Spontaneous Degradation of the "Forever Chemicals" Perfluoroalkyl and Polyfluoroalkyl Substances (PFASs) on Water Droplet Surfaces.

Because of their innate chemical stability, the ubiquitous perfluoroalkyl and polyfluoroalkyl substances (PFASs) have been dubbed "forever chemicals" and have attracted considerable attention. However, their stability under environmental conditions has not been widely verified. Herein, perfluorooctanoic acid (PFOA), a widely used and detected PFAS, was found to be spontaneously degraded in aqueous microdroplets under room temperature and atmospheric pressure conditions. This unexpected fast degradation occurred via a unique multicycle redox reaction of PFOA with interfacial reactive species on the droplet surface. Similar degradation was observed for other PFASs. This study extends the current understanding of the environmental fate and chemistry of PFASs and provides insight into aid in the development of effective methods for removing PFASs.

Heterogeneous hydrochlorination of lipids mediated by fatty acids in an indoor environment.

Fatty acids from cooking fumes and hypochlorous acid (HOCl) released from indoor cleaning adversely affect respiratory health, but the molecular-level mechanism remains unclear. Here, the effect of cooking oil fumes [palmitic acid (PA), oleic acid (OA), and linoleic acid (LA)] on lung model phospholipid (POPG) hydrochlorination mediated by HOCl at the air-water interface of the hanged droplets was investigated. Interfacial hydrochlorination of POPG was impeded by OA and LA, while that of POPG was facilitated by PA. The effect on POPG hydrochlorination increased with the decrease in oil fume concentration. A potential mechanism with respect to the chain length of these oil fumes, regardless of their saturation, was proposed. PA with a short carbon chain looses the POPG packing and leads to the exposure of the C=C double bonds of POPG, whereas OA and LA with a long carbon chain hinder HOCl from reaching the C=C bonds of POPG. These results for short chain and low concentration dependence suggest that the decay of oil fumes or the conversion of short-chain species by indoor interfacial chemistry might be adverse to lung health. These results provide insights into the relationship between indoor multicomponent pollutants and the respiratory system.

Probabilistic risk assessment of dietary exposure to benzophenone derivatives in cereals in Taiwan.

Benzophenone (BP) and BP derivatives (BPDs) are widely used as ultraviolet (UV) stabilizers in food packaging materials and as photoinitiators in UV-curable inks for printing on food-contact materials. However, our knowledge regarding the sources and risks of dietary exposure to BP and BPDs in cereals remains limited, which prompted us to conduct this study. We measured the levels of BP and nine BPDs-BP-1, BP-2, BP-3, BP-8, 2-hydroxybenzophenone, 4-hydroxybenzophenone, 4-methylbenzophenone (4-MBP), methyl-2-benzoylbenzoate, and 4-benzoylbiphenyl-in three types of cereals (rice flour, oatmeal, and cornflakes; 180 samples in total). A Bayesian Markov-chain Monte Carlo (MC) simulation approach was used for deriving the posterior distributions of BP and BPD residues. This approach helped in addressing the uncertainty in probabilistic distribution for the sampled data under the detection limit. Through an MC simulation, we calculated the daily exposure levels of dietary BP and BPDs and corresponding health risks. The results revealed the ubiquitous presence of BP, BP-3, and 4-MBP in cereals. Older adults (aged >65 years) had the highest (97.5 percentile) lifetime carcinogenic risk for BP exposure through cereals (9.41 × 10-7), whereas children aged 0-3 years had the highest (97.5 percentile) hazard indices for BPD exposure through cereals (2.5 × 10-2). Nevertheless, across age groups, the lifetime carcinogenic risks of BP exposure through cereals were acceptable, and the hazard indices for BPD exposure through cereals were <1. Therefore, BPD exposure through cereals may not be a health concern for individuals in Taiwan.

Interfacial Extraction to Trap and Characterize the Criegee Intermediates from Phospholipid Ozonolysis.

Criegee intermediates (CIs) play a significant role in cell membrane peroxidation, but their identification remains elusive at the molecular level. Herein, we combined interfacial extraction and sonic spray ionization mass spectrometry to study the oxidation reaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) mediated by ozone (O3) at/near the surface of a hung water droplet. On-line interfacial extraction and ionization provided a snapshot of the short-lived CIs. Experiments in which the content of water was varied provided evidence for the formation of CIs, which has not been previously observed. Capture experiments using 5,5-dimethyl-pyrroline N-oxide (DMPO) indicated that CIs could be selectively characterized, and the extracted ion current (EICs) of CIs vs DMPO-CI adducts further confirmed the successful observation of CIs. Theoretical calculation suggested that surface ozonolysis of POPG was mainly mediated by anti-CI. These results open a new route for aqueous surface reactive species identification, and benefit toward the understanding of disease development associated with cell oxidative stress mediated by CIs.

DRAGON: Harnessing the power of DNA repair for accelerating genome evolution in Corynebacterium glutamicum.

Hypermutation is a robust phenotype characterized by high elevation of spontaneous mutation rates, which has been shown to facilitate rapid adaptation to the stressful environments by hitchhiking with favorable mutations. Accumulating evidence argues that deficient DNA repair can give rise to hypermutation events in bacteria. Here, we provided a comprehensive survey of DNA repair systems to identify promising targets ensuring high DNA fidelity in Corynebacterium glutamicum. Four effective DNA repair factors, including nucS, tag, xpb, and dinP, were found to be strongly associated with the occurrence of hypermutable phenotypes, and these targets were then engineered to establish a CRISPRi-based all-in-one plasmid system for genome mutagenesis. On the basis of these findings, we presented a novel evolutionary engineering method named "DNA repair-assisted genome evolution (DRAGON)". As a proof-of-concept, DRAGON strategy was successfully applied to facilitate rapid acquisition of microbial robustness in C. glutamicum, such as increased tolerances towards kanamycin, acidic pH and high L-serine, showing its promise and potential for rapid strain improvement. Overall, our study will offer new insights into the understanding of DNA repair and evolutionary adaptation in C. glutamicum.

Reaction acceleration in microdroplet mass spectrometry: Inlet capillary and solvent composition effects.

RATIONALE: Microdroplet chemistry has attracted tremendous interest in recent years. We have previously reported that microdroplet mass spectrometry (MS) achieves reaction acceleration. Here we systematically investigated the effect of capillary heating of MS inlet and solvent polarity of microdroplets on the conversion ratios of dehydration and phosphorylation reactions. METHODS: The micron-sized droplets generated by high-speed gas encapsulated the compounds. The conversion ratios of dehydration and phosphorylation reactions were investigated at different capillary temperatures of MS inlet between 30°C and 300°C. Subsequently, the effects of solvent polarity of different microdroplets (acetonitrile, acetonitrile/water [v/v: 9:1], and water) on microdroplet reactions were investigated. RESULTS: The microdroplets could be used as reaction vessels for rapid dehydration and phosphorylation reactions. Microdroplet MS is characterized by the completion of the reaction in microseconds. The increase in capillary temperature increased the conversion ratio of dehydration reactions but had little effect on phosphorylation reactions. The stability of compounds supports this phenomenon. In addition, the increase in solvent polarity in microdroplets promoted the dehydration reaction but inhibited the nucleophilic substitution reaction (phosphorylation reaction). CONCLUSIONS: Microdroplet MS achieved an acceleration of the reaction, which was attributed to capillary temperature, microdroplet solvents, and the stability of reaction products. This finding suggested that the inlet capillary and solvent system should be considered in the study and interpretation of microdroplet MS.

Alteration of twinfilin1 expression underlies opioid withdrawal-induced remodeling of actin cytoskeleton at synapses and formation of aversive memory.

Exposure to drugs of abuse induces alterations of dendritic spine morphology and density that has been proposed to be a cellular basis of long-lasting addictive memory and heavily depend on remodeling of its underlying actin cytoskeleton by the actin cytoskeleton regulators. However, the actin cytoskeleton regulators involved and the specific mechanisms whereby drugs of abuse alter their expression or function are largely unknown. Twinfilin (Twf1) is a highly conserved actin-depolymerizing factor that regulates actin dynamics in organisms from yeast to mammals. Despite abundant expression of Twf1 in mammalian brain, little is known about its importance for brain functions such as experience-dependent synaptic and behavioral plasticity. Here we show that conditioned morphine withdrawal (CMW)-induced synaptic structure and behavior plasticity depends on downregulation of Twf1 in the amygdala of rats. Genetically manipulating Twf1 expression in the amygdala bidirectionally regulates CMW-induced changes in actin polymerization, spine density and behavior. We further demonstrate that downregulation of Twf1 is due to upregulation of miR101a expression via a previously unrecognized mechanism involving CMW-induced increases in miR101a nuclear processing via phosphorylation of MeCP2 at Ser421. Our findings establish the importance of Twf1 in regulating opioid-induced synaptic and behavioral plasticity and demonstrate its value as a potential therapeutic target for the treatment of opioid addiction.

Phosphatidylinositol-4-phosphate 5-kinase type 1α attenuates Aß production by promoting non-amyloidogenic processing of amyloid precursor protein.

Alzheimer's disease (AD) is characterized by a chronic decline in cognitive function and is pathologically typified by cerebral deposition of amyloid-ß peptide (Aß). The production of Aß is mediated by sequential proteolysis of amyloid precursor protein (APP) by ß- and γ-secretases, and has been implicated as the essential determinant of AD pathology. Previous studies have demonstrated that the level of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in the membrane may potentially modulate Aß production. Given that PI(4,5)P2 is produced by type 1 phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks), we sought to determine whether the level of PIP5K type Iα (PIP5K1A) can affect production of Aß by modulating the lipid composition of the membrane. Using a HEK-derived cell line that constitutively expresses yellow fluorescent protein-tagged APP (APP-YFP), we demonstrated that overexpression of PIP5K1A results in significant enhancement of non-amyloidogenic APP processing and a concomitant suppression of the amyloidogenic pathway, leading to a marked decrease in secreted Aß. Consistently, cells overexpressing PIP5K1A exhibited a significant redistribution of APP-YFP from endosomal compartments to the cell surface. Our findings suggest that PIP5K1A may play a critical role in governing Aß production by modulating membrane distribution of APP, and as such, the pathway may be a valuable therapeutic target for AD.

DNA Damage Response/TP53 Pathway Is Activated and Contributes to the Pathogenesis of Dilated Cardiomyopathy Associated With LMNA (Lamin A/C) Mutations.

RATIONALE: Mutations in the LMNA gene, encoding LMNA (lamin A/C), are responsible for laminopathies. Dilated cardiomyopathy (DCM) is a major cause of mortality and morbidity in laminopathies. OBJECTIVE: To gain insights into the molecular pathogenesis of DCM in laminopathies. METHODS AND RESULTS: We generated a tet-off bigenic mice expressing either a WT (wild type) or a mutant LMNA (D300N) protein in cardiac myocytes. LMNAD300N mutation is associated with DCM in progeroid syndromes. Expression of LMNAD300N led to severe myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. Administration of doxycycline suppressed LMNAD300N expression and prevented the phenotype. Whole-heart RNA sequencing in 2-week-old WT and LMNAD300N mice led to identification of ≈6000 differentially expressed genes. Gene Set Enrichment and Hallmark Pathway analyses predicted activation of E2F (E2F transcription factor), DNA damage response, TP53 (tumor protein 53), NFκB (nuclear factor κB), and TGFß (transforming growth factor-ß) pathways, which were validated by Western blotting, quantitative polymerase chain reaction of selected targets, and immunofluorescence staining. Differentially expressed genes involved cell death, cell cycle regulation, inflammation, and epithelial-mesenchymal differentiation. RNA sequencing of human hearts with DCM associated with defined LMNA pathogenic variants corroborated activation of the DNA damage response/TP53 pathway in the heart. Increased expression of CDKN2A (cyclin-dependent kinase inhibitor 2A)-a downstream target of E2F pathway and an activator of TP53-provided a plausible mechanism for activation of the TP53 pathway. To determine pathogenic role of TP53 pathway in DCM, Tp53 gene was conditionally deleted in cardiac myocytes in mice expressing the LMNAD300N protein. Deletion of Tp53 partially rescued myocardial fibrosis, apoptosis, proliferation of nonmyocyte cells, left ventricular dilatation and dysfunction, and slightly improved survival. CONCLUSIONS: Cardiac myocyte-specific expression of LMNAD300N, associated with DCM, led to pathogenic activation of the E2F/DNA damage response/TP53 pathway in the heart and induction of myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. The findings denote the E2F/DNA damage response/TP53 axis as a responsible mechanism for DCM in laminopathies and as a potential intervention target.

Pengzhenrongella sicca gen. nov., sp. nov., a new member of suborder Micrococcineae isolated from High Arctic tundra soil.

A yellow bacterial strain, designated LRZ-2T, was isolated from High Arctic tundra near the settlement Ny-Ålesund in the Svalbard Archipelago, Norway. The cells were Gram-stain-positive, aerobic and non-sporulating. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain LRZ-2T represented a novel member of the suborder Micrococcineae. Its nearest phylogenetic neighbours were the members of the genus Luteimicrobium, with 16S rRNA gene sequence similarity of 95.3-96.9 %. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain LRZ-2T and its closely related strains were 77.4-74.3 % and 21.4-19.6 %, respectively. The DNA G+C content was 72.4 mol%. The peptidoglycan type of the isolate was A4ß with an interpeptide bridge comprising l-ornithine and d-glutamic acid. The predominant menaquinone was MK-9 (H4) and the major fatty acids were anteiso-C15 : 0, C16 : 0, anteiso-C15 : 1 A, anteiso-C17 : 0 and iso-C15 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylinositol dimannoside, unidentified phosphoglycolipid, four unidentified phospholipids and two unidentified polar lipids. Strain LRZ-2T showed a 16S rRNA gene signature pattern consisting of nucleotides at positions 120 (A), 131-231 (C-G), 196 (C), 342-347 (C-G), 444-490 (A-U), 580-761 (C-G), 602-636 (C-G), 670-736 (A-U), 822-878 (G-C), 823-877 (G-C), 826-874 (C-G), 827 (U), 843 (C), 950-1231 (U-A), 1047-1210 (G-C), 1109 (C), 1145 (G), 1309-1328 (G-C), 1361 (G) and 1383 (C), which clearly distinguished it from all genera previously reported in the suborder Micrococcineae. On the basis of the phylogenetic, phenotypic and chemotaxonomic data, strain LRZ-2T is considered to represent a novel species of a new genus, for which the name Pengzhenrongella sicca gen. nov., sp. nov. is proposed. The type strain of Pengzhenrongella sicca is LRZ-2T (=CCTCC AB 2012163T=DSM 100332T).

Molecular cloning and sequence analysis of a mitogen-activated protein kinase gene in the Antarctic yeast Rhodotorula mucilaginosa AN5.

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascades play important roles in various signaling transduction networks of biotic and abiotic stress responses. However, MAPK signaling pathways in cold-active yeast Rhodotorula mucilaginosa have not been reported comprehensively. METHODS AND RESULTS: In the present study, MAPK gene (RmMAPK) was first cloned and characterized from Antarctic sea ice yeast R. mucilaginosa AN5. The full length of the RmMAPK gene is 1086 bp and encodes a 361 amino acids protein with a predicted molecular mass of 40.9 kDa and a pI of 5.25. The RmMAPK contains 11 MAPK conserved subdomains and the phosphorylation motif TGY located in the activation loop of the kinase. Quantitative real-time PCR and western blot assay revealed that the expression and phosphorylation level of RmMAPK up-regulated rapidly and significantly when yeast cells were subjected to low temperature (4 °C), high salinity (120‰ NaCl) and heavy metal (2 mmol/L CuCl2). CONCLUSIONS: All data suggested that the MAPK cascades might act as a key function in response to extreme stresses, such as low temperature, high salinity and heavy metal.

The Fungal Endophyte Epicoccum dendrobii as a Potential Biocontrol Agent Against Colletotrichum gloeosporioides.

Anthracnose caused by Colletotrichum gloeosporioides is one of most serious fungal diseases on Chinese fir (Cunninghamia lanceolata). Eight fungal endophytes were isolated from a young heathy branch of Chinese fir and screened against the pathogen in vitro. One isolate, designated as SMEL1 and subsequently identified as Epicoccum dendrobii based on morphological and phylogenetic analyses, suppressed mycelial growth of Colletotrichum gloeosporioides on dual-culture plates. Additionally, E. dendrobii metabolites significantly decreased the biomass of Colletotrichum gloeosporioides. E. dendrobii was able to enter the internal tissues of the host plant via stomatal cells. Metabolites of E. dendrobii significantly inhibited conidial germination and appressorium formation, which at least partly explained why the endophyte significantly inhibited lesion development caused by Colletotrichum gloeosporioides on various host plants. We further confirmed that some components with antifungal activity could be extracted from E. dendrobii using ethyl acetate as an organic solvent. To our knowledge, this is the first report of E. dendrobii as a potential biocontrol agent against a fungal phytopathogen.

Distribution and environmental risk assessment of trace metals in sludge from multiple sources in Taiwan.

This study evaluated the level of the contaminant of the heavy metals in sludge from different sources and the ecological risk criteria associated with it was also analyzed to establish its reuse in agriculture. The sludge samples were collected from the water plant (WTP), wastewater treatment plant (WWTP), and industrial water treatment plant (IPT) in Taiwan. The inductively coupled plasma mass spectrometry was used to measure the trace metals in sludge. The pollution level and ecological risk criteria for heavy metals in sludge were also used to evaluate its reuse in agriculture. The result shows the average concentrations of trace metals in sludge for three groups (WTP, WWTP, and ITP). Significant correlations were found between concentrations of Zn-Ag (p < 0.001). The higher values of Igeo showed in ITP, indicated Hg to be a major pollutant. In Taiwan, the regulations did not establish the reuse of sludge in agriculture. However, the concentration level of trace metals in sludge was particularly lower than the regular levels in most groups, like WTP and WWTP groups. The industrial sludge was not recommended for the use in agriculture. The results of this study can be used for regular monitoring to establish a reference for sludge management and application to agriculture.

The Covalent and Coordination Co-Driven Assembly of Supramolecular Octahedral Cages with Controllable Degree of Distortion.

Discovering and constructing novel and fancy structures is the goal of many supramolecular chemists. In this work, we propose an assembly strategy based on the synergistic effect of coordination and covalent interactions to construct a set of octahedral supramolecular cages and adjust their degree of distortion. Our strategy innovatively utilizes the addition of sulfur atoms of a metal sulfide synthon, [Et4N][Tp*WS3] (A), to an alkynyl group of a pyridine-containing linker, resulting in a novel vertex with low symmetry, and of Cu(I) ions. By adjusting the length of the linker and the position of the reactive alkynyl group, the control of the deformation degree of the octahedral cages can be realized. These supramolecular cages exhibit enhanced third-order nonlinear optical (NLO) responses. The results offer a powerful strategy to construct novel distorted cage structures as well as control the degree of distortion of supramolecular geometries.

AIM2 inflammasome contributes to brain injury and chronic post-stroke cognitive impairment in mice.

Although over one-third of stroke patients may develop post-stroke cognitive impairment (PSCI), the mechanisms underlying PSCI remain unclear. We explored here, the involvement of post-stroke inflammasomes in long-term PSCI development, using a 45 min-middle cerebral artery occlusion (MCAO)/reperfusion-induced PSCI model. Immunohistological assessment on day 1, 3, and 7 was followed by cognitive function test 28 days post-stroke. Evaluation of inflammasome sensor gene expression in aged mouse brains showed dominant expression of absent in melanoma 2 (Aim2) in 6-, 12-, and 18-month-old mouse brains. AIM2 mRNA and protein increased until 7 days post-stroke. PSCI decreased anxiety in elevated plus maze test and impaired spatial learning and memory functions in Morris water maze test 28 days post-stroke. AIM2 and other inflammasome subunit immunoreactivities, including those for caspase-1, interleukin (IL)-1ß, and IL-18, were higher in the hippocampus and cortex of the PSCI than in those of the sham group 7 days post-stroke. AIM2 immunoreactivity of the PSCI group was primarily co-localized with Iba-1 (microglial marker) and CD31 (endothelial cell marker) immunoreactivities but not NeuN (neuronal marker) and GFAP (astrocyte marker) immunoreactivities, suggesting that microglia or endothelial cell-induced AIM2 production mediated PSCI pathogenesis. Additionally, inflammasome-induced pyroptosis might contribute to acute and chronic neuronal death after stroke. AIM2 knockout (KO) and Ac-YVAD-CMK-induced caspase-1 inhibition in mice significantly improved cognitive function and reversed brain volume in the hippocampus relative to those in stroke mice. Conclusively, AIM2 inflammasome-mediated inflammation and pyroptosis likely aggravated PSCI; therefore, targeting and controlling AIM2 inflammasome could potentially treat PSCI.

Three-Dimensional Wind Measurement Based on Ultrasonic Sensor Array and Multiple Signal Classification.

The wind power industry continues to experience rapid growth worldwide. However, the fluctuations in wind speed and direction complicate the wind turbine control process and hinder the integration of wind power into the electrical grid. To maximize wind utilization, we propose to precisely measure the wind in a three-dimensional (3D) space, thus facilitating the process of wind turbine control. Natural wind is regarded as a 3D vector, whose direction and magnitude correspond to the wind's direction and speed. A semi-conical ultrasonic sensor array is proposed to simultaneously measure the wind speed and direction in a 3D space. As the ultrasonic signal transmitted between the sensors is influenced by the wind and environment noise, a Multiple Signal Classification algorithm is adopted to estimate the wind information from the received signal. The estimate's accuracy is evaluated in terms of root mean square error and mean absolute error. The robustness of the proposed method is evaluated by the type A evaluation of standard uncertainty under a varying signal-to-noise ratio. Simulation results validate the accuracy and anti-noise performance of the proposed method, whose estimated wind speed and direction errors converge to zero when the SNR is over 15 dB.

Antimicrobial Peptides Display Strong Synergy with Vancomycin Against Vancomycin-Resistant E. faecium, S. aureus, and Wild-Type E. coli.

There is an urgent and imminent need to develop new antimicrobials to fight against antibiotic-resistant bacterial and fungal strains. In this study, a checkerboard method was used to evaluate the synergistic effects of the antimicrobial peptide P-113 and its bulky non-nature amino acid substituted derivatives with vancomycin against vancomycin-resistant Enterococcus faecium, Staphylococcus aureus, and wild-type Escherichia coli. Boron-dipyrro-methene (BODIPY) labeled vancomycin was used to characterize the interactions between the peptides, vancomycin, and bacterial strains. Moreover, neutralization of antibiotic-induced releasing of lipopolysaccharide (LPS) from E. coli by the peptides was obtained. Among these peptides, Bip-P-113 demonstrated the best minimal inhibitory concentrations (MICs), antibiotics synergism, bacterial membrane permeabilization, and supernatant LPS neutralizing activities against the bacteria studied. These results could help in developing antimicrobial peptides that have synergistic activity with large size glycopeptides such as vancomycin in therapeutic applications.

Dry and wet seasonal variation of total mercury, inorganic mercury, and methylmercury formation in estuary and harbor sediments.

This study analyzed the seasonal variations and the spatial distributions of total mercury (THg), inorganic divalent mercury (IHg), and methylmercury (MeHg) in sediments of river mouth (RM), main channel (MC), and entrance (E) of the Port of Kaohsiung, Taiwan. The THg, IHg, and MeHg concentrations were, respectively, 198-9130, 2.6-3164, and <0.3-42.6 µg/kg in the wet season and 362-2264, 11.0-790, and 3.3-65.6 µg/kg in the dry season. As for seasonal variations, the concentrations of THg and IHg for RM sediment were higher in the wet season than in the dry season, whereas for MC and E was converse. Generally, MeHg in sediment was higher in the dry season than in the wet season. THg and IHg were mainly transported from the river, whereas MeHg was generated by onsite microbes transforming the local available IHg. Results indicated that the formation of MeHg in sediment may be mainly influenced by the concentration of IHg and seasonal variations.

Purification and characterization of a novel wild-type α-amylase from Antarctic sea ice bacterium Pseudoalteromonas sp. M175.

A novel wild-type α-amylase named wtAmy175 from Pseudoalteromonas sp. M175 strain was purified through ammonium sulphate precipitation, DEAE cellulose, and Sephadex G-75 sequentially (25.83-fold, 7.67%-yield) for biochemical characterization. SDS-PAGE and zymographic activity staining of purified enzyme showed a single band with a predicted molecular mass of about 61 kDa. The optimum temperature and pH for enzyme activity were 30 °C and 7.5, respectively. Additionally, the enzyme exhibited high activity and remarkable stability in 0-10 mM SDS. The values of Km and Vmax for soluble starch as substrate were 2.47 mg/ml and 0.103 mg/ml/min, respectively. Analysis of hydrolysis products of soluble starch and maltooligosaccharides showed that wtAmy175 cleaved the interior and the terminal α-1,4-glycosidic linkage in starch, and had transglycosylation activity. The result of fluorescence spectroscopy showed that wtAmy175 had strong binding affinity with soluble starch. In brief, this study discovered the first wild-type α-amylase so far with several distinctive properties of cold activity, SDS-resistance, and the mixed activity of α-amylase and α-glucosidase, suggesting that wtAmy175 possess high adaptive capability to endure harsh industrial conditions and would be an excellent candidate in detergent and textile industries.

Enhanced insulin production and reprogramming efficiency of mesenchymal stem cells derived from porcine pancreas using suitable induction medium.

BACKGROUND: Genetic reprogramming is a powerful method for altering cell properties and inducing differentiation. However, even if the same gene is reprogrammed, the results vary among cells. Therefore, a better possible strategy involves treating cells with factors that further stimulate differentiation while using stem cells with the same tissue origin. This study aimed to increase induction efficiency and insulin production in reprogrammed cells using a combination of factors that promote cell differentiation. METHODS: Porcine pancreatic cells were cultured to obtain mesenchymal stem cells expressing pancreatic cell-specific markers through sequential passages. The characteristics of these cells were identified, and the M3 gene (Pdx1, Ngn3, MafA) was reprogrammed to induce differentiation into insulin-producing cells. Additionally, the differentiation efficiency of insulin-producing cells was compared by treating reprogrammed cells with a differentiation-promoting factor. RESULTS: Mesenchymal stem cells isolated from porcine pancreatic tissues expressed exocrine cell markers, including amylase and cytokeratin 18, and most cells continuously expressed the beta cell transcription factors Ngn3 and NeuroD. Reprogramming of the M3 gene resulted in differentiation into insulin-producing cells. Moreover, significantly increased insulin and glucagon expressions were observed in the suitable induction medium, and the characteristic beta cell transcription factors Pdx1, Ngn3, and MafA were expressed at levels as high as those in pancreatic islet cells. CONCLUSIONS: Differentiation into insulin-producing cells represents an alternative therapy for insufficient pancreatic islet cells when treating diabetes. Therefore, cells with the characteristics of the target cell should be used to improve differentiation efficiency by creating an environment that promotes reprogramming and differentiation.