Rapid and Efficient Investigation of Ciliate Nuclear Development During Conjugation Through Optimizing Hoechst33342 Staining Workflow

Ciliates are eukaryotic unicellular organisms with complex morphology and developmental processes,including asexual and sexual processes.Conjugation is a form of sexual process that renews genetic materials.However,visualizing conjugation in ciliates is a challenge due to the complexity and dynamics of the process,while traditional staining methods are often insufficient for the research.This study introduces a new method for visualizing developmental progression in the nuclei during conjugation using Hoechst33342 staining.It describes how to proceed from cell culture,conjugation induction and synchronization,staining preparation,and observation to statistical analysis.The combination of fluorescent staining with the‘volume-fixing'technique eliminates the fixation and dehydration steps,thus reducing the overall operation time to just 20 minutes.This method offers several advantages over traditional staining techniques for studying the nuclei during conjugation.It improves image quality and workflow efficiency and enables real-time observation of live cell states.Potential solutions to challenges that may arise during experimental procedures are introduced and references and guidelines for cytological research are provided in this paper.

Bioinspired Passive Tactile Sensors Enabled by Reversible Polarization of Conjugated Polymers

Tactile perception plays a vital role for the human body and is also highly desired for smart prosthesis and advanced robots.Compared to active sensing devices,passive piezoelectric and triboelectric tactile sensors consume less power,but lack the capability to resolve static stimuli.Here,we address this issue by utilizing the unique polarization chemistry of conjugated polymers for the first time and propose a new type of bioinspired,passive,and bio-friendly tactile sensors for resolving both static and dynamic stimuli.Specifically,to emulate the polarization process of natural sensory cells,conjugated polymers(including poly(3,4-ethylenedioxythiophen e):poly(styrenesulfonate),polyaniline,or polypyrrole)are controllably polarized into two opposite states to create artificial potential differences.The controllable and reversible polarization process of the conjugated polymers is fully in situ characterized.Then,a micro-structured ionic electrolyte is employed to imitate the natural ion channels and to encode external touch stimulations into the variation in potential difference outputs.Compared with the currently existing tactile sensing devices,the developed tactile sensors feature distinct characteristics including fully organic composition,high sensitivity(up to 773 mV N^(−1)),ultralow power consumption(nW),as well as superior bio-friendliness.As demonstrations,both single point tactile perception(surface texture perception and material property perception)and two-dimensional tactile recognitions(shape or profile perception)with high accuracy are successfully realized using self-defined machine learning algorithms.This tactile sensing concept innovation based on the polarization chemistry of conjugated polymers opens up a new path to create robotic tactile sensors and prosthetic electronic skins.

Localization of stationary pronuclei during conjugation of Paramecium as indicated by immunofluorescence staining

After the third prezygotic division during conjugation of Paramecium caudatum,migratory and stationary pronuclei are produced.The migratory pronuclei remain in the paroral region tightly against the conjugating boundaries;while the stationary pronuclei are located beside the migratory pronuclei.To date,however,it is not clear what causes this close side-by-side localization between migratory and stationary pronuclei.In the current study,immunofluorescence staining with monoclonal antibody of anti-α tubulin indicated that ＂U＂ or ＂V＂ shaped spindles connected the migratory and stationary pronuclei during the third prezygotic division.This observation accounts for the close localization between these two types of pronuclei.

New details indicated by different stainings during conjugation of ciliated protozoa Paramecium

During conjugation of Paramecium caudatum, nuclear events occur in a scheduled program. Morphological studies on nuclear behavior during conjugation of P. caudatum have been performed since the end of the 19th century. Here we report on new details concerning the conjugation of P. caudatum through the staining of conjugating ceils with protargol, carbol fuchsin solution, Hoechst 33342 and immunofluorescence labeling with monoclonal antibody of anti-a tubulin. 1） The crescent nucleus is a characteristic of the meiotic prophase of P. caudatum, has an unstained area. We stained this area with protargol, which was separated from the chromatin area and was not detected by the other stainings. 2） In regards to the four meiotic products, it has long been considered that only one product enters the paroral cone region （PC） and survives after meiosis. However, our protargol and immunofluorescence labeling results indicated that PC entrance of the meiotic product happened before the completion of meiosis instead of after. 3） In our previous study, protargol staining indicated the presence of a swollen structure around the central part of the ＂U＂ and ＂V＂ shaped spindles connecting the two types of prospective pronuclei. However, immunofluorescence labeling with anti-a tubulin antibodies gave a different image from protargol. All these observations form the basis for further studies of their molecular mechanisms.

Characterization and Comparison of Ochratoxin A-Ovalbumin(OTA-OVA) Conjugation by Three Methods

In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten （ochratoxin A, OTA） was coupled to ovalbumin （OVA） by an active ester reaction. To develop a technique for detecting the conjugation, the hapten-protein conjugate （OTA-OVA） was characterized thoroughly by immunoarray technology, ultraviolet （UV） spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS）, respectively. The molecular weight of OTA-OVA was 50 350.141 Da, and the molecular weight of OVA was 44 887.506 Da, which were determined by MALDI-TOF-MS, respectively. In OTA-OVA, the molecular coupling ratio was 13：1 by MALDI-TOF-MS while the molecular coupling ratio was 10：1 by UV. In this experiment, UV and MALDI-TOF-MS were selected as the efficient methods to evaluate the cross-linking effect and calculate the molecular coupling ratio.

Wavefront correction of Ti:sapphire terawatt laser with varying precision of phase conjugation between deformable mirror and wavefront sensor

The phase conjugation between the deformable mirror and the wavefront sensor in the aberration correction of a terawatt Ti：sapphire laser is studied experimentally and theoretically in this paper. At varying values of phase- conjugation precision, we focus the corresponding beams into spots of the same size of 5.1 μm × 5.3 μm with a f/4 parabola in the 32 TW/36 fs Ti：sapphire laser system. The results show that the precision of conjugation can induce an intensity modulation but does not significantly affect the wavefront correction.

OPTICAL PHASE CONJUGATION RESPONSE OF PHOTOINDUCED POLYMER FILMS CONTAINING AZOBENZENE MOIETIES WITH CHIRAL GROUP

An optically active monomer containing azobenzene moieties with chiral group (s-2-methyl-butyl), 4-[2-(methacryloyloxy)ethyloxy] -4'-(s-2-methyl-1-butyloxycarbonyl) azobenzene (M1) was synthesized. Polymer (PM1) possessing optical phase conjugated response was obtained by homopolymerization of the optically active monomer (M1) using free radical polymerization. The polymer was very soluble in common solvents and good optical quality films could be easily fabricated by spin coating. The optical phase conjugated responses of the polymer PM1 were measured by degenerate four-wave mixing (DFWM). In comparison with polymer containing no chiral group, it was found from the preliminary measurement of photoisomeric change that optical phase conjugated response of the PM1 in the long-range order hexagonal symmetry microstructure could be easily controlled by choosing the appropriate polarization direction of the irradiating beams (514.5 nm) and the irradiating number, presumably due to the chiral group in the PM1 molecular structure. For the case of the polymer investigated here, a chiral group side chain was introduced to increase optical phase conjugated response intensity with different polarization directions of the irradiating beams, which aims originally at searching for a new photoactive material.

Transposon mutagenesis of Psychrobacter cryohalolentis PAMC 21807 by tri-parental conjugation

Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.

Strategies for Precise Engineering and Conjugation of Antibody Targeted-nanoparticles for Cancer Therapy

Improvements in the diagnosis and treatment of cancer are urgently needed for use in nanotechnology.Nanoparticles(NPs)can reduce the side effects of traditional chemotherapy by sustained release of loaded drugs and increase therapeutic efficiency.NPs can also enhance endothelial permeation retention by size effect and its accumulation in tumor cells through passive targeting.Furthermore,it is critical to treat cancer with a controlled targeted drug which can be specifically delivered into tumor cells and released there,resulting in a targeted therapy to eradicate tumor cells while sparing normal cells.To this end,antibody-mediated targeting therapy has been developed,but imperfections in antibodies(Abs)limit this therapy.Therefore,the combination of NPs and Abs has been highly valued in recent years,because conjugating special Abs on the surface of NPs can increase targeting efficiency,enabling selective delivery of anti-cancer drugs to tumor cells.In this mini-review,we would like to enumerate the strategies for the conjugation of Abs to the surface of the NPs as well as the precise engineering of targeted NPs.The application of targeting antibody fragments in this drug delivery system will also be discussed.

Conjugation of a zwitterionic polymer with dimethyl chains to lipase significantly increases the enzyme activity and stability

Enzyme-polymer conjugates are complex molecules with great practical significance.This work was designed to develop a novel enzyme-polymer conjugate by covalently coupling a zwitterionic polymer with side dimethyl chains(pID)to Candida rugosa lipase(CRL)via the reaction between the anhydrides of polymer chains with the amino groups of the enzyme.The resulting two CRL-pID conjugates with different pID grafting densities were investigated in term of the catalytic activity,stability and structural changes.In comparison with native CRL,both the CRL conjugates displayed 2.2 times higher activity than the native enzyme,and showed an increase in the maximum reaction rate(V_(max))and a decrease in the Michaelis constant(K_(m)),thus resulting in about three-fold increases in the catalytic efficiency(k_(cat)/K_(m)).These are mainly attributed to the activation of lipase by the hydrophobic alky side chains.Moreover,the thermostability and pH tolerance of the lipase conjugates were significantly enhanced due to the stabilizing effect of the zwitterion moieties.For instance,a five-fold increase of the enzyme half-life at 50℃ for the high-pID conjugated CRL was observed.Spectroscopic studies reveal that the pID conjugation protected the enzyme in the changes in its microenvironment and conformation,well correlating with enhanced activity and stability of lipase conjugates.The findings indicate that enzyme conjugation to the zwitterionic polymer is promising for improving enzyme performance and deserves further development.

Boundary Control for 2 ×2 Elliptic Systems with Conjugation Conditions

In this paper, we consider 2 × 2 non-cooperative elliptic system involving Laplace operator defined on bounded, continuous and strictly Lipschitz domain of Rn. First we prove the existence and uniqueness for the state of the system under conjugation conditions;then we discuss the existence of the optimal control of boundary type with Neumann conditions, and we find the set of equations and inequalities that characterize it.

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione:Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions

Unsymmetrical bisacridines(UAs) are a novel potent class of antitumor-active therapeutics.A significant route of phase II drug metabolism is conjugation with glutathione(GSH),which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes.The aim of this work was to investigate the GSHmediated metabolic pathway of a representative UA,C-2028.GSH-supplemented incubations of C-2028 with rat,but not with human,liver cytosol led to the formation of a single GSH-related metabolite.Interestingly,it was also revealed with rat liver microsomes.Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450(CYP450) inhibitor 1-aminobenzotriazole.Therefore,the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate.In turn,incubations of C-2028 and GSH with human recombinant glutathione S-transferase(GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form,respectively.These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule.Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction.Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry.Mechanisms for their formation were proposed.The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important.This may affect the patient’s drug clearance due to GST activity,loss of GSH,or the interactions with GSH-conjugated drugs.Moreover,GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

Theoretical study on phase conjugation in weakly injected vertical-cavity surface-emitting lasers

This paper gives a detailed theoretical investigation on phase conjugation induced by nearly degenerate fourwave mixing in single mode vertical-cavity surface-emitting lasers （VCSELs） with weak optical injection. Considering VCSELs that can work in linearly polarized or elliptically polarized states, it derives the theoretical expression of the conjugated field by small signal analysis based on the vectoral rate equations-the spin-flip model. For linearly polarized state, VCSELs show similar conjugate spectra to edge-emitting semiconductor lasers. For the elliptically polarized state, dichroism and birefringence parameters as well as the spin-flip rate can change the conjugate spectra. Especially, when frequency detuning of the probe and pump waves is between the positive and negative relaxation oscillation frequency, changes are evident. For specific values of parameters, conjugate efficiency between 20 dB to 40 dB are obtained.

Improving the performance of arylamine-based hole transporting materials in perovskite solar cells： Extending π-conjugation length or increasing the number of side groups？

In this work, we prepared three simple arylamine-based hole transporting materials from commercially available starting materials. The effect of extending z-conjugation length or increasing the number of side groups compared with reference compound on the photophysical, electrochemical, hole mobility properties and performance in perovskite solar cells were further studied. It is noted that these two kinds of molecular modifications can significantly lower the HOMO level and improve the hole mobility, thus improving the hole injection from valence band of perovskite. On the other hand, the compound with more side groups showed higher hole injection efficiency due to lower HOMO level and higher hole mo- bility compared with the compound with extending π-conjugation length. The perovskite solar cells with the modified molecules as hole transporting materials showed a higher efficiency of 15.40% and 16.95%, respectively, which is better than that of the reference compound （13.18%）. Moreover, the compound with increasing number of side groups based devices showed comparable photovoltaic performance with that of conventional spiro-OMeTAD （16.87%）.

Discrete H~∞ Control Via Conjugation

Discrete H ∞ control theory were obtained by conjugation,chain scattering representation and (J,J′) lossless factorization.The existence of the solution is equivalent to the existence of two positive solutions of two Riccati equations.

Symmetric Identities from an Invariant in Partition Conjugation and Their Applications in <i>q</i>-Series

For every partition and its conjugation , there is an important invariant , which denotes the number of different parts. That is , . We will derive a series of symmetric q-identities from the invariant in partition conjugation by studying modified Durfee rectangles. The extensive applications of the several symmetric q-identities in q-series ?[1] will also be discussed. Without too much effort one can obtain much well-known knowledge as well as new formulas by proper substitutions and elementary calculations, such as symmetric identities, mock theta functions, a two-variable reciprocity theorem, identities from Ramanujan’s Lost Notebook and so on.

On Conjugation Partitions of Sets of Trinucleotides

We prove that a trinucleotide circular code is self-complementary if and only if its two conjugated classes are complement of each other. Using only this proposition, we prove that if a circular code is self-complementary then either both its two conjugated classes are circular codes or none is a circular code.

Intersubband Third-Order Nonlinearities in a GaAs/AIGaAs Step Quantum Well Structure by Phase Conjugation Method

In this letter,we present the observation of phase conjugation at 9.27μm in a GaAs/AlGaAs multiple step quantum well structure.The response is caused by the nearly resonant intersubband transition.The magnitude of X^((3))determined by this phase conjugation method is about 8×10^(-5)esu and the phase conjugate reflectivityηis about 6×10^(-3)uncorrected for Fresnel reflections.

Coherent Mutually Pumped Phase Conjugation Induced with High-Efficiency by Self-Pumped Phase Conjugate Reflection

A new scheme of coherent mutually pumped phase conjugate(MPPC)reflection that depends on the formation of self-pumped phase conjugate reflection of another beam has been demonstrated in a Cu:KNSBN crystal.The dependences of the MPPC reflectivity on the intensity ratio of the two incident beams and on the incident position of the signal beam were measured.We obtain phase conjugate reflectivities of greater than 90%at 488nm.

Phase Conjugation of Squeezed Light

Phase conjugation in degenerate four-wave mixing with a squeezed input beam is investigated.We show that the phase conjugate mode is always super-Poissonian and never squeezed,but the combination mode may be squeezed to an extent even greater than the input probe wave.