Catheter-Induced Left Ventricular Perforation and Cardiac Tamponade During Left Heart Catheterization.

Iatrogenic ventricular perforation of the myocardial wall is a rare but life-threatening complication. It has been described using pulmonary artery catheter, pigtail catheter, and Judkins catheter. Straight wires and catheters can be used to cross the aortic valve for left ventriculogram; however, the risk of perforation is higher compared with J-tip wires. Prompt recognition of cardiac tamponade and pericardial drain insertion is vital, but surgical patch repair may be required for definitive treatment. This case highlights the importance of increased vigilance and prompt management of cardiac tamponade with the use of high-risk equipment during cardiac catheterization.

Very late bioprosthetic aortic valve thrombosis.

A 79-year-old man with a history of bioprosthetic aortic valve (AV) replacement in 2008 and atrial fibrillation was admitted with acute pulmonary oedema. Transthoracic and transoesophageal echocardiograms revealed significantly elevated AV gradients and thickened AV leaflets. These findings were suggestive of bioprosthetic valve thrombosis (BVT). The patient was treated with intravenous heparin and commenced on vitamin K antagonist. BVT remains an under recognised cause of late prosthetic valve dysfunction. A lack of awareness of BVT occurring beyond 3 months post-implantation is likely to account for this. Furthermore, structural valve degeneration is the most common mechanism of late prosthetic valve dysfunction. Recognising the difference between the two aetiologies is crucial as the management plan differs significantly. Here, we report a case of very late bioprosthetic AV thrombosis diagnosed 8 years after implantation. This was successfully treated with systemic anticoagulation, thereby avoiding the need for redo cardiac surgery.

Audiovisual Lexical Retrieval Deficits Following Left Hemisphere Stroke.

Binding sensory features of multiple modalities of what we hear and see allows formation of a coherent percept to access semantics. Previous work on object naming has focused on visual confrontation naming with limited research in nonverbal auditory or multisensory processing. To investigate neural substrates and sensory effects of lexical retrieval, we evaluated healthy adults (n = 118) and left hemisphere stroke patients (LHD, n = 42) in naming manipulable objects across auditory (sound), visual (picture), and multisensory (audiovisual) conditions. LHD patients were divided into cortical, cortical⁻subcortical, or subcortical lesions (CO, CO⁻SC, SC), and specific lesion location investigated in a predictive model. Subjects produced lower accuracy in auditory naming relative to other conditions. Controls demonstrated greater naming accuracy and faster reaction times across all conditions compared to LHD patients. Naming across conditions was most severely impaired in CO patients. Both auditory and visual naming accuracy were impacted by temporal lobe involvement, although auditory naming was sensitive to lesions extending subcortically. Only controls demonstrated significant improvement over visual naming with the addition of auditory cues (i.e., multisensory condition). Results support overlapping neural networks for visual and auditory modalities related to semantic integration in lexical retrieval and temporal lobe involvement, while multisensory integration was impacted by both occipital and temporal lobe lesion involvement. The findings support modality specificity in naming and suggest that auditory naming is mediated by a distributed cortical⁻subcortical network overlapping with networks mediating spatiotemporal aspects of skilled movements producing sound.

Caspase-2 modulates osteoclastogenesis through down-regulating oxidative stress.

The loss of caspase-2 (Casp-2) in mice results in an osteopenic phenotype associated with increased numbers of osteoclasts in vivo. In this study, we show that Casp-2 is involved in osteoclastogenesis. Protein levels of Casp-2 decrease during the differentiation of macrophages to osteoclasts. Furthermore, siRNA-mediated Casp-2 knockdown in osteoclast precursors or differentiation of bone marrow macrophage (BMM) precursors from Casp2(-/-) mice results in increased osteoclast numbers and tartrate-resistant acid phosphatase (TRAP) activity. Casp2(-/-) osteoclasts are larger in size compared to wild-type osteoclasts and exhibited increased numbers of nuclei, perhaps due to increased precursor fusion. The loss of Casp-2 did not alter earlier stages of differentiation, but had a greater consequence on later stages involving NFATc1 auto-amplification and pre-osteoclast fusion. We have previously shown that the loss of Casp-2 results in increased oxidative stress in the bone. Reactive oxygen species (ROS) is known to play a critical role in late osteoclast differentiation and we show that total ROS and specifically, mitochondrial ROS, significantly increased in Casp2(-/-) BMM precursors after RANKL administration, with a concomitant reduction in FoxO3a and its target antioxidant enzymes, catalase and superoxide 2 (SOD2). Because mitochondrial ROS has been identified as a putative regulator of the later stages of differentiation, the heightened ROS levels in Casp2(-/-) cells likely promote precursor fusion and increased osteoclast numbers. In conclusion, our results indicate a novel role of Casp-2 in the osteoclast as a modulator of total and mitochondrial ROS and osteoclast differentiation.

A nonapoptotic role for CASP2/caspase 2: modulation of autophagy.

CASP2/caspase 2 plays a role in aging, neurodegeneration, and cancer. The contributions of CASP2 have been attributed to its regulatory role in apoptotic and nonapoptotic processes including the cell cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in the cells. Previously, our lab demonstrated CASP2-mediated modulation of autophagy during oxidative stress. Here we report the novel finding that CASP2 is an endogenous repressor of autophagy. Knockout or knockdown of CASP2 resulted in upregulation of autophagy in a variety of cell types and tissues. Reinsertion of Caspase-2 gene (Casp2) in mouse embryonic fibroblast (MEFs) lacking Casp2 (casp2(-/-)) suppresses autophagy, suggesting its role as a negative regulator of autophagy. Loss of CASP2-mediated autophagy involved AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and autophagy-related proteins, indicating the involvement of the canonical pathway of autophagy. The present study also demonstrates an important role for loss of CASP2-induced enhanced reactive oxygen species production as an upstream event in autophagy induction. Additionally, in response to a variety of stressors that induce CASP2-mediated apoptosis, casp2(-/-) cells demonstrate a further upregulation of autophagy compared with wild-type MEFs, and upregulated autophagy provides a survival advantage. In conclusion, we document a novel role for CASP2 as a negative regulator of autophagy, which may provide important insight into the role of CASP2 in various processes including aging, neurodegeneration, and cancer.

Caspase-2 maintains bone homeostasis by inducing apoptosis of oxidatively-damaged osteoclasts.

Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging.

Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a ß-thujaplicinol analogue.

EFV (efavirenz) and ß-thujaplicinol [2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one] have contrasting effects on the RNase H activity of HIV-1 RT (reverse transcriptase). EFV binds in the non-nucleoside inhibitor-binding pocket and accelerates this activity, whereas ß-thujaplicinol binds in the RNase H active site and inhibits it. We have used pre-steady-state kinetic analyses to gain an insight into the mechanism by which EFV and a ß-thujaplicinol analogue [19616 (2,7-dihydroxy-2,4,6-cyclo-heptatrien-1-one)] modulate RT RNase H activity. Our data show that EFV and 19616 have no effect on polymerase-dependent RNase H cleavages. However, both compounds significantly affected the rates of polymerase-independent RNase H cleavages. In regard to the latter, we found no evidence that the bound RNA/DNA template/primer substrate restricted 19616 from interacting with RT. In light of these data, we propose a model in which 19616 binds to the RNase H active site of RT after the primary polymerase-dependent RNase H cleavage has occurred and stabilizes the 3'-end of the DNA primer in the polymerase active site thus blocking the enzyme's ability to carry out the polymerase-independent cleavages. By contrast, EFV destabilizes the 3'-end of the DNA primer in the DNA polymerase active site and promotes RT-mediated polymerase-independent cleavages. Consistent with this model, we show antagonism between EFV and 19616.

Loss of caspase-2 accelerates age-dependent alterations in mitochondrial production of reactive oxygen species.

Mitochondria are known to be a major source and target of oxidative stress. Oxidative stress increases during aging and is suggested to underlie in part the aging process. We have previously documented an increase in endogenous caspase-2 (casp2) activity in hepatocytes obtained from old (28 months) vs. young mice (5 months). More recently, we have shown that casp2 is activated by oxidative stress and is critical for mitochondrial oxidative stress-induced apoptosis. Since casp2 appears integral to mitochondrial oxidative stress-induced apoptosis, in this study we determined whether loss of casp2 altered the production of mitochondrial reactive oxygen radicals (mROS) as a function of age in intact living hepatocytes. To stimulate mitochondrial metabolic activity, we added a mixture of pyruvate and glutamate to hepatocytes while continuously monitoring endogenous mROS production in the presence or absence of rotenone and/or antimycin A. Our data demonstrate that mROS production and neutralization are compromised in hepatocytes of old mice. Interestingly, casp2 deficient hepatocytes from middle age mice (12 months) had similar mROS neutralization kinetics to those of hepatocytes from old WT mice. Rotenone had no effect on mROS metabolism, whereas antimycin A significantly altered mROS production and metabolism in an age-dependent fashion. Our results indicate that: (1) hepatocytes from young and old mice respond differently to dysfunction of the mitochondrial electron transport chain; (2) age-dependent alterations in mROS metabolism are likely regulated by complex III; and (3) absence of casp2 accelerates age-dependent changes in terms of pyruvate/glutamate-induced mROS metabolism.

N348I in HIV-1 reverse transcriptase counteracts the synergy between zidovudine and nevirapine.

The efficacy of regimens that include both zidovudine and nevirapine can be explained by the synergistic interactions between these drugs. N348I in HIV-1 reverse transcriptase confers decreased susceptibility to zidovudine and nevirapine. Here, we demonstrate that N348I reverses the synergistic inhibition of HIV-1 by zidovudine and nevirapine. Also, the efficiency of zidovudine-monophosphate excision in the presence of nevirapine is greater for N348I HIV-1 reverse transcriptase compared with the wild-type enzyme. These data help explain the frequent selection of N348I in regimens that contain zidovudine and nevirapine, and suggest that the selection of N348I should be monitored in resource-limited settings where these drugs are routinely used.

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs.

ß-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

Synthesis, antiviral activity, cytotoxicity and cellular pharmacology of l-3'-azido-2',3'-dideoxypurine nucleosides.

Microwave-assisted optimized transglycosylation reactions were used to prepare eleven modified l-3'-azido-2',3'-dideoxypurine nucleosides. These l-nucleoside analogs were evaluated against HIV and hepatitis B virus. The l-3'-azido-2',3'-dideoxypurines nucleosides were metabolized to nucleoside 5'-triphosphates in primary human lymphocytes, but exhibited weak or no antiviral activity against HIV-1. The nucleosides were also inactive against HBV in HepG2 cells. Pre-steady state kinetic experiments demonstrated that the l-3'-azido-2',3'-dideoxypurine triphosphates could be incorporated by purified HIV-1 reverse transcriptase, although their catalytic efficiency (k(pol)/K(d)) of incorporation was low. Interestingly, a phosphoramidate prodrug of l-3'-azido-2',3'-dideoxyadenosine exhibited anti-HIV-1 activity without significant toxicity.

Synthesis and anti-HIV evaluation of 3'-triazolo nucleosides.

A series of hitherto unknown 3'-α-[1,2,3]-substituted triazolo-2',3'-dideoxypyrimidine nucleoside analogues of the anti-HIV 3'-azido-3'-deoxythymidine (AZT) were synthesized through catalyzed alkyne-azide 1,3-dipolar cycloaddition (Huisgen reaction). Those 3'-[1,2,3]-triazolo analogues bearing an azido alkyl chain were evaluated for their anti-HIV activity against HIV-1 in primary human lymphocytes as well as for their cytotoxicity in different cells. None of them inhibit HIV replication (EC(50) > 20 µM); two of them were converted to their triphosphate form to evaluate their HIV-RT inhibition.

Safety of same day discharge following percutaneous coronary intervention.

BACKGROUND: There is a body of literature reporting the safety of discharging patients the same day as percutaneous coronary revascularisation. Nevertheless, overnight stay continues to be the general standard of care. METHODS: Over a single calendar year, 130 patients having elective, percutaneous coronary revascularisation were discharged home the day of the procedure with the majority of procedures using radial access. Patients were observed post procedure for six hours and if no problems occurred, discharge was undertaken. The purpose of the study was to assess complications in the 24 hours following discharge. RESULTS: Within the following 24 hours post discharge, there were no complications reported including bleeding, recurrent ischaemia, or hospitalisation. CONCLUSION: Same day discharge following elective percutaneous revascularisation appears both efficacious and safe with a low risk of post discharge complications.

A knockout of the caspase 2 gene produces increased resistance of the nigrostriatal dopaminergic pathway to MPTP-induced toxicity.

This study investigated the effect of a knockout of the caspase 2 gene on the sensitivity of murine nigral dopaminergic neurons to 1-methyl-4-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. Female wild type (WT), heterozygous caspase 2 NL (HET) and homozygous caspase 2 null (NL) mice were treated with cumulative dosages of 0, 10, 15 or 20 mg/kg MPTP free base. Without MPTP treatment, one week later dopamine (DA) levels were not significantly different in HET or NL versus WT mice. Twenty mg/kg MPTP reduced striatal DA in WT and HET (p<0.01) but not NL mice. This same MPTP dosage regimen also induced a significantly greater decrease in tyrosine hydroxylase immunopositive (TH+) protein in striata of WT compared to NL mice (p<0.001). Subsequently, WT and NL mice were treated daily with 20 mg/kg MPTP for 3 days and 25 mg/kg MPTP for 2 additional days, and TH+ neurons in the substantia nigra (SN) were estimated using unbiased stereology. When compared to untreated WT, the numbers of TH+ neurons were significantly lower in the SN of untreated NL mice (p<0.05). Treatment with the MPTP regimen significantly reduced TH+ neurons in WT mice but not NL mice. In primary mesencephalic cultures both the cell bodies and the neuronal processes of TH immunopositive (TH+) neurons from NL embryos were significantly (p<0.001) more resistant to 10 µM MPP+ compared to WT. Following MPP+ treatment, features of apoptotic cell death were also significantly (p<0.001) more prevalent in nuclei of TH+ neurons in cultures prepared from WT versus NL mouse pups. These results suggest that caspase 2 may play a role in modulating the MPTP-induced damage to the nigrostriatal dopaminergic system.

Loss of caspase-2-dependent apoptosis induces autophagy after mitochondrial oxidative stress in primary cultures of young adult cortical neurons.

Mitochondrial dysfunctions have been associated with neuronal apoptosis and are characteristic of neurodegenerative conditions. Caspases play a central role in apoptosis; however, their involvement in mitochondrial dysfunction-induced neuronal apoptosis remains elusive. In the present report using rotenone, a complex I inhibitor that causes mitochondrial dysfunction, we determined the initiator caspase and its role in cell death in primary cultures of cortical neurons from young adult mice (1-2 months old). By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor that irreversibly binds to and traps the active caspase, we identified caspase-2 as an initiator caspase activated in rotenone-treated primary neurons. Loss of caspase-2 inhibited rotenone-induced apoptosis; however, these neurons underwent a delayed cell death by necrosis. We further found that caspase-2 acts upstream of mitochondria to mediate rotenone-induced apoptosis in neurons. The loss of caspase-2 significantly inhibited rotenone-induced activation of Bid and Bax and the release of cytochrome c and apoptosis inducing factor from mitochondria. Rotenone-induced downstream activation of caspase-3 and caspase-9 were also inhibited in the neurons lacking caspase-2. Autophagy was enhanced in caspase-2 knock-out neurons after rotenone treatment, and this response was important in prolonging neuronal survival. In summary, the present study identifies a novel function of caspase-2 in mitochondrial oxidative stress-induced apoptosis in neurons cultured from young adult mice.

Synthesis and Anti-HIV-1 Activity of a Novel Series of Aminoimidazole Analogs.

There is still an urgent need to develop nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) with a high-genetic barrier to resistance that facilitate patient adherence and allow durable suppression of HIV-1 replication. In this study, we describe the synthesis of a novel series of N-aminoimidazole (NAIM) analogs. Each of the NAIM analogs display potent activity against wild-type recombinant purified HIV-1 RT as well as RTs containing the K103N or Y181C resistance mutations. The analogs, however, do not exhibit significant antiviral activity in cell culture and were, in general, cytotoxic. Nevertheless, these data suggest that the NAIM backbone may provide a suitable scaffold from which inhibitors active against NNRTI-resistant HIV-1 could be developed.

The acyclic 2,4-diaminopyrimidine nucleoside phosphonate acts as a purine mimetic in HIV-1 reverse transcriptase DNA polymerization.

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.