Cerebral ischemia is accompanied by mitochondrial integrity destruction. Thus, reversion of mitochondrial damage holds great potential for cerebral ischemia therapy. As a crucial Bcl-2 family member, pro-apoptotic Bax protein is a main effector of mitochondrial permeabilization and plays an important role in mitochondrial homeostasis. However, there is still a lack of an effective cerebral protective strategy through selectively targeting Bax. In this study, we reported that natural small-molecule protosappanin A (PTA) showed a significant mitochondrial protective effect on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cells injury through increasing ATP production and maintaining mitochondrial DNA (mtDNA) content. The mechanism study revealed that PTA selectively induced pro-apoptotic protein Bax degradation, without affecting other Bcl-2 family members such as Bcl-2, Bcl-xl, Bad, Puma, Bid, Bim, and Bik. In addition, we found that PTA promoted the association of autophagosomal marker LC3B to Bax for its degradation via an autophagy-dependent manner but not the ubiquitin-proteasome pathway. Collectively, our findings offered a new pharmacological strategy for maintaining mitochondrial function by inducing autophagic degradation of Bax and also provided a novel drug candidate against ischemic neuronal injury.

Keywords: Bax; Ischemic neuronal injury; autophagic degradation; mitochondrial homeostasis; protosappanin A (PTA).

One of the major problems found when developing a 3D recognition system involves the choice of keypoint detector and descriptor. To help solve this problem, we present a new method for the detection of 3D keypoints on point clouds and we perform benchmarking between each pair of 3D keypoint detector and 3D descriptor to evaluate their performance on object and category recognition. These evaluations are done in a public database of real 3D objects. Our keypoint detector is inspired by the behavior and neural architecture of the primate visual system. The 3D keypoints are extracted based on a bottom-up 3D saliency map, that is, a map that encodes the saliency of objects in the visual environment. The saliency map is determined by computing conspicuity maps (a combination across different modalities) of the orientation, intensity, and color information in a bottom-up and in a purely stimulus-driven manner. These three conspicuity maps are fused into a 3D saliency map and, finally, the focus of attention (or keypoint location) is sequentially directed to the most salient points in this map. Inhibiting this location automatically allows the system to attend to the next most salient location. The main conclusions are: with a similar average number of keypoints, our 3D keypoint detector outperforms the other eight 3D keypoint detectors evaluated by achieving the best result in 32 of the evaluated metrics in the category and object recognition experiments, when the second best detector only obtained the best result in eight of these metrics. The unique drawback is the computational time, since biologically inspired 3D keypoint based on bottom-up saliency is slower than the other detectors. Given that there are big differences in terms of recognition performance, size and time requirements, the selection of the keypoint detector and descriptor has to be matched to the desired task and we give some directions to facilitate this choice.

Decreased expression of proapoptotic genes can lead to the chemoresistenance in cancer therapy. Carboxyl-terminal binding protein 1 (CtBP1), a transcriptional corepressor with multiple oncogenic effects, has been previously identified to suppress the expression of two proapoptotic genes [BAX (BCL2 associated X) and BIM (Bcl-2 interacting mediator of cell death)] by assembling a complex with the Forkhead box O3 (FOXO3a) transcription factor and the p300 histone acetyltransferase. However, the upstream regulatory signaling of the CtBP1-p300-FOXO3a complex is obscure, and the effects of changing this signaling on chemosensitivity in osteosarcoma are unknown. Herein, we discovered that the downregulation of HIPK2 (Homeodomain-interacting protein kinase 2) was essential for the function of the CtBP1-p300-FOXO3a complex. Downregulation of HIPK2 prevented the phosphorylation and subsequent degradation of CtBP1, thereby allowing the assembly of the CtBP1-p300-FOXO3a complex and suppression of the expression of proapoptotic genes, such as BAX, BIM, BIK (Bcl-2 interacting killer) and NOXA/PMAIP1 (Phorbol-12-myristate-13-acetate-induced protein 1). Overexpression of HIPK2 promoted the phosphorylation of CtBP1 and the degradation of CtBP1 by proteasomes, thereby preventing the formation of the CtBP1-p300-FOXO3a complex. The abolition of CtBP1 transrepression increased the expression of proapoptotic genes to induce apoptosis and increase chemosensitivity in osteosarcoma cells. Taken together, our in vitro and in vivo results revealed that overexpression of HIPK2 could remove the CtBP1-mediated transrepression of proapoptotic genes, indicating a new therapeutic option for the treatment of osteosarcoma.

Keywords: CtBP1; HIPK2; chemosensitivity; osteosarcoma; proapoptotic genes.

The objective of this study was to determine the frequency of expression of GRP78 and BIK/NBK proteins, as well as to evaluate their correlation with prognostic factors for clinical use in patients with locally advanced breast cancer (LABC) or preoperative chemotherapy (PC). An observational, analytical and retrospective study that evaluated the expression of BIK/NBK and GRP78 by means of immunohistochemistry in paraffin-embedded tumour tissue samples obtained before the start of PC was executed. GRP78 was positive in 93.3% and BIK/NBK was positive in 40% of the samples analysed. Correlation of GRP78 expression with Ki67 expression (Rho = 0.732, p = .039), and of BIK/NBK expression with Ki67 expression (Rho = 0.777, p = .023) and number of residual lymph nodes (Rho = 0.619, p = .014) was observed. Given the correlation observed, association studies of the expression of GRP78 and BIK/NBK with clinical and pathological response, recurrence and survival should be carried out in a greater number of patients with LABC or PC. Impact Statement What is already known on this subject? There are reports of the overexpression of GRP78 in various types of cancer cell lines and tumour biopsies, including breast cancer, with participation in resistance to adjuvant chemotherapeutic agents (topoisomerase inhibitors including Adriamycin). On the other hand, BIK/NBK has been observed expressed associated with poor outcomes in breast cancer. There are no studies of the expression of both biomarkers in patients with preoperative chemotherapy, including locally advanced breast cancer (LABC) or their correlation with prognosis markers of clinical use. What the results of this study add? We observed a correlation of higher expression percentage of GRP78 and BIK/NBK with higher expression of Ki67, a marker of poor prognosis in breast cancer, in addition to a significant correlation of higher percentage of BIK/NBK expression with a greater number of residual nodes in patients with LABC or PC. What the implications are of these findings for clinical practice and/or further research? There is a potential prognostic value of GRP78 and BIK/NBK in the pathological response that merits further study addressing their interaction with other proteins, their cellular location in relation to the clinical stages of breast cancer, and their association with prognosis (clinical and pathological response, recurrence, disease free period and survival) in a greater number of patients with LABC or PC.

Apoptosis among intestinal epithelial cells contributes to necrotizing enterocolitis (NEC), a severe intestinal disease that particularly affects premature infants. β-arrestin-2, an important regulator of G-protein-coupled receptors, is expressed in intestinal epithelial cells, where its activation promotes apoptosis. We found that β-arrestin-2 was overexpressed in both human and murine NEC samples. β-arrestin-2-deficient mice were protected from endoplasmic reticulum stress and NEC development. The endoplasmic reticulum-resident chaperone BiP was found to promote intestinal epithelial cell survival. Pretreatment of intestinal epithelial cells or mice with the BiP inhibitor HA15 increased cell apoptosis and promoted NEC development. β-arrestin-2 bound to BiP and promoted its polyubiquitination and degradation, thereby facilitating the release of the pro-apoptotic molecule BIK from BiP. Silencing β-arrestin-2 downregulated apoptosis by increasing BiP levels, which suppressed endoplasmic reticulum stress. This study suggests that β-arrestin-2 induces NEC development by inhibiting BiP, thereby triggering apoptosis in response to endoplasmic reticulum stress. Thus, novel therapeutic strategies to inhibit β-arrestin-2 may enhance the treatment of NEC.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with multiple molecular mechanisms. To investigate and contrast the molecular processes differing between bronchiolitis and emphysema phenotypes of COPD, we downloaded the GSE69818 microarray data set from the Gene Expression Omnibus (GEO), which based on lung tissues from 38 patients with emphysema and 32 patients with bronchiolitis. Then, weighted gene coexpression network analysis (WGCNA) and differential coexpression (DiffCoEx) analysis were performed, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG) analysis. Modules and hub genes for bronchiolitis and emphysema were identified, and we found that genes in modules linked to neutrophil degranulation, Rho protein signal transduction and B cell receptor signalling were coexpressed in emphysema. DiffCoEx analysis showed that four hub genes (IFT88, CCDC103, MMP10 and Bik) were consistently expressed in emphysema patients; these hub genes were enriched, respectively, for functions of cilium assembly and movement, proteolysis and apoptotic mitochondrial changes. In our re-analysis of GSE69818, gene expression networks in relation to emphysema deepen insights into the molecular mechanism of COPD and also identify some promising therapeutic targets.

OBJECTIVE

This study aimed to identify the mechanism of how MG-132 stimulates cell death in SEB-1 sebocytes.

METHODS

TUNEL staining and annexin-FITC/PI flow cytometry were utilized to examine the apoptotic cell number of SEB-1 sebocytes and HaCaT keratinocytes upon MG-132 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. MTT assay and CCK-8 assay monitored the proliferative rate and viability of both cell lines with different treatment. Western blotting (WB) and qPCR were performed to detect the expression of TRAIL and members of Bcl-2 family at protein and gene level. Additionally, RNA interfering was used to knockdown the mRNA transcription of TRAIL and BIK gene.

RESULTS

MG-132 treatment enhanced cell death in SEB-1 sebocytes but not in HaCaT keratinocytes. Meanwhile, TRAIL concentrations in SEB-1 sebocytes treated with MG-132 were markedly elevated. Furthermore, treatment with TRAIL or the TRAIL receptor-specific monoclonal antibody AY4 at various doses stimulated cell death in SEB-1 sebocytes in a time- and dose-dependent manner. Silencing of TRAIL restored the cell viability of SEB-1 cells to a normal level after MG-132 treatment. Combined treatment of SEB-1 sebocytes with TRAIL and MG-132 synergistically triggered cell death, suppressed cell proliferation and survival, and promoted BIK expression. Furthermore, BCL2 Interacting Killer (BIK) knockdown via RNA interference participated in the recovery of cell survival reduced by treatment with TRAIL and MG-132.

CONCLUSIONS

These findings suggest that treatment with the selective proteasome suppressor MG-132 and TRAIL induces cell death in sebocytes through upregulation of BIK, a member of the Bcl-2 family.

A device BIK for active suppression of infection has been used against Herpes virus infection type 1 and 2 in 72 patients with keratouveitis. The immunological and genotypic monitoring show that patients with herpetic keratouveitis after BIK treatment have lower titers of antiherpetic antibodies, normal cellular and humoral immunity, weaker viremia and reduced quantity of viral material in corneal tissue. This positive effect persisted for 8-12 months. Thus, BIK device provides a definite, prolonged and stable therapeutic effect in patients with herpetic keratouveitis.

BACKGROUND

Heat causes airway damage during inhalation injury because of bronchial epithelial cell damage. Accumulating evidence shows that mitochondrial uniporter (MCU) is involved in cell damage. We investigated the MCU activity after heat treatment and assessed whether Astragaloside-IV (AS-IV) suppresses heat-induced apoptosis in bronchial epithelial cells by inhibiting the activation of the mitochondrial Ca2+ uniporter (MCU), mitochondrial depolarisation and reactive oxygen species (ROS) production.

METHODS

The bronchial epithelial cell line 16HBE14o- was heat treated, and cell apoptosis was induced in vitro and in vivo. AS-IV was inorganically administered to Wistar rats twice a day after thermal inhalation injury, and 16HBE140- cells were treated with AS-IV after incubation at 47°C for 5 min. Protein expression was determined using Western blotting and commercial kits, apoptosis with TUNEL staining, mitochondrial channel activity by patch clamp, reactive oxygen species by MitoSOXTM fluorescence, ATP levels and enzyme activities by commercial kits as well as mitochondrial respiration and calcium by fluorescence.

RESULTS

AS-IV markedly inhibited heat-induced apoptosis, as indicated by the increased expression of the pro-apoptotic genes Bak, Bik and Bmf and increased expression of the apoptosis markers Bax, cleaved parp, cleaved caspase3 and cytochrome C. We found that MCU activation promoted mitochondrial Ca2+ overload, ATP depletion, mitochondrial ROS production and cytochrome c release and rapidly induced apoptosis. However, AS-IV treatment reduced excessive MCU activation and led to resistance against mitochondrial Ca2+ overload and excessive cytochrome C release; these effects were blocked by the MCU activator spermine. AS-IV treatment elevated ATP production and decreased ROS activity.

CONCLUSIONS

MCU plays crucial roles in heat-induced mitochondrial apoptosis in 16HBE140- cells, suggesting a potential target for AS-IV treatment.

Purpose: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity.

Materials and methods: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4^{-/ -} mice.

Results: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed.

Conclusion: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.

Keywords: NADPH oxidase 4; Toxoplasma gondii; human retinal pigment epithelium; mitochondrial apoptosis; reactive oxygen species; silver nanoparticles.

EU-7 zeolite with Si/Al ratio of 15 is identified as a BIK type zeolite. The framework charge is compensated with Cs+ cations located in 8-ring channels at regular distances. Even partially exchanged, Cu-loaded EU-7 is active in NH3-SCR and withstands hydrothermal aging at 900 °C needed for application in diesel particulate filters.

Star-PAP is a noncanonical poly(A) polymerase and required for the expression of a select set of mRNAs. However, the pathological role of Star-PAP in cancer largely remains unknown. In this study, we observed decreased expression of Star-PAP in breast cancer cell lines and tissues. Ectopic Star-PAP expression inhibited proliferation as well as colony-forming ability of breast cancer cells. In breast cancer patients, high levels of Star-PAP correlated with an improved prognosis. Moreover, by regulating the expression of BIK (BCL2-interacting killer), Star-PAP induced apoptosis of breast cancer cells through the mitochondrial pathway. The growth of breast cancer xenografts in NOD/SCID mice was also inhibited by the doxycycline-induced Star-PAP overexpression. Furthermore, Star-PAP sensitized breast cancer cells to chemotherapy drugs both in vitro and in vivo. In mammary epithelial cells, Star-PAP knockdown partially transformed these cells and induced them to undergo epithelial-mesenchymal transition (EMT). These findings suggested that Star-PAP possesses tumor-suppressing activity and can be a valuable target for developing new cancer therapeutic strategies.

A novel acridone derivative, N-(2-(dimethylamino)ethyl)-1-((3-methoxybenzyl)amino)-5- nitro-9-oxo-9,10-dihydro-acridine-4-carboxamide (8q), which was synthesized in our lab, showed potent anti-leukaemia activity against CCRF-CEM cells. Moreover, in silico predictions showed that 8q conformed to the rule of five and displayed low toxicity. However, the mechanism of anti-leukaemia action remains unclear. The aim of this research was to reveal the probable anti-leukaemia mechanism of 8q on CCRF-CEM cells. Flow cytometry assay demonstrated that 8q induced apoptosis. The expression of caspase family proteins results showed that 8q could only promote cleaved caspase-3, 7 and 9 expressions without affecting cleaved caspase-8 protein, hinting that 8q induced mitochondrial-mediated apoptosis. Further, we detected 3 indicators of mitochondrial lesions, including increased of Cyt-C release, with a decrease in MMP and ATP levels. Next, metabolomics were introduced to assist in the research of the anti-leukaemia mechanism of 8q. The metabolomics results showed that 100 nM 8q could increase the level of GSH, and decrease its oxidation products. These indicated 8q could influence the ROS, which derived by mitochondria. Then we examined the effect of 8q on intracellular ROS levels. What is particularly interesting is that 8q inhibited cell ROS stress at low concentration and stimulated ROS stress at high concentration. The pro-apoptosis mechanisms of 8q were then explored. 8q significantly decreased anti-apoptotic proteins Bcl-2 and Bcl-xL expression, whereas it up-regulated the pro-apoptotic proteins Bax, Bak, Bad, Bik and Puma expression. In addition, 8q dramatically inhibited the expression of FASN, which is related to fatty acid metabolism. Furthermore, PI3K, AKT and FOXO1 were inactivated, and the expression of total AKT was also inhibited by 8q treatment, which promoted intrinsic apoptosis. In conclusion, these findings demonstrate that 8q can induce mitochondrial lesions and promote mitochondrial-mediated pathway apoptosis by regulating the expression of Bcl-2 family proteins and inhibiting the activity of the PI3K/AKT/FOXO1 signaling pathway.

Obesity and insulin resistance affect metabolic reactions, but their ensuing contributions to macrophage metabolism remain insufficiently understood. We investigated the contributions of berberine and metformin combination to the inhibition of sebocyte apoptosis in high-fat diet-induced diabetic hamsters and an insulin-treated human cell line. Golden hamsters were fed a high-glucose high-fat diet and administered a 6-week treatment with a combination of metformin and two concentrations of berberine (100 or 50 mg·kg^-1 ). Body weights of treated hamsters were remarkably reduced compared with those of controls. Histological examination indicated that berberine repressed liver fat accumulation. Moreover, insulin and glucose concentrations were noticeably decreased by the combination treatments. In glucose tolerance tests, hamsters receiving berberine displayed higher tolerance to glucose, compared with the control group. Sebocytes isolated from high-fat diet-induced diabetic hamsters and insulin-treated human sebocytes displayed elevated cell death rates, which were attenuated by berberine and metformin treatments. Further studies showed that the effects of metformin and berberine on cellular apoptosis were mediated via the Bik pathway. Thus, berberine may effectively decrease circulating glucose levels, ameliorate insulin resistance, reduce body weight, and attenuate sebocyte apoptosis in diabetic hamsters, potentially decreasing vulnerability to the cardiovascular complications of diabetes. SIGNIFICANCE OF THE STUDY: The present data indicate that insulin stimulates changes in the expression levels of cell death-associated proteins, which participate in sebaceous gland diseases during obesity or diabetes. The anti-apoptotic effects of BBR and MET in sebaceous gland cells are regulated partially by Bik expression. To the best of our knowledge, this study is the first to suggest cell death counteracting effects of BBR in hamster and human sebocytes as well as to propose BBR as an innovative therapeutic agent for insulin-related sebaceous gland diseases, including acne.

Background: Bilirubin has long been exclusively considered as a potentially dangerous sign of liver diseases, but it is currently regarded as a reliable signaling molecule as well.

Objective: This study investigated the effects of unconjugated bilirubin on survival, proliferation, apoptotic and cell arrest capacities of melanoma SKMEL-3 and non-melanoma A431 skin cancer cells in comparison with normal human dermal fibroblast (HDF) cells.

Methods: The MTT assay test was used to identify survival and the IC50 at various concentrations of bilirubin on SKMEL-3, A431, and HDF cells for 24h and 48h. The comet assay technique was used to investigate genotoxicity effects and flow cytometry was run to investigate apoptotic and cell arresting effects of bilirubin on the cells. The gene expression of cyclin D1, cyclin E1, survivin, Bcl-2, and p53 was investigated by qRT-PCR. The molecular docking of bilirubin on CDKs (Cyclin-dependent kinases 2, 4, and 6) and pro-apoptotic factors Bad, Bak, Bax, Bid, Bik, and Bim were done by Autodock software version 2.

Results: The IC50 of bilirubin on HDF, A431, and SKMEL-3 cells were 125, 115, and 95 μM at 24h and 115, 100, and 75 μM at 48h, respectively. Although cell arrest in the G1 phase occurred in all cells, bilirubin induced the genotoxicity and apoptosis in SKMEL-3 and A431 cancer cells more pronouncedly than those in normal HDF cells.

Conclusion: Bilirubin led to cell arrest in the G1 phase in SKMEL-3, A431, and HDF cells. Additionally, bilirubin induced apoptotic pathways in SKMEL-3 and A431 cancer cells.

Keywords: Bilirubin; CDK; HDF cells; apoptosis; cell arresting; skin cancer.

In recent years, immunohistochemistry as applied to the Bcl-2 family of proteins has represented a burgeoning area of interest to cancer researchers. The majority of studies have focused on the original member Bcl-2, first identified via its involvement in the common t(14;18) chromosomal translocation in B-cell lymphomas (1). However, since this discovery, preclinical and clinical interest in Bcl-2 has dramatically increased owing to (a) its recognition as the first of a new class of oncogene able to prolong survival by inhibiting programmed cell death (apoptosis) and (b) the discovery of many additional related genes/proteins some of which, like Bcl-2, inhibit apoptosis, whereas others, such as Bax, conversely promote cell death (2) (Table 1). Table 1 Bcl-2 Family Members Pro-apoptotic Anti-apoptotic Bax Bcl-2 Bcl-Xs Bcl-Xl Bak Bcl-w Bad Bfl-1 Bid Brag-1 Bik Mcl-1 Hrk A1.

In patients with BRAF-mutated melanoma specific inhibitors of BRAFV600E and MEK1/2 frequently induce initial tumor reduction, frequently followed by relapse. As demonstrated previously, BRAFV600E-inhibition induces apoptosis only in a fraction of treated cells, while the remaining arrest and survive providing a source or a niche for relapse. To identify factors contributing to the differential initial response towards BRAF/MEK inhibition, we established M14 melanoma cell line-derived single cell clones responding to treatment with BRAF inhibitor vemurafenib and MEK inhibitor trametinib predominantly with either cell cycle arrest (CCA-cells) or apoptosis (A-cells). Screening for differentially expressed apoptosis-related genes revealed loss of BCL2-Interacting Killer (BIK) mRNA in CCA-cells. Importantly, ectopic expression of BIK in CCA-cells resulted in increased apoptosis rates following vemurafenib/trametinib treatment, while knockdown/knockout of BIK in A-cells attenuated the apoptotic response. Furthermore, we demonstrate reversible epigenetic silencing of BIK mRNA expression in CCA-cells. Importantly, HDAC inhibitor treatment associated with re-expression of BIK augmented sensitivity of CCA-cells towards vemurafenib/trametinib treatment both in vitro and in vivo. In conclusion, our results suggest that BIK can be a critical mediator of melanoma cell fate determination in response to MAPK pathway inhibition.

Precise 10 K X-band EPR measurements and subsequent spin-Hamiltonian analysis by direct matrix diagonalization methods are reported for a Ti3+ (S = 1/2) center in tetragonal zircon (zirconium silicate, ZrSiO4). A special, and previously unobserved, feature of the supposedly uniaxial spectrum is the marked angular dependence of the titanium hyperfine lines in the perpendicular, ab crystal plane. As discussed, this can only arise from the presence of high-spin nuclear terms of dimension BIk, SIk (k = 3, 5) in the spin Hamiltonian. Parameters arising from these terms were determined to have magnitudes very much larger than observed previously in first-row transition ions. The consequences of precise determination of these high-spin parameters are significant and several: a precise determination of the nuclear quadrupole tensor leading to a ratio 47P/49P in excellent agreement with the ratio derived from the corresponding nuclear quadrupole moments; an apparent anisotropy in the nuclear Zeeman interaction which can be identified with anisotropy in the chemical shielding "tensor"; a marked hyperfine anomaly. The origin and significance of these observations are discussed.

Ocimum minimum L. (fine-leaved basil) is cultivated along the Douro Litoral Region in Portugal. In June 2009, a disease was observed in fine-leaved basil fields in three geographically separated locations: Maia, Rio Tinto, and São Mamede Infesta. Affected plants showed wilt symptoms, chlorotic leaves, and stem necrosis. Discolored vascular tissue was observed when the stems were cut longitudinally. For isolation, crown and stem sections (10 cm long) were surface disinfected for 1 min in 1.5% NaOCl and washed twice with sterile distilled water. The sections were cut longitudinally and small pieces of discolored vascular tissue were plated onto potato dextrose agar (PDA) amended with streptomycin sulfate (0.5 g liter^-1). Plates were incubated at 25°C in the dark. Fusarium colonies were consistently isolated from symptomatic plants sampled from the three different locations and transferred to PDA and Spezieller Nährstoffarmer agar (SNA) culture media for morphological species identification (2). After 10 days of incubation at 25°C, all isolates were identified as F. oxysporum. A PCR-based assay was conducted with nine single-spored isolates (F2, F3, F4, F7, F8, F9, F10, F11, and F13) using the F. oxysporum f. sp. basilici specific primer pair, Bik 1 and Bik 2 (1). A single DNA fragment of 382 bp was amplified in all isolates, which confirmed the identification of F. oxysporum f. sp. basilici. Pathogenicity of all nine isolates was determined on 2-month-old fine-leaved basil seedlings growing in sterile peat moss. Plants were inoculated by watering the roots with 20 ml of a conidial suspension (10⁶ conidia ml^-1) harvested from 3-week-old cultures grown on PDA. Thirty-six replicates (each one in individual pots) for each isolate were used, with an equal number of control plants. Plants were maintained in a greenhouse at 15 to 20°C. Within 2 weeks of inoculation, all inoculated plants wilted and exhibited severe leaf and stem necrosis. The fungus was reisolated from vascular tissues of the crown area and the stems of symptomatic seedlings, fulfilling Koch's postulates. Control plants remained healthy. To our knowledge, this is the first report of F. oxysporum f. sp. basilici infecting fine-leaved basil in Portugal. References: (1) A. Chiocchetti et al. Plant Dis. 85:607, 2001. (2) J. F. Leslie et al. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

OBJECTIVE

Determine if a nucleoside reverse transcriptase inhibitors (NRTI)-free regimen affected mitochondrial DNA (mtDNA) levels in peripheral blood mononuclear cells (PBMCs) of patients enrolled in BIKS-2 trial.

METHODS

Antiretroviral (ARV) naïve (N=13) and NRTI experienced (N=7) patients, received lopinavir/ritonavir, a boosted protease inhibitor, and efavirenz, a non-nucleoside reverse transcriptase inhibitor from Month (M) 0 to M12 (1-year BIKS trial) and from M12 to M36 (2-year BIKS-2 trial). MtDNA was quantified at M12, M24 and M36 via real-time PCR assay.

RESULTS

From M12 to M36, the 20 patients have maintained undetectable plasma HIV-1 RNA, gained CD4 cells and had no side effects attributable to these drugs. Median mtDNA contents were constant: 478.6 at M12, 478.6 at M24 and 324.4 copies/cell at M36 (pM12-M36=0.5). Because M0 data is missing, these results were compared to those of two groups of age matched individuals: healthy donors and HIV-infected patients before and after exposure to NRTIs. Healthy donors have higher contents (871), followed by patients never treated (602), than by BIKS patients where 7 had toxic NRTIs (478.6) and at last by patients exposed for six months to the most toxic combination (ddI-d4T) (85 copies/cell).

CONCLUSIONS

Lopinavir/ritonavir+efavirenz did not affect mtDNA contents in PBMCs.

Molecular (photo)switch: A W-Co photomagnetic discrete complex can be prepared through the self-assembly of preformed building blocks. [Co(bik)3][{W(CN)8}3{Co(bik)2}3]⋅2 H2O⋅13 CH3CN (see figure) exhibits a thermally-induced electron-transfer-coupled spin transition between the two states: Co(HS)(II)-W(V)↔Co(LS)(III)-W(IV). It also shows photomagnetic effects at low temperature.

The recognition of helical BH3 domains by Bcl-2 homology (BH) receptors plays a central role in apoptosis. The residues that determine specificity or promiscuity in this interactome are difficult to predict from structural and computational data. Using a cell free split-luciferase system, we have generated a 276 pairwise interaction map for 12 alanine mutations at the binding interface for three receptors, Bcl-xL, Bcl-2, and Mcl-1, and interrogated them against BH3 helices derived from Bad, Bak, Bid, Bik, Bim, Bmf, Hrk, and Puma. This panel, in conjunction with previous structural and functional studies, starts to provide a more comprehensive portrait of this interactome, explains promiscuity, and uncovers surprising details; for example, the Bcl-xL R139A mutation disrupts binding to all helices but the Bad-BH3 peptide, and Mcl-1 binding is particularly perturbed by only four mutations of the 12 tested (V220A, N260A, R263A, and F319A), while Bcl-xL and Bcl-2 have a more diverse set of important residues depending on the bound helix.

Here, we describe the characteristics of a Pt-blue complex [Pt4 (2-atp)8 (H2 O)(OH)] (2-atp: 2-aminothiophenol) as a prodrug for its DNA-binding properties and its use in cancer therapy. The nature of the interaction between the Pt-blue complex and DNA was evaluated based on spectroscopic measurements, the electronic absorption spectra, thermal behavior, viscosity, fluorometric titration, and agarose gel electrophoresis. Our results suggested that the compound was able to partially intercalate DNA and appeared to induce both single- and double-stranded breaks (DBS) on DNA in vitro, but no DSBs in cells. The ability of the compound to induce DNA damage was dependent on reactive oxygen species (ROS) in vitro. There was also elevated formation of ROS and SOD expression in response to drug treatment in cell culture. The complex was found to be more cytotoxic to cancer cells in comparison with noncancer controls using WST-1 assay. The mean of cell death was determined to be apoptosis as assessed via biochemical, morphological, and molecular observations, including DNA condensation/fragmentation analysis, live cell imaging microscopy, TUNEL analyses, and increase in the levels of pro-apoptotic genes such as Bag3, Bak, Bik, Bmf, and Hrk. Hence, the Pt-blue complex under study grants premise for further studies.