Taxol induces caspase-independent cytoplasmic vacuolization and cell death through endoplasmic reticulum (ER) swelling in ASTC-a-1 cells

High concentration of taxol was found to induce programmed cell death (PCD) and cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. To elucidate the relationship between the PCD and cytoplasm vacuolization, confocal fluorescence microscopy was performed on the cytoplasm vacuolization, endoplasmic reticulum (ER) and mitochondria swelling after taxol treatment in living cells. erRFP plasmid was used to probe the ER distribution, and SCAT3 plasmid was used to monitor the caspase-3 activation in living cells. Our results showed that taxol induced concentration-dependent and caspases-independent cytoplasm vacuolization and cell death through ER and mitochondria swelling. Live confocal imaging of ASTC-a-1 cells stably expressing SCAT3 further verified that taxol-induced cytoplasm vacuolization and cell death was caspase-3-independent. In conclusion, we found for the first time that taxol induces a paraptosis-like PCD in the ASTC-a-1 cells by cytoplasm vacuolization due to the swelling of both ER and mitochondria without activating the caspase enzymes.     

Circulation of Fluorescently Labelled Phage in a Murine Model

Interactions between bacteriophages and mammals strongly affect possible applications of bacteriophages. This has created a need for tools that facilitate studies of phage circulation and deposition in tissues. Here, we propose red fluorescent protein (RFP)-labelled E. coli lytic phages as a new tool for the investigation of phage interactions with cells and tissues. The interaction of RFP-labelled phages with living eukaryotic cells (macrophages) was visualized after 20 min of co-incubation. RFP-labeled phages were applied in a murine model of phage circulation in vivo. Phages administered by three different routes (intravenously, orally, rectally) were detected through the course of time. The intravenous route of administration was the most efficient for phage delivery to multiple body compartments: 20 min after administration, virions were detected in lymph nodes, lungs, and liver; 30 min after administration, they were detectable in muscles; and 1 h after administration, phages were detected in spleen and lymph nodes. Oral and rectal administration of RFP-labelled phages allowed for their detection in the gastrointestinal (GI) tract only.     

Bioresponsive micro-to-nano albumin-based systems for targeted drug delivery against complex fungal infections

As a typical human pathogenic fungus, Cryptococcus neoformans is a life-threatening invasive fungal pathogen with a worldwide distribution causing ∼700,000 deaths annually. Cryptococcosis is not just an infection with multi-organ involvement, intracellular survival and extracellular multiplication of the fungus also play important roles in the pathogenesis of C. neoformans infections. Because adequate accumulation of drugs at target organs and cells is still difficult to achieve, an effective delivery strategy is desperately required to treat these infections. Here, we report a bioresponsive micro-to-nano (MTN) system that effectively clears the C. neoformans in vivo. This strategy is based on our in-depth study of the overexpression of matrix metalloproteinase 3 (MMP-3) in infectious microenvironments (IMEs) and secreted protein acidic and rich in cysteine (SPARC) in several associated target cells. In this MTN system, bovine serum albumin (BSA, a natural ligand of SPARC) was used for the preparation of nanoparticles (NPs), and then microspheres were constructed by conjugation with a special linker, which mainly consisted of a BSA-binding peptide and an MMP-3-responsive peptide. This MTN system was mechanically captured by the smallest capillaries of the lungs after intravenous injection, and then hydrolyzed into BSA NPs by MMP-3 in the IMEs. The NPs further targeted the lung tissue, brain and infected macrophages based on the overexpression of SPARC, reaching multiple targets and achieving efficient treatment. We have developed a size-tunable strategy where microspheres “shrink” to NPs in IMEs, which effectively combines active and passive targeting and may be especially powerful in the fight against complex fungal infections.     

KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons

Dense-core vesicles (DCVs) are responsible for transporting, processing, and secreting neuropeptide cargos that mediate a wide range of biological processes, including neuronal development, survival, and learning and memory. DCVs are synthesized in the cell body and are transported by kinesin motor proteins along microtubules to pre- and postsynaptic release sites. Due to the dependence on kinesin-based transport, we sought to determine if the kinesin-3 family member, KIF1A, transports DCVs in primary cultured hippocampal neurons, as has been described for invertebrate neurons. Two-color, live-cell imaging showed that the DCV markers, chromogranin A-RFP and BDNF-RFP, move together with KIF1A-GFP in both the anterograde and retrograde directions. To demonstrate a functional role for KIF1A in DCV transport, motor protein expression in neurons was reduced using RNA interference (shRNA). Fluorescently tagged DCV markers showed a significant reduction in organelle flux in cells expressing shRNA against KIF1A. The transport of cargo driven by motors other than KIF1A, including mitochondria and the transferrin receptor, was unaffected in KIF1A shRNA expressing cells. Taken together, these data support a primary role for KIF1A in the anterograde transport of DCVs in mammalian neurons, and also provide evidence that KIF1A remains associated with DCVs during retrograde DCV transport.     

Imaging cancer dynamics in vivo at the tumor and cellular level with fluorescent proteins

Whole-body imaging with fluorescent proteins has been shown to be a powerful technology to follow the dynamics of metastatic cancer. Whole-body imaging of fluorescent protein-expressing-cancer cells enables the facile determination of efficacy of candidate anti-tumor and anti-metastatic agents in mouse models. GFP-expressing transgenic mice transplanted with the RFP-expressing cancer cells enable the distinction of cancer and host cells and the efficacy of drugs on each type of cell. This is particularly useful for imaging tumor angiogenesis. Cancer-cell trafficking through the cardiovascular and lymphatic systems is the critical means of spread of cancer. The use of fluorescent proteins to differentially label cancer calls in the nucleus and cytoplasm and high-powered imaging technology are used to visualize the nuclear-cytoplasmic dynamics of cancer-cell trafficking in both blood vessels and lymphatic vessels in the live animal. This technology has furthered our understanding of the spread of cancer at the subcellular level in the live mouse. Fluorescent proteins thus enable both macro and micro imaging technology and thereby provide the basis for the new field of in vivo cell biology.     

Novel strategy to selectively label excitatory and inhibitory neurons in the cerebral cortex of mice

Revealing the connections of neuronal systems is critical for understanding how they function. The vast majority of neurons in all cortical areas consist of excitatory cells whose activity is controlled by inhibitory cells. Distribution and projection patterns of inhibitory and excitatory cells are key information to understand the organization of the nervous system. To investigate axonal projections, we developed a method to uniquely distinguish excitatory axons from inhibitory ones in the cortex using transgenic mice expressing Cre recombinase in the Ca2+/calmodulin-dependent protein kinase IIalpha-containing neurons. These animals were injected by an adenoviral vector engineered so that it directs red fluorescent protein expression in non-Cre-expressing cells, and green fluorescent protein in Cre-positive neurons. We demonstrated in vitro and in vivo that GFP-expressing neurons are GABA-immunonegative (excitatory), while the RFP-expressing cells are either GABAergic neurons or glial cells. One week after the viral vector injection RFP and GFP signals overlapped in a subset of cells but after 1 month, the two signals showed total segregation. Six months post-inoculation, GFP-labelling was clearly visible in axons but RFP remained only in somata and proximal dendrites. This technique can thus be used to differentiate excitatory axonal projections from inhibitory ones, and represent a unique tool in neuronal circuit analysis.     

Assessment of HPV 16 and HPV 18 antibody responses by pseudovirus neutralization,Merck cLIA and Merck total IgG LIA immunoassays in a reduced dosage quadrivalent HPV vaccine trial

We assessed HPV 16 and 18 antibody responses of female subjects enrolled in a 2- vs. 3-dose quadrivalent HPV (Q-HPV) vaccine trial (ClinicalTrials.gov NCT00501137) using the Merck competitive Luminex (cLIA) and total IgG Luminex (TIgG) immunoassays, and a pseudovirus neutralizing antibody (PsV NAb) assay. Subjects were enrolled in one of three groups: (1) 9–13 yr, 2 doses of Q-HPV at 0, 6 months (n = 259); (2) 9–13 yr, 3 doses at 0, 2, 6 months (n = 260); and (3) 16–26 yr, 3 doses at 0, 2, 6 months (n = 305). Sera were collected from all subjects at baseline, months 7 and 24, and from half the subjects at months 18 and 36. High correlation was observed between all three assays. At month 36, HPV 16 antibodies remained detectable in all subjects by all assays, whereas 86.4%, 99.6% and 100% of subjects respectively were HPV 18 cLIA, TIgG and PsV NAb (partial neutralization endpoint) seropositive. The proportion seropositive for HPV 18 by cLIA at 36 months was not significantly different for 2-dose girls vs. 3-dose adults (85.9% vs. 79.4%; p = 0.51), whereas the proportion for 3-dose girls was significantly higher than for 3-dose adults (95.3% vs. 79.4%; p < 0.01). The HPV 18 seropositive proportions by the TIgG and PsV NAb (partial neutralization endpoint) assays were the same for all subjects. High baseline HPV 16 and HPV 18 seropositivity was observed for the TIgG assay and it is unclear if all the detected TIgG antibodies are type-specific and/or neutralizing. For the PsV NAb assay, 90% and partial neutralization geometric mean titres were consistently 2–8-fold higher than for 100% neutralization, which enabled detection of HPV 18 NAb in subjects who lost detectable cLIA antibodies over time. We conclude that the PsV NAb assay is more sensitive than the cLIA, and likely more specific than the TIgG assay.     

Clinical analysis of multibacillary leprosy patients after 1-year fixed World Health Organization recommended multidrug therapy at Yangon General Hospital, Myanmar

This study included 200 randomly selected multibacillary leprosy cases who had completed 1 year of fixed World Health Organization recommended multidrug therapy (WHO-MDT) without prior dapsone (DDS) monotherapy. The time interval after release from treatment varied from a few months to 8 years. All cases were clinically reviewed in 2006 by comparison with their old clinical records. Reactions, particularly reversal reactions, occurred frequently among patients who had completed MDT within the last 3 years. It was difficult to distinguish relapse cases and late reversal reactions in skin smear-negative multibacillary cases. Based on bacteriological and histological analyses, one patient was confirmed to have relapsed 1 year after release from treatment. The overall relapse rate was 0.5%. No drug resistance mutations were detected by polymerase chain reaction or dot blot hybridization. The present study indicates that it is important to follow up patients for several years after completion of MDT in order to detect possible lepra reactions and relapses.     

溃疡型浅表淋巴结结核50例临床分析

目的治疗组和对照组观察溃疡型淋巴结结核的临床疗效。方法随机抽样将溃疡型淋巴结结核的病人分为治疗组和对照组。治疗组采用清创＋INH＋RFP外敷;对照组采用清创＋RFP外敷。结果治疗组和对照组病人伤口的愈合时间分别为15—70d和17～74d,差异无统计学意义（P〉0．05）。结论伤口的愈合与清创是否彻底关系密切,不受局部单用RFP或联合用药的影响。