Abundant Alkaline Phosphatase Research Articles

A one-tube dual-readout biosensor for detection of nucleic acids and non-nucleic acids using CRISPR-ALP tandem assay.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have been considered a next-generation molecular diagnosis tool. Single-readout mode has been extensively employed in massive CRISPR/Cas12a-based biosensors. In this work, we propose a one-tube dual-readout biosensor (CRISAT) for the first time for the detection of ultrasensitive nucleic acids and non-nucleic acids developed by harnessing CRISPR-ALP tandem assay. In the presence of a target, Cas12a is activated to randomly cut the single-stranded hyDNA sequence of MB@hyDNA-cALP, thus releasing abundant alkaline phosphatase (ALP) in the supernatant solution. By using 4-aminophenol phosphate as the substrate of ALP, p-aminophenol is produced, which then reacts with N-[3-(trimethoxysilyl)propyl]ethylenediamine or diethylenetriamine to generate silicon-containing polymer carbon dots (Si PCDs) or polymer carbon dots (PCDs) in situ, which can be observed by the naked eye or detected using a fluorescent device in the same solution. Using this strategy, a fluorescence and colorimetry dual-readout nanoplatform for CRISPR-based biosensors can be rationally developed. We ascertain the applicability of CRISAT by detecting the SARS-CoV-2 pseudovirus, achieving superior sensitivity and specificity. With simple modification of crRNAs, the CRISAT platform can also be employed to detect monkeypox virus (MPXV) and non-nucleic acids of adenosine triphosphate (ATP). This work shows great potential for the detection of nucleic acids and non-nucleic acids.

The potential role of sediment organic phosphorus in algal growth in a low nutrient lake

The role of sediment–bound organic phosphorus (Po) as an additional nutrient source is a component of internal P budgets in lake system that is usually neglected. Here we examined the relative importance of sediment Po to internal P load and the role of bioavailable Po in algal growth in Lake Erhai, China. Lake Erhai sediment extractable Po accounted for 11–43% (27% average) of extractable total P, and bioavailable Po accounted for 21–66% (40%) of Po. The massive storage of bioavailable Po represents an important form of available P, essential to internal loads. The bioavailable Po includes mainnly labile monoester P and diester P was identified in the sequential extractions by H2O, NaHCO3, NaOH, and HCl. 40% of H2O−Po, 39% of NaHCO3−Po, 43% of NaOH−Po, and 56% of HCl−Po can be hydrolyzed to labile monoester and diester P, suggesting that the bioavailability of Po fractions was in decreasing order as follows: HCl−Po > NaOH−Po > H2O−Po > NaHCO3−Po. It is implied that traditional sequential fractionation of Po might overestimate the availability of labile Po in sediments. Furthermore, analysis of the environmental processes of bioavailable Po showed that the stabler structure of dissloved organic matter (DOM) alleviated the degradation and release of diester P, abundant alkaline phosphatase due to higher algal biomass promoted the degradation of diester P. The stability of DOM structure and the degradation of diester P might responsible for the spatial differences of labile monoester P. The biogeochemical cycle of bioavailable Po replenishs available P pools in overlying water and further facilitate algal growth during the algal blooms. Therefore, to control the algal blooms in Lake Erhai, an effective action is urgently required to reduce the accumulation of Po in sediments and interrupt the supply cycle of bioavailable Po to algal growth.

Label-Free Detection of Protein Tyrosine Phosphatase 1B (PTP1B) by Using a Rationally Designed Förster Resonance Energy Transfer (FRET) Probe.

A highly selective detection method of native protein tyrosine phosphatase 1B (PTP1B) is described using a target specific probe equipped with 1-naphthylamine (λex =330 nm, λem =445 nm). Irradiation of a mixture of PTP1B and Probe 1 with ultraviolet light of 280 nm (corresponding to PTP1B excitation maximum) resulted in significant fluorescence increase at 445 nm, following FRET characteristics. This phenomenon does not occur with other closely related phosphatases or cellular abundant alkaline phosphatase (APP). Probe 1, the most potent and selective probe, was found to competitively inhibit PTP1B (Ki ≈42 nm), whereas APP inhibition was found to be in the low micromolar range. Furthermore, Probe 1 discriminates between PTP1B and several other phosphatases. Here, we report real-time label-free FRET detection of pure PTP1B as well as induced human PTP1B in Escherichia coli cell lysate. In contrast to 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a representative fluorescence turn-on PTP substrate, our FRET probe successfully differentiated human cervical carcinoma cell lysate, SiHa, which has a high expression level of PTP1B, from PTP1B-knockdown SiHa cell lysate (that is, siRNA was used for PTP1B knockdown).

Response of sediment organic phosphorus composition to lake trophic status in China

Organic phosphorus (Po) constitutes the most important fraction of P in lake sediments, and the compositional properties of Po affect its behavior in lake ecosystems. In this study, 31P NMR, FT-IR spectroscopy, and UV–visible absorbance spectroscopy were combined to identify the dynamic composition of sediment Po across two sets of lakes in China ranging from oligotrophic to eutrophic, and their possible effects on lake eutrophication were evaluated. The results showed that sediment Po content (accounting for 24–75% of TP) was positively correlated with trophic status in both Eastern Plain and Yun-Gui Plateau lakes of China, and the linear relationship was more stable compared to total P (TP), implying that sediment Po may be a superior indicator of trophic status than TP. The Po component, phosphonate accounted for only 0.4% or less of Po, while the monoester P and diester P, accounted for 2–24% and 0.5–5% of Po, respectively, and were the main factors causing Po to increase with the increasing trophic status. The factors were closely related to the enhanced organic sewage load and intensification of contemporary sedimentation of phytoplankton. As trophic status increased, sediment Po might integrate into larger amounts of aromatic substances and functional groups, which could enhance the stability of Po in sediments. Furthermore, sediments from lakes with higher trophic status exhibited a higher degree of humification and molecular weights, which impart resistance to biodegradation, and therefore, reduced the risk of sediment Po release. However, the massive accumulation of bioavailable Po (monoester and diester P) allows possible degradation, supporting algal growth and maintains eutrophic status because there is abundant alkaline phosphatase in eutrophic lakes. Thus, to control lake eutrophication more effectively, targeted actions are urgently required to reduce the accumulation and degradation of Po in lake sediment.

Alkaline phosphatase binds tenaciously to titanium; implications for biological surface evaluation following bone implant retrieval

Enhancing the performance and longevity of titanium (Ti) implants continues to be a significant developmental theme in contemporary biomaterials design. Our specific focus pertains to the surface functionalisation of Ti using the bioactive lipid, lysophosphatidic acid (LPA) and certain phosphatase-resistant analogues of LPA. Coating survivorship to a plethora of testing regimens is required to align with due regulatory process before novel biomaterials can enter clinical trials. One of the key acceptance criteria is coating retention to the physical stresses experienced during implantation. In assessing coating stability to insertion into porcine bone we found that a subsequent in vitro assessment to confirm coating persistence was masked by abundant alkaline phosphatase (ALP) contamination adsorbed to the metal surface. Herein we report that ALP can bind to Ti in a matter of minutes by simply immersing Ti samples in aqueous solutions of the enzyme. We strongly discourage the in vitro monitoring of osteoblast and stromal cell ALP expression when assessing bioactive coating survivorship following Ti implant retrieval form native bone tissue.

1,25-Dihydroxyvitamin D3 and extracellular calcium promote mineral deposition via NPP1 activity in a mature osteoblast cell line MLO-A5

While vitamin D supplementation is common, the anabolic mechanisms that improve bone status are poorly understood. Under standard mineralising conditions including media ionised calcium of 1.1 mM, 1,25-dihydroxyvitamin D3 (1,25D) enhanced differentiation and mineral deposition by the mature osteoblast/pre-osteocyte cell line, MLO-A5. This effect was markedly increased with a higher ionised calcium level (1.5 mM). Gene expression analyses revealed that 1,25D-induced mineral deposition was associated with induction of Enpp1 mRNA, coding for nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and NPP1 protein levels. Since MLO-A5 cells express abundant alkaline phosphatase that was not further modified by 1,25D treatment or exposure to increased calcium, this finding suggested that the NPP1 production of pyrophosphate (PPi) may provide alkaline phosphatase with substrate for the generation of inorganic phosphate (Pi). Consistent with this, co-treatment with Enpp1 siRNA or a NPP1 inhibitor, PPADS, abrogated 1,25D-induced mineral deposition. These data demonstrate that 1,25D stimulates osteoblast differentiation and mineral deposition, and interacts with the extracellular calcium concentration. 1,25D regulates Enpp1 expression, which presumably, in the context of adequate tissue non-specific alkaline phosphatase activity, provides Pi to stimulate mineralisation. Our findings suggest a mechanism by which vitamin D with adequate dietary calcium can improve bone mineral status.

Alkaline phosphatases in microbialites and bacterioplankton from Alchichica soda lake, Mexico.

Dissolved organic phosphorus utilization by different members of natural communities has been closely linked to microbial alkaline phosphatases whose affiliation and diversity is largely unknown. Here we assessed genetic diversity of bacterial alkaline phosphatases phoX and phoD, using highly diverse microbial consortia (microbialites and bacterioplankton) as study models. These microbial consortia are found in an oligo-mesotrophic soda lake with a particular geochemistry, exhibiting a low calcium concentration and a high Mg:Ca ratio relative to seawater. In spite of the relative low calcium concentration in the studied system, our results highlight the diversity of calcium-based metallophosphatases phoX and phoD-like in heterotrophic bacteria of microbialites and bacterioplankton, where phoX was the most abundant alkaline phosphatase found. phoX and phoD-like phylotypes were more numerous in microbialites than in bacterioplankton. A larger potential community for DOP utilization in microbialites was consistent with the TN:TP ratio, suggesting P limitation within these assemblages. A cross-system comparison indicated that diversity of phoX in Lake Alchichica was similar to that of other aquatic systems with a naturally contrasting ionic composition and trophic state, although no phylotypes were shared among systems.

Growth and competition for different forms of organic phosphorus by the dinoflagellate Prorocentrum donghaiense with the dinoflagellate Alexandrium catenella and the diatom Skeletonema costatum s.l.

Growth and competition between the dinoflagellates Prorocentrum donghaiense and Alexandrium catenella and the diatom Skeletonema costatum sensu lato in response to different forms of phosphorus were investigated in mixed cultures. The results indicated that S. costatum s.l. outcompeted P. donghaiense to become dominant owing to high growth rates, irrespective of the KH2PO4 content or any dissolved organic phosphorus (DOP) substrate. However, P. donghaiense densities in the DOP substrates, particularly lecithin, grew to 2–4 times higher than those in KH2PO4. The growth rates of P. donghaiense and A. catenella were comparable. A. catenella densities decreased when the phosphorus substrate was changed from inorganic to organic, and P. donghaiense outcompeted A. catenella only when lecithin was added. Abundant alkaline phosphatase (AP) was expressed when the external phosphate was lower than 0.1 μmol l−1. The highest bulk AP activities in the lecithin treatments suggested that lecithin might be more difficult to hydrolyze by these algae compared with glucose-6-phosphate and ribonucleic acid. We suggested that the advantage which P. donghaiense was shown to have in utilizing DOP for growth is what allowed it to become abundant or even dominant in natural communities when phosphate was depleted.

Epiblast isolation by a new four stage method (peeling) from whole bovine cloned blastocysts

We have established a new 4 stage epiblast isolation method from whole bovine cloned blastocysts without using immunosurgery. The new "peeling" method consists of dissolution of the zona pellucida (first stage), elimination of mural trophoblast (second stage), isolation of primitive endoderm and epiblast from polar trophoblast (third stage), and isolation of epiblast from primitive endoderm (fourth stage). The bovine cloned blastocyst consists of 4 different types of cells showing abundant alkaline phosphatase activity. The epiblast origin of isolated cells was confirmed by in vitro differentiation of isolated cells to tubulin beta3-positive neurons and by embryoid body formation. The bovine cloned blastocyst origin of isolated epiblasts was confirmed by microsatellite analysis and mitochondrial DNA sequencing analysis. This new method might accelerate establishment of somatic cell nuclear transfer derived embryonic stem cell lines from bovine and other mammals.

Histochemical and molecular analyses of distraction osteogenesis in a mouse model.

A tibial lengthening scheme in the mouse was used to study the molecular and cellular events regulating tissue regeneration during distraction osteogenesis. Here, we report on the surgical technique and frame design and describe the histochemical and molecular aspects of distraction during different phases of treatment. A total of 26 mice were used in this study. The treatment protocol was divided into a latency period of 7 days, a phase of active distraction that lasted 10 days with a distraction rate of 0.42 mm/day, and a maturation phase of 9 days. During latency, the distraction site resembled a stabilized fracture callus on both a histochemical and a molecular level. During active distraction, the gap was characterized by a central fibrous interzone bordered by primary matrix fronts, regenerate bone aligned with the distraction force, parallel columns of vascular sinusoids, and a medullary cavity. Alkaline phosphatase activity was detected in the endosteal and periosteal surfaces of the bone ends. Tartrate resistant acid phosphatase staining revealed that osteoclasts remodeled the bone regenerate as it formed. Collagen type I was expressed in the periosteum and the primary matrix front during distraction, whereas collagen type-II transcripts were localized to discrete regions on the periosteal surfaces, immediately adjacent to the osteotomy ends. Collagen type-II transcripts were not detected in the fibrous interzone. During the maturation phase, cells within the fibrous interzone expressed collagen type I and exhibited abundant alkaline phosphatase activity, suggesting that they had begun to terminally differentiate. Collectively, these data demonstrate the utility of a mouse model to study the molecular and cellular bases for the regeneration and remodeling of tissue.

The human auricular chondrocyte. Responses to growth factors.

The first human auricular (elastic cartilage) chondrocyte cell culture model is presented. These chondrocytes, harvested from fresh human cadaver auricular cartilage, are easily grown in culture. They produce type II collagen and abundant alkaline phosphatase. Like growth plate chondrocytes, human auricular chondrocytes respond mitogenically to both transforming growth factor-beta and basic fibroblast growth factor by increasing proliferation twofold. The growth factors exert a synergistic effect on thymidine uptake in this model, with maximal stimulation occurring at the dose combination of basic fibroblast growth factor, 10 ng/mL, and transforming growth factor-beta, 3 ng/mL. Human nasal septal (hyaline cartilage) chondrocytes do not increase proliferation in response to these two growth factors. Human auricular chondrocytes also increase matrix production in response to transforming growth factor-beta, as indicated by increased proteoglycan production and increased collagen synthesis. The increased matrix production combined with increased proliferative rate elicited with transforming growth factor-beta and basic fibroblast growth factor indicates that human elastic cartilage chondrocytes harvested from the external ear are better suited for in vitro cartilage implant growth than human hyaline cartilage chondrocytes. Further biochemical and molecular biological characterization of the human auricular chondrocyte cell culture model is currently under way. Additional work is also under way to generate multilayer culture growth on absorbable frameworks, in the pursuit of the long-term goal of in vitro production of autogenous cartilage implants.

The skin of primates. XXXIV. The skin of the golden spider monkey (Ateles geoffroyi)

AbstractThe skin of the golden spider monkey (Ateles geoffroyi) has many histological and histochemical similarities to that of the woolly monkey (Lagothrix lagotricha) and howler monkey (Alouatta caraya); however, this monkey possesses certain peculiar properties such as large sebaceous glands, a combined distributional pattern of eccrine and apocrine sweat glands, and abundant alkaline phosphatase in the sebaceous glands, apocrine and eccrine sweat glands. In brief, the anatomical and histochemical properties of the skin of this animal are more similar to those of the howler monkey than to the woolly monkey. In addition, the skin of these three Ceboids falls phylogenetically between that of the Cercopithecoidea and Pithecoidea.