Intracranial EEG reveals a time- and frequency-specific role for the right inferior frontal gyrus and primary motor cortex in stopping initiated responses.

Inappropriate response tendencies may be stopped via a specific fronto/basal ganglia/primary motor cortical network. We sought to characterize the functional role of two regions in this putative stopping network, the right inferior frontal gyrus (IFG) and the primary motor cortex (M1), using electocorticography from subdural electrodes in four patients while they performed a stop-signal task. On each trial, a motor response was initiated, and on a minority of trials a stop signal instructed the patient to try to stop the response. For each patient, there was a greater right IFG response in the beta frequency band ( approximately 16 Hz) for successful versus unsuccessful stop trials. This finding adds to evidence for a functional network for stopping because changes in beta frequency activity have also been observed in the basal ganglia in association with behavioral stopping. In addition, the right IFG response occurred 100-250 ms after the stop signal, a time range consistent with a putative inhibitory control process rather than with stop-signal processing or feedback regarding success. A downstream target of inhibitory control is M1. In each patient, there was alpha/beta band desynchronization in M1 for stop trials. However, the degree of desynchronization in M1 was less for successfully than unsuccessfully stopped trials. This reduced desynchronization on successful stop trials could relate to increased GABA inhibition in M1. Together with other findings, the results suggest that behavioral stopping is implemented via synchronized activity in the beta frequency band in a right IFG/basal ganglia network, with downstream effects on M1.

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

Host risk factors for rifamycin-resistant clostridium difficile infection: A case control study

Objective. To evaluate the host risk factors associated with rifamycin-resistant Clostridium difficile (C. diff) infection in hospitalized patients compared to rifamycin-susceptible C.diff infection.^ Background. C. diff is the most common definable cause of nosocomial diarrhea affecting elderly hospitalized patients taking antibiotics for prolonged durations. The epidemiology of Clostridium difficile associated disease is now changing with the reports of a new hypervirulent strain causing hospital outbreaks. This new strain is associated with increased disease severity and mortality. The conventional therapy for C. diff includes metronidazole and vancomycin but high recurrence rates and treatment failures are now becoming a major concern. Rifamycin antibiotics are being developed as a new therapeutic option to treat C. diff infection after their efficacy was established in a few in vivo and in vitro studies. There are some recent studies that report an association between the hypervirulent strain and emerging rifamycin resistance. These findings assess the need for clinical studies to better understand the efficacy of rifamycin drugs against C. diff.^ Methods. This is a hospital-based, matched case-control study using de-identified data drawn from two prospective cohort studies involving C. diff patients at St Luke's Hospital. The C. diff isolates from these patients are screened for rifamycin resistance using agar dilution methods for minimum inhibitory concentrations (MIC) as part of Dr Zhi-Dong Jiang's study. Twenty-four rifamycin-rifamycin resistant C. diff cases were identified and matched with one rifamycin susceptible C. diff control on the basis of ± 10 years of age and hospitalization 30 days before or after the case. De-identified data for the 48 subjects was obtained from Dr Kevin Garey's clinical study at St Luke's Hospital enrolling C. diff patients. It was reviewed to gather information about host risk factors, outcome variables and relevant clinical characteristic.^ Results. Medical diagnosis at the time of admission (p = 0.0281) and history of chemotherapy (p = 0.022) were identified as a significant risk factor while hospital stay ranging from 1 week to 1 month and artificial feeding were identified as an important outcome variable (p = 0.072 and p = 0.081 respectively). Horn's Index assessing the severity of underlying illness and duration of antibiotics for cases and controls showed no significant difference.^ Conclusion. The study was a small project designed to identify host risk factors and understand the clinical implications of rifamycin-resistance. The study was underpowered and a larger sample size is needed to validate the results.^

Thermo -modulation of MuSVts110 splicing by inhibitory RNA sequences

Viral systems have contributed tremendously to the understanding of eukaryotic molecular biology. The proportional pattern of retroviral RNA expression offers many clues into the alternative splicing of cellular transcripts. The MuSVts110 virus presents an unusual expression system, where the mechanistic combination of RNA splicing and cellular transformation can be physiologically manipulated. Splicing of MuSVts110 pre-mRNA occurs inefficiently (30%-50%) at 33$\sp\circ$C or below and is subdued at 39$\sp\circ$C ($<$5%). Like most alternatively spliced cellular and retroviral transcripts, the MuSVts110 pre-mRNA contains cis-acting intron and exon sequences that attenuate splicing. These include a splicing inhibitory sequence at the 3$\prime$ end of the MuSVts110 v-mos exon, called the E2 Distal Element (E2DE), and a sub-optimal 3$\prime$ splice site. The E2DE directly inhibits MuSVts110 RNA splicing in a sequence-specific fashion at 39$\sp\circ$C but not at 28$\sp\circ$C, potentially through the association of cellular factors. Inefficient MuSVts110 splicing is pre-dominantly attributed to the utilization of multiple weak branchpoint sequences located between $-113$ and $-34$ nucleotides upstream of the 3$\prime$ splice site. The molecular control of MuSVts110 splicing, represented primarily by scattered multiple inefficient branchpoint sequences that are conditionally modulated by the E2DE at higher growth temperatures, is discussed. ^

Regulation of phosphatidylinositide turnover in the myometrium by cAMP: Implications for the control of uterine contraction

Regulation of uterine quiescence involves the integration of the signaling pathways regulating uterine contraction and relaxation. Uterine contractants increase intracellular calcium through receptor/GαqPLC coupling, resulting in contraction of the myometrium. Elevation of cAMP concentration has been correlated with relaxation of the myometrium. However, the mechanism of cAMP action in the uterus is unclear. ^ Both endogenous and exogenous increases in cAMP inhibited oxytocin-stimulated phosphatidylinositide turnover in an immortalized pregnant human myometrial cell line (PHM1-41). This inhibition was reversed by cAMP-dependent protein kinase (PKA) inhibitors, suggesting the involvement of PKA. cAMP inhibited phosphatidyinositide turnover stimulated by different agonists in different cell lines. These data suggest that the cAMP inhibitory mechanism is neither cell nor receptor dependent, and inhibits Gαq/PLCβ1 and PLCβ3 coupling. ^ The subcellular localization of PKA occurs via PKA binding to A-Kinase-Anchoring-Proteins (AKAP), and peptides that inhibit this association have been developed (S-Ht31). S-Ht31 blocked cAMP-stimulated PKA activity and decreased PKA concentration in PHM1-41 cell plasma membranes. S-Ht31 reversed the ability of CPT-cAMP, forskolin and relaxin to inhibit phosphatidylinositide turnover in PHM1-41 cells. Overlay analysis of both PHM1-41 cell and nonpregnant rat myometrium found an AKAPs of 86 kDa and 150 kDa associated with the plasma membrane, respectively. These data suggest that PKA anchored to the plasma membrane via AKAP150/PKA anchoring is involved in the cAMP inhibitory mechanism. ^ CPT-cAMP and isoproterenol inhibited phosphatidylinositide turnover in rat myometrium from days 12 through 20 of gestation. In contrast, neither agent was effective in the 21 day pregnant rat myometrium. The decrease in the cAMP inhibitory mechanism was correlated with a decrease in PKA and an increase in protein phosphatase 2B (PP2B) concentration in rat myometrial plasma membranes on day 21 of gestation. In myometrial total cell homogenates, both PKA and PP2B concentration increased on day 21. S-Ht31 inhibited cAMP inhibition of phosphatidylinositide turnover in day 19 pregnant rat myometrium. Both PKA and PP2B coimmunoprecipitated with an AKAP150 in a gestational dependent manner, suggesting this AKAP localizes PKA and PP2B to the plasma membrane. ^ These data presented demonstrate the importance of the cAMP inhibitory mechanism in regulating uterine contractility. ^