Screening potential polyhydroxyalkanoate-producing bacteria from wastewater sludge.

We applied fluorescence staining of Nile red, polymerase chain reaction (PCR), and carbon substrate utilization and pressure tolerance analysis to execute three-stage screening for potential polyhydroxyalkanoate (PHA) producers in the sludge samples of 21 large-scale wastewater treatment plants of city and industrial parks in Taiwan area. Total 35,429 colonies were grown on 196 plates, the screened 30 strains were subjected to 16S rRNA analysis, and 18 identified genera belonged to Proteobacteria (67%), Firmicutes (17%), and Actinomycetota (16%). The PHA accumulation results revealed that nine genera (50% of 18 screened) produced PHAs by limiting the nitrogen source and excess single carbon sources of glucose in an aerobic status. The PHA accumulation percentage was 1.44-58.77% at dry cell weight, and the theoretical yield from glucose was 0.52-58.76%. Our results indicate that our triple-screening method is promising for identifying a high biodiversity of PHA-accumulating bacteria from activated sludge for future industrial applications.

A mouse seminal vesicle-secreted lysozyme c-like protein modulates sperm capacitation.

Lysozyme (LYZ) c-like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c-type LYZ family, the seminal vesicle-secreted LYZ c-like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N-linked glycans. SVLLP is structurally similar to c-type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c-type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin-induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm-oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor.

Unibody core-shell smart polymer as a theranostic nanoparticle for drug delivery and MR imaging.

Developing novel multifunctional nanoparticles (NPs) with robust preparation, low cost, high stability, and flexible functionalizability is highly desirable. This study provides an innovative platform, termed unibody core-shell (UCS), for this purpose. UCS is comprised of two covalent-bonded polymers differed only by the functional groups at the core and the shell. By conjugating Gd(3+) at the stable core and encapsulating doxorubicin (Dox) at the shell in a pH-sensitive manner, we developed a theranostic NPs (UCS-Gd-Dox) that achieved a selective drug release (75% difference between pH 7.4 and 5.5) and MR imaging (r1 = 0.9 and 14.5 mm(-1) s(-1) at pH 7.4 and 5.5, respectively). The anti-cancer effect of UCS-Gd-Dox is significantly better than free Dox in tumor-bearing mouse models, presumably due to enhanced permeability and retention effect and pH-triggered release. To the best of our knowledge, this is the simplest approach to obtain the theranostic NPs with Gd-conjugation and Dox doping.

One-pot synthesis of fluorescent BSA-Ce/Au nanoclusters as ratiometric pH probes.

A facile, one-pot synthetic approach has been developed for the preparation of BSA-Ce/Au NCs. The fluorescence intensities of BSA-Ce/Au NCs at 410 and 650 nm are pH dependent and independent, respectively. The fluorescence intensity ratio (I410/I650) is linear against pH values from 6.0 to 9.0. These stable and biocompatible BSA-Ce/Au NCs have been used as ratiometric probes for monitoring local pH values inside HeLa cells.

Detection of adenosine 5'-triphosphate by fluorescence variation of oligonucleotide-templated silver nanoclusters.

Oligonucleotide-templated Ag nanoclusters (DNA-Ag NCs) prepared from AgNO3 using an oligonucleotide (5'-TAACCCCTAACCCCT-3') as a template and NaBH4 as a reducing agent have been used for sensing of adenosine 5'-triphosphate (ATP). The fluorescence intensity and emission wavelength of DNA-Ag NCs are dependent on the pH value and ATP concentration. At pH 3.0 and 11.0, ATP shows greater effects on fluorescence of the DNA-Ag NCs. Upon increasing ATP concentration from 10 to 50µM, their emission wavelength at pH 3.0 shifts from 525 to 585nm. At pH 11.0, their fluorescence intensity (510nm) increases upon increasing ATP concentration. The circular dichroism (CD), electrospray ionization-mass spectrometry (ESI-MS), absorption, and fluorescence results indicate that ATP and pH affect the interactions between DNAs and Ag atoms, resulting in changes in their fluorescence. The DNA-Ag NCs allow detection of ATP over a concentration range of 0.1-10µM, with a limit of detection 33nM. Practicality of the DNA-Ag NCs probe has been validated with the determination of ATP concentrations in the lysate of MDA-MB-231 breast carcinoma cells.

Carbon dots prepared from ginger exhibiting efficient inhibition of human hepatocellular carcinoma cells.

Fluorescent carbon nanodots (C-dots; 4.3 ± 0.8 nm) from fresh tender ginger juice provide high suppression of the growth of human hepatocellular carcinoma cells (HepG2), with low toxicity to normal mammary epithelial cells (MCF-10A) and normal liver cells (FL83B). The inhibition is selective to HepG2 over other tested cancer cells, including human lung cancer cell line (A549), human breast cancer cell line (MDA-MB-231), and human cervical cancer cell line (HeLa). Western blot results reveal that the C-dots up-regulate the expression of p53 protein only in the HepG2 cell line. The 50% inhibiting concentration (IC50) value of the C-dots on HepG2 cells is 0.35 mg mL-1. Image cytometry results show significant uptake of C-dots by HepG2 cells that induce intracellular production of reactive oxygen species (ROS, 18.2-fold increased), while other cells remain almost the same in ROS levels after treatment with C-dots (1.11 mg mL-1). The C-dots trigger the pro-apoptotic factor to promote HepG2 cell apoptosis. The C-dots effectively inhibit the growth of tumors in nude mice (104 ± 14 vs. 3.7 ± 0.2 mg with and without treatment within 14 days).

Synthesis of fluorescent gold nanodot-liposome hybrids for detection of phospholipase C and its inhibitor.

We report the synthesis of fluorescent 11-mercaptoundecanoic acid-gold nanodot-liposome (11-MUA-Au ND/Lip) hybrids by incorporation of gold nanoparticles (∼3 nm) and 11-MUA molecules in hydrophobic phospholipid membranes that self-assemble to form small unilamellar vesicles. A simple and homogeneous fluorescence assay for phospholipase C (PLC) was developed on the basis of the fluorescence quenching of 11-MUA-Au ND/Lip hybrids in aqueous solution. The fluorescence of the 11-MUA-Au ND/Lip hybrids is quenched by oxygen (O2) molecules in solution, and quenching is reduced in the presence of PLC. PLC catalyzes the hydrolysis of phosphatidylcholine units from Lip to yield diacylglycerol (DAG) and phosphocholine (PC) products, leading to the decomposition of Lip. The diacylglycerol further interacts with 11-MUA-Au NDs via hydrophobic interactions, leading to inhibition of O2 quenching. The 11-MUA-Au ND/Lip probe provides a limit of detection (at a signal-to-noise ratio of 3) of 0.21 nM for PLC, with high selectivity over other proteins, enzymes, and phospholipases. We have validated the practicality of using this probe for the determination of PLC concentrations in breast cancer cells (MCF-7 and MDA-MB-231 cell lines) and nontumor cells (MCF-10A cell line), revealing that the PLC activity in the first two is at least 1.5-fold higher than that in the third. An inhibitor assay using 11-MUA-Au ND/Lip hybrids demonstrated that tricyclodecan-9-yl potassium xanthate (D609) inhibits PLC (10 nM) with an IC50 value of 3.81 ± 0.22 µM. This simple, sensitive, and selective approach holds great potential for detection of PLC in cancer cells and for the screening of anti-PLC drugs.

Synthesis of aluminum oxide supported fluorescent gold nanodots for the detection of silver ions.

Photoluminescent gold nanodots (Au NDs) on aluminum oxide nanoparticles (Al2O3 NPs) with the emission wavelengths ranging from 510 to 630 nm are unveiled. Orange Al2O3 NP@AuNDs show high selectivity and sensitivity towards Ag(+) ions by metallophilic Ag(+)-Au(+) interactions and induced fluorescence quenching of Au NDs.

Synthesis of photoluminescent Au ND-PNIPAM hybrid microgel for the detection of Hg2+.

Poly(N-isopropylacrylamide) microgels (PNIPAM MGs) incorporated with photoluminescent gold nanodots (Au NDs) have been prepared and employed for the detection of mercury ions (Hg(2+)). Each of the PNIPAM MGs (hydrodynamic diameter 615 ± 15 nm) contains several Au NDs (diameter 1.8 ± 0.2 nm) in the Au ND-PNIPAM MGs. Like Au NDs, Au ND-PNIPAM MGs exhibit an absorption band at 375 nm that is assigned for ligand to metal charge transfer mixed with metal centered (ds/dp) states and photoluminescence at 520 nm originated from Au ND/polynuclear gold(I)-thiolate (core/shell) complexes. Purification of Au ND-PNIPAM MGs relative to Au NDs is much easier through a simple centrifugation/wash process. On the basis of Hg(2+)-induced photoluminescence quenching due to the formation of Au-Hg amalgam and formation of Au ND-PNIPAM MGs aggregates, the signal response of Au ND-PNIPAM MGs against Hg(2+) concentration is linear over a range from 2 to 20 nM (r = 0.9945). This selective approach provides limits of detection for Hg(2+) (at a signal-to-noise ratio of 3) of 1.9 and 1.7 nM in phosphate buffer solutions (5 mM, pH 7.0) with and without containing 500 mM NaCl, respectively. This selective and sensitive Au ND-PNIPAM MG probe has been applied to the determination of the concentration of Hg in a representative fish sample, showing its practical potential for monitoring of Hg levels in complicated biological and environmental samples.

Highly efficient inhibition of human immunodeficiency virus type 1 reverse transcriptase by aptamers functionalized gold nanoparticles.

We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45-Au NPs shows inhibitory efficiency in the retroviral replication cycle with a decreasing infectivity (40.2%).

Sensitive pH probes of retro-self-quenching fluorescent nanoparticles.

Polymeric fluorescent nanoparticles, R6GDARs, containing rhodamine 6G within 1,3-phenylenediamine resin are prepared using the extensive Stöber method. The R6GDAR is capable of sensing intracellular pH in living cells, with the fluorescence intensity increasing upon decreasing the pH values from 8.0 to 3.0. This fluorescence enhancement at low pH is based on the "retro-self-quenching" mechanism, where the protonation of the R6GDAR backbones expands the particle structure, leading to increase in the separation among concentrated R6G molecules as well as their release. Fluorescence time-course measurement shows that even individual R6GDARs have high sensitivity to report the environmental pH at the single particle level. Compared to other existing pH sensors, R6GDARs offer a wider working pH range (5 pH units), higher sensitivity, and greater photostability. R6GDARs have been demonstrated to be sensitive to map local pH values inside MCF7 and MDA-MB-231 cells, with extremely low cell toxicity. R6GDARs serve as an excellent pH sensing probe for cellular microenvironments.

Extremely high inhibition activity of photoluminescent carbon nanodots toward cancer cells.

Highly water-soluble, biocompatible, and photoluminescent carbon nanodots (C-dots) having an average diameter 3.4 ± 0.8 nm and a quantum yield 4.3% are obtained from used green tea through grinding, calcination and centrifugation. The as-prepared C-dots are stable in high-ionic-strength media (e.g. 500 mM NaCl) and under light irradiation, allowing images of MCF-10A, MCF-7 and MDA-MB-231 cells to be recorded. The C-dots are mostly localized in the cell membranes and cytoplasms, with evidence of excitation-wavelength cell images. Relative to catechin, the C-dots provide greater inhibition efficiency of the growth of MCF-7 and MDA-MB-231 cancer cells, with lower toxicity for the MCF-10A normal cells. The inhibitory activity of C-dots is associated with the generation of greater amounts of reactive oxygen species. A prothrombin time (PT) assay of plasma samples reveals excellent biocompatibility of the C-dots. To the best of our knowledge, for the first time the C-dots provide inhibition efficiencies up to 80% and 82% for MCF-7 and MDA-MB-231 cancer cells, respectively, showing their high potential as cancer inhibitors.

Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug.

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS. Further, mouse SVS I-III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I-III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I-III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.

Exclusive expression of a membrane-bound Spink3-interacting serine protease-like protein TESPL in mouse testis.

We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. In contrast, Prss2 was constitutively expressed in many tissues including testis. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Further, orthology group for mouse TESPL was identified in the conserved gene family of eutherian testis serine protease 5.