Novel Humanized and Highly Efficient Bispecific Antibodies Mediate Killing of Prostate Stem Cell Antigen-Expressing Tumor Cells by CD8+ and CD4+ T Cells

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3-anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8(+) and CD4(+) T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4(+) T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.     

Draft genome sequence for Microbacterium laevaniformans strain OR221, a bacterium tolerant to metals, nitrate, and low pH

Microbacterium laevaniformans strain OR221 was isolated from subsurface sediments obtained from the Field Research Center (FRC) in Oak Ridge, TN. It was characterized as a bacterium tolerant to heavy metals, such as uranium, nickel, cobalt, and cadmium, as well as nitrate and low pH. We present its draft genome sequence.     

Towards toxicity detection using a lab-on-chip based on the integration of MOEMS and whole-cell sensors

A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Electro-Mechanical-System) modulator, and solid-state photo-detectors was implemented for the first time. Whole-cell sensors were genetically engineered to express a bioluminescent reporter (lux) as a function of the lac promoter. The MOEMS modulator was designed to overcome the inherent low frequency noise of solid-state photo-detectors by means of a previously reported modulation technique, named IHOS (Integrated Heterodyne Optical System). The bio-reporter signals were modulated prior to photo-detection, increasing the SNR of solid-state photo-detectors at least by three orders of magnitude. Experiments were performed using isopropyl-beta-d-thiogalactopyranoside (IPTG) as a preliminary step towards testing environmental toxicity. The inducer was used to trigger the expression response of the whole-cell sensors testing the sensitivity of the lab-on-chip. Low intensity bio-reporter optical signals were measured after the whole-cell sensors were exposed to IPTG concentrations of 0.1, 0.05, and 0.02 mM. The experimental results reveal the potential of this technology for future implementation as an inexpensive massive method for rapid environmental toxicity detection.     

An acoustic method for the analysis of bacterial cells

The application of a biological electroacoustic sensor based on a lateral electric-field-excited piezoelectric resonator for the study of bacterial cells that interact with specific bacteriophages, mini-antibodies, and polyclonal antibodies was successfully demonstrated. The determined lower limit of microbialcell detection was approximately of 10³ to 10⁴ cells/mL for the duration of the assay of 10 min. The possibility of bacterial-cell detection via interaction with specific agents in the presence of extraneous microbiota was shown. The method allowed us to determine the spectrum of lytic activity of bacteriophages and the sensitivity of microbial cells to bacteriophages. The results of the study showed that application of a sensor piezoelectric lateral-field resonator is a promising technique for the detection and identification of microbial cells and determination of their phage resistance in microbiology, medicine, and veterinary medicine. Furthermore, the results of the experiments made it possible to understand the mechanisms of the processes that occur in a suspension of bacterial cells in the presence of various biological agents. The method also may provide useful information regarding biophysical mechanisms of interactions that occur in microbial populations.     

Wavelet-synchrony of Brain Evoked Responses during Listening to Naturalistic Stimuli in the Prognosis of Consciousness Recovery (Study 1: Wavelet-synchrony during Listening to Instrumental Music)

The features and prognostic value of wavelet-synchrony of evoked responses during the listening to realistic stimuli (music) were revealed in the study. Musical passages were selected according the patients’ preferences by their relations consultation. Wavelet-connections were calculated for each melody in the range from 0 to 1000 ms from stimuli. Total 22 examinees in 12 patients being in unconsciousness state were carried out. Resting state values of wavelet-connectivity were poorer in vegetative patients in comparison to healthy subjects regardless of consciousness recovery degree in future while wavelet-connections in mutism were higher in comparison to healthy subjects. Absolute value of wavelet-synchrony was lower than in norm. In patients with compete consciousness recovery in the future the maximal values of wavelet-synchrony were revealed in the temporal-occipital area while in patients who in the future have been revealed in the lesser degree the maximal values of wavelet-synchrony are shown in frontal-central areas. In chronic unconsciousness patients during the listening to instrumental music the maximal wavelet-connections values were determined in the right frontal area.     

Pixel classification method in optical coherence tomography for tumor segmentation and its complementary usage with OCT microangiography

A novel machine‐learning method to distinguish between tumor and normal tissue in optical coherence tomography (OCT) has been developed. Pre‐clinical murine ear model implanted with mouse colon carcinoma CT‐26 was used. Structural‐image‐based feature sets were defined for each pixel and machine learning classifiers were trained using “ground truth” OCT images manually segmented by comparison with histology. The accuracy of the OCT tumor segmentation method was then quantified by comparing with fluorescence imaging of tumors expressing genetically encoded fluorescent protein KillerRed that clearly delineates tumor borders. Because the resultant 3D tumor/normal structural maps are inherently co‐registered with OCT derived maps of tissue microvasculature, the latter can be color coded as belonging to either tumor or normal tissue. Applications to radiomics‐based multimodal OCT analysis are envisioned.      

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.     

<Emphasis Type="Italic">Gallaecimonas mangrovi</Emphasis> sp. nov., a novel bacterium isolated from mangrove sediment

A Gram-stain negative, rod-shaped, non-motile, strictly aerobic bacterium HK-28^T was isolated from a mangrove sediment sample in Haikou city, Hainan Province, China. Strain HK-28^T was able to grow at 10–45 °C (optimum 25–30 °C), pH 5.0–8.5 (optimum 6.0–7.0) and 0.5–12.0% (w/v) NaCl (optimum 1.0–3.0%, w/v). The major cellular fatty acids were C_16:0, Summed Feature 8 (C_18:1 ω7c and/or C_18:1 ω6c), Summed Feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_17:0, C_12:0 3-OH and C_17:1ω8c. Ubiquinone-8 (Q-8) was the predominant respiratory quinone. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, four unidentified phospholipids, two unidentified glycolipid, an unidentified glycophospholipid, an unidentified aminolipid and an unidentified lipid. The DNA G+C content was 50.2 mol%. Accoroding to 16S rRNA gene sequence similarities, strain HK-28^T shared 97.1 and 96.7% sequence similarities to the validly named species Gallaecimonas xiamenensis MCCC 1A01354^T and Gallaecimonas pentaromativorans MCCC 1A06435^T, respectively, and shared lower sequence similarities (<?92.0%) to all other genera. Phylogenetic analysis showed strain HK-28^T was clustered with G. pentaromativorans MCCC 1A06435^T and G. xiamenensis MCCC 1A01354^T. Strain HK-28^T showed low DNA–DNA relatedness with G. xiamenensis MCCC 1A01354^T (28.3?±?1.5%) and G. pentaromativorans MCCC 1A06435^T (25.2?±?2.4%). On the basis of phenotypic, chemotaxonomic and genotypic characteristics, strain HK-28^T is considered to represent a novel species in the genus Gallaecimonas, for which the name Gallaecimonas mangrovi sp. nov. is proposed. The type strain is HK-28^T (=?KCTC 62177^T?=?MCCC 1K03441).     

Effect of Aromatic Compounds on Cellular Fatty Acid Composition of Rhodococcus opacus

In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of R. opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R. opacus to lipophilic aromatic compounds.