectiveness of parent support to alleviate hypothalamic-pituitary-adrenal (HPA) axis stress responses of children (ages 9-10, N = 40) and adolescents (ages 15-16, N = 41). We experimentally manipulated the provision of parent support during the speech preparation period before a modified Trier Social Stress Test (TSST) and examined its effect on levels of salivary cortisol secreted in response to this laboratory stressor. Analyses revealed a significant interaction of condition and age group such that social support from the parent (versus a stranger) significantly eliminated the cortisol stress response in children, but had no effect on the response among adolescents.

Risk-taking and sensation-seeking propensity in postinstitutionalized early adolescents.

BACKGROUND: Youth with histories of institutional/orphanage care are at increased risk for externalizing and internalizing problems during childhood and adolescence. Although these problems have been well described, the related adolescent behaviors of risk-taking and sensation-seeking have not yet been explored in this population. This study examined risk-taking and sensation-seeking propensity, and associations with conduct problems and depressive symptoms, in early adolescents who were adopted as young children from institutional care. METHODS: Risk-taking and sensation-seeking propensities of 12- and 13-year-old postinstitutionalized (PI; n = 54) adolescents were compared with two groups: youth who were internationally adopted early from foster care (PFC; n = 44) and nonadopted youth (NA; n = 58). Participants were recruited to equally represent pre/early- and mid/late-pubertal stages within each group. Participants completed the youth version of the Balloon Analogue Risk Task (Lejuez et al., ) and the Sensation-Seeking Scale for Children (Russo et al., ). Parents completed clinical ratings of participants' conduct problems and depressive symptoms. RESULTS: PI adolescents demonstrated lower risk-taking than PFC and NA peers. Pre/early-pubertal PI youth showed lower sensation-seeking, while mid/late-pubertal PI youth did not differ from other groups. PI adolescents had higher levels of conduct problems but did not differ from the other youth in depressive symptoms. In PI youth only, conduct problems were negatively correlated with risk-taking and positively correlated with sensation-seeking, while depressive symptoms were negatively correlated with both risk-taking and sensation-seeking. CONCLUSIONS: Early institutional care is associated with less risk-taking and sensation-seeking during adolescence. The deprived environment of an institution likely contributes to PI youth having a preference for safe choices, which may only be partially reversed with puberty. Whether this reflects hyporesponsiveness to rewards and how it relates to psychopathology are discussed.

The effect of early deprivation on executive attention in middle childhood.

BACKGROUND: Children reared in deprived environments, such as institutions for the care of orphaned or abandoned children, are at increased risk for attention and behavior regulation difficulties. This study examined the neurobehavioral correlates of executive attention in post institutionalized (PI) children. METHODS: The performance and event-related potentials (ERPs) of 10- and 11-year-old internationally adopted PI children on two executive attention tasks, go/no-go and Flanker, were compared with two groups: children internationally adopted early from foster care (PF) and nonadopted children (NA). RESULTS: Behavioral measures suggested problems with sustained attention, with PIs performing more poorly on go trials and not on no-go trials of the go/no-go and made more errors on both congruent and incongruent trials on the Flanker. ERPs suggested differences in inhibitory control and error monitoring, as PIs had smaller N2 amplitude on go/no-go and smaller error-related negativity on Flanker. CONCLUSIONS: This pattern of results raises questions regarding the nature of attention difficulties for PI children. The behavioral errors are not specific to executive attention and instead likely reflect difficulties in overall sustained attention. The ERP results are consistent with neural activity related to deficits in inhibitory control (N2) and error monitoring (error-related negativity). Questions emerge regarding the similarity of attention regulatory difficulties in PIs to those experienced by non-PI children with ADHD.

Growth delay as an index of allostatic load in young children: predictions to disinhibited social approach and diurnal cortisol activity.

The goal of this study was to examine whether growth delay can serve as an index of allostatic load during early development, as it is well known that the activity of stress-mediating systems inhibits growth. The participants were children adopted internationally from institutional care (n = 36), children adopted internationally from foster care (n = 26), and nonadopted children (n = 35). For the adopted children, height for age and weight for height were assessed at adoption; for all children, disinhibited social approach (DSA; termed elsewhere as "indiscriminate friendliness") and diurnal cortisol were assessed at 6-8 years (M = 6.9 years). For internationally adopted children in general, and postinstitutionalized children specifically, linear growth delay assessed at the time of adoption was associated with more dysregulated behavior in response to an unfamiliar adult (i.e., greater DSA) and a more dysregulated diurnal cortisol rhythm (i.e., higher late afternoon and evening values). Further, among the most growth-delayed children, higher cortisol levels later in the day were correlated with DSA. The potential for using growth delay as an allostatic load indicator and the possible problems and limitations in its use in child populations are discussed.

A reengineered hospital discharge program to decrease rehospitalization: a randomized trial.

BACKGROUND: Emergency department visits and rehospitalization are common after hospital discharge. OBJECTIVE: To test the effects of an intervention designed to minimize hospital utilization after discharge. DESIGN: Randomized trial using block randomization of 6 and 8. Randomly arranged index cards were placed in opaque envelopes labeled consecutively with study numbers, and participants were assigned a study group by revealing the index card. SETTING: General medical service at an urban, academic, safety-net hospital. PATIENTS: 749 English-speaking hospitalized adults (mean age, 49.9 years). INTERVENTION: A nurse discharge advocate worked with patients during their hospital stay to arrange follow-up appointments, confirm medication reconciliation, and conduct patient education with an individualized instruction booklet that was sent to their primary care provider. A clinical pharmacist called patients 2 to 4 days after discharge to reinforce the discharge plan and review medications. Participants and providers were not blinded to treatment assignment. MEASUREMENTS: Primary outcomes were emergency department visits and hospitalizations within 30 days of discharge. Secondary outcomes were self-reported preparedness for discharge and frequency of primary care providers' follow-up within 30 days of discharge. Research staff doing follow-up were blinded to study group assignment. RESULTS: Participants in the intervention group (n = 370) had a lower rate of hospital utilization than those receiving usual care (n = 368) (0.314 vs. 0.451 visit per person per month; incidence rate ratio, 0.695 [95% CI, 0.515 to 0.937]; P = 0.009). The intervention was most effective among participants with hospital utilization in the 6 months before index admission (P = 0.014). Adverse events were not assessed; these data were collected but are still being analyzed. LIMITATION: This was a single-center study in which not all potentially eligible patients could be enrolled, and outcome assessment sometimes relied on participant report. CONCLUSION: A package of discharge services reduced hospital utilization within 30 days of discharge. FUNDING: Agency for Healthcare Research and Quality and National Heart, Lung, and Blood Institute, National Institutes of Health.

Chondroitin sulfate perlecan enhances collagen fibril formation. Implications for perlecan chondrodysplasias.

Inactivation of the perlecan gene leads to perinatal lethal chondrodysplasia. The similarity to the phenotypes of the Col2A1 knock-out and the disproportionate micromelia mutation suggests perlecan involvement in cartilage collagen matrix assembly. We now present a mechanism for the defect in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional to the content of the 4,6-disulfated disaccharide in the different cartilage extracts, with growth plate cartilage glycosaminoglycan being the most efficient enhancer. These findings demonstrate a role for perlecan chondroitin sulfate side chains in cartilage extracellular matrix assembly and provide an explanation for the perlecan-null chondrodysplasia.