Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2.

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

A Resonance Raman Marker Band Characterizes the Slow and Fast Form of Cytochrome c Oxidase.

Cytochrome c oxidase (CcO) in its as-isolated form is known to exist in a slow and fast form, which differ drastically in their ability to bind oxygen and other ligands. While preparation methods have been established that yield either the fast or the slow form of the protein, the underlying structural differences have not been identified yet. Here, we have performed surface enhanced resonance Raman (SERR) spectroscopy of CcO immobilized on electrodes in both forms. SERR spectra obtained in resonance with the heme a3 metal-to-ligand charge transfer (MLCT) transition at 650 nm displayed a sharp vibrational band at 748 or 750 cm-1 when the protein was in its slow or fast form, respectively. DFT calculations identified the band as a mode of the His-419 ligand that is sensitive to the oxygen ligand and the protonation state of Tyr-288 within the binuclear complex. Potential-dependent SERR spectroscopy showed a redox-induced change of this band around 525 mV versus Ag/AgCl exclusively for the fast form, which coincides with the redox potential of the Tyr-O/Tyr-O- transition. Our data points to a peroxide ligand in the resting state of CcO for both forms. The observed frequencies and redox sensitivities of the Raman marker band suggest that a radical Tyr-288 is present in the fast form and a protonated Tyr-288 in the slow form.

Intramolecular Proton Transfer Controls Protein Structural Changes in Phytochrome.

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module. The focus of this work is the Pfr-to-Pr photoconversion of the bathy bacteriophytochrome Agp2 in which Pfr is the thermodynamically stable state. Using spectroscopic techniques, we studied the structural and functional consequences of substituting Arg211, Tyr165, His278, and Phe192 close to the biliverdin (BV) chromophore. In Pfr, substitutions of these residues do not affect the BV structure. The characteristic Pfr properties of bathy phytochromes, including the protonated propionic side chain of ring C (propC) of BV, are preserved. However, replacing Arg211 or Tyr165 blocks the photoconversion in the Meta-F state, prior to the secondary structure transition of the tongue and without deprotonation of propC. The Meta-F state of these variants displays low photochemical activity, but electronic excitation causes ultrafast alterations of the hydrogen bond network surrounding the chromophore. In all variants studied here, thermal back conversion from the photoproducts to Pfr is decelerated but substitution of His278 or Phe192 is not critical for the Pfr-to-Pr photoconversion. These variants do not impair deprotonation of propC or the α-helix/ß-sheet transformation of the tongue during the Meta-F-to-Pr decay. Thus, we conclude that propC deprotonation is essential for restructuring of the tongue.

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria, using α-humulene as a model compound.

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden-Meyerhof-Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.

Genome-scale evaluation of core one-carbon metabolism in gammaproteobacterial methanotrophs grown on methane and methanol.

Methylotuvimicrobium alcaliphilum 20Z is a promising platform strain for bioconversion of one-carbon (C1) substrates into value-added products. To carry out robust metabolic engineering with methylotrophic bacteria and to implement C1 conversion machinery in non-native hosts, systems-level evaluation and understanding of central C1 metabolism in methanotrophs under various conditions is pivotal but yet elusive. In this study, a genome-scale integrated approach was used to provide in-depth knowledge on the metabolic pathways of M. alcaliphilum 20Z grown on methane and methanol. Systems assessment of core carbon metabolism indicated the methanol assimilation pathway is mostly coupled with the efficient Embden-Meyerhof-Parnas (EMP) pathway along with the serine cycle. In addition, an incomplete TCA cycle operated in M. alcaliphilum 20Z on methanol, which might only supply precursors for de novo synthesis but not reducing powers. Instead, it appears that the direct formaldehyde oxidation pathway supply energy for the whole metabolic system. Additionally, a comparative transcriptomic analysis in multiple gammaproteobacterial methanotrophs also revealed the transcriptional responses of central metabolism on carbon substrate change. These findings provided a systems-level understanding of carbon metabolism and new opportunities for strain design to produce relevant products from different C1-feedstocks.

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z.

BACKGROUND: Methanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product. RESULTS: In this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain. CONCLUSIONS: This study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

Role of the Propionic Side Chains for the Photoconversion of Bacterial Phytochromes.

Bacteriophytochromes harboring a biliverdin IXα (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analyzed the role of propionic substituents of BV in the transformations between parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from Agrobacterium fabrum. Both proteins form adducts with BV monoesters (BVM), esterified at propionic side chain B (PsB) or C (PsC), but in each case, only one monoester adduct is reactive. In the reactive Agp2-BVM-B complex (esterified at ring B), the Pfr dark state displays the structural properties characteristic of bathy phytochromes, including a protonated PsC. As in native Agp2, PsC is deprotonated in the final step of the Pfr phototransformation. However, the concomitant α-helix/ß-sheet secondary structure change of the tongue is blocked at the stage of unfolding of the coiled loop region. This finding and the shift of the tautomeric equilibrium of BVM toward the enol form are attributed to the drastic changes in the electrostatic potential. The calculations further suggest that deprotonation of PsC and the protonation state of His278 control the reactivity of the enol tautomer, thereby accounting for the extraordinarily slow thermal reversion. Although strong perturbations of the electrostatic potential are also found for Agp1-BVM, the consequences for the Pr-to-Pfr phototransformation are less severe. Specifically, the structural transition of the tongue is not impaired and thermal reversion is even accelerated. The different response of Agp1 and Agp2 to monoesterification of BV points to different photoconversion mechanisms.

A comparative transcriptome analysis of the novel obligate methanotroph Methylomonas sp. DH-1 reveals key differences in transcriptional responses in C1 and secondary metabolite pathways during growth on methane and methanol.

BACKGROUND: Methanotrophs play an important role in biotechnological applications, with their ability to utilize single carbon (C1) feedstock such as methane and methanol to produce a range of high-value compounds. A newly isolated obligate methanotroph strain, Methylomonas sp. DH-1, became a platform strain for biotechnological applications because it has proven capable of producing chemicals, fuels, and secondary metabolites from methane and methanol. In this study, transcriptome analysis with RNA-seq was used to investigate the transcriptional change of Methylomonas sp. DH-1 on methane and methanol. This was done to improve knowledge about C1 assimilation and secondary metabolite pathways in this promising, but under-characterized, methane-bioconversion strain. RESULTS: We integrated genomic and transcriptomic analysis of the newly isolated Methylomonas sp. DH-1 grown on methane and methanol. Detailed transcriptomic analysis indicated that (i) Methylomonas sp. DH-1 possesses the ribulose monophosphate (RuMP) cycle and the Embden-Meyerhof-Parnas (EMP) pathway, which can serve as main pathways for C1 assimilation, (ii) the existence and the expression of a complete serine cycle and a complete tricarboxylic acid (TCA) cycle might contribute to methane conversion and energy production, and (iii) the highly active endogenous plasmid pDH1 may code for essential metabolic processes. Comparative transcriptomic analysis on methane and methanol as a sole carbon source revealed different transcriptional responses of Methylomonas sp. DH-1, especially in C1 assimilation, secondary metabolite pathways, and oxidative stress. Especially, these results suggest a shift of central metabolism when substrate changed from methane to methanol in which formaldehyde oxidation pathway and serine cycle carried more flux to produce acetyl-coA and NADH. Meanwhile, downregulation of TCA cycle when grown on methanol may suggest a shift of its main function is to provide de novo biosynthesis, but not produce NADH. CONCLUSIONS: This study provides insights into the transcriptomic profile of Methylomonas sp. DH-1 grown on major carbon sources for C1 assimilation, providing in-depth knowledge on the metabolic pathways of this strain. These observations and analyses can contribute to future metabolic engineering with the newly isolated, yet under-characterized, Methylomonas sp. DH-1 to enhance its biochemical application in relevant industries.

Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane.

Methane is considered a next-generation feedstock, and methanotrophic cell-based biorefinery is attractive for production of a variety of high-value compounds from methane. In this work, we have metabolically engineered Methylomicrobium alcaliphilum 20Z for 2,3-butanediol (2,3-BDO) production from methane. The engineered strain 20Z/pBudK.p, harboring the 2,3-BDO synthesis gene cluster (budABC) from Klebsiella pneumoniae, accumulated 2,3-BDO in methane-fed shake flask cultures with a titer of 35.66 mg/L. Expression of the most efficient gene cluster was optimized using selection of promoters, translation initiation rates (TIR), and the combination of 2,3-BDO synthesis genes from different sources. A higher 2,3-BDO titer of 57.7 mg/L was measured in the 20Z/pNBM-Re strain with budA of K. pneumoniae and budB of Bacillus subtilis under the control of the Tac promoter. The genome-scale metabolic network reconstruction of M. alcaliphilum 20Z enabled in silico gene knockout predictions using an evolutionary programming method to couple growth and 2,3-BDO production. The ldh, ack, and mdh genes in M. alcaliphilum 20Z were identified as potential knockout targets. Pursuing these targets, a triple-mutant strain ∆ldh ∆ack ∆mdh was constructed, resulting in a further increase of the 2,3-BDO titer to 68.8 mg/L. The productivity of this optimized strain was then tested in a fed-batch stirred tank bioreactor, where final product concentrations of up to 86.2 mg/L with a yield of 0.0318 g-(2,3-BDO) /g-CH4 were obtained under O2-limited conditions. This study first demonstrates the strategy of in silico simulation-guided metabolic engineering and represents a proof-of-concept for the production of value-added compounds using systematic approaches from engineered methanotrophs.

Biological conversion of methane to chemicals and fuels: technical challenges and issues.

Methane is a promising next-generation carbon feedstock for industrial biotechnology due to its low price and huge availability. Biological conversion of methane to valuable products can mitigate methane-induced global warming as greenhouse gas. There have been challenges for the conversion of methane into various chemicals and fuels using engineered non-native hosts with synthetic methanotrophy or methanotrophs with the reconstruction of synthetic pathways for target products. Herein, we analyze the technical challenges and issues of potent methane bioconversion technology. Pros and cons of metabolic engineering of methanotrophs for methane bioconversion, and perspectives on the bioconversion of methane to chemicals and liquid fuels are discussed.

Influence of Cavity Lining on the 3-Year Clinical Outcome of Posterior Composite Restorations: A Randomized Controlled Clinical Trial.

The purpose of this randomized, split-mouth-designed controlled and single-blinded clinical study was to evaluate the 3-year clinical performance of Class I and Class II resin composite restorations placed with or without cavity lining with a flowable composite. Fifty patients with treatment needs in two premolars or molars were included. One of the teeth was restored using the nanohybrid composite (Grandio®SO, control group), in the test group a high viscosity flowable composite was additionally applied as a first layer. In both groups, the same self-etch adhesive system was applied. Clinical evaluation after 3 years was carried out using the modified USPHS/Ryge criteria. At the 3-year follow-up the recall rate was 92%. Four restorations failed in the test group (8.7%), three due to the loss of vitality and one after fracture. The control group exhibited a cumulative success rate of 100%, while the test group achieved a success rate of 91.3%. This led to significant differences in the annual failure rate (AFR) between the two groups, with rates of 0% and 2.9% (p < 0.05; Mann-Whitney U-test). After 3 years the cumulative survival rate including all restorations was 95.7%. Statistical analysis revealed significant differences for the parameters: tooth vitality, marginal discoloration, success rate, and AFR. The other parameters exhibited no significant differences. Consequently, the nanohybrid composite demonstrated excellent performance over a 3-year period, whereas the utilization of a flowable composite for the cavity lining did not appear to exert a beneficial influence on clinical outcomes.

Spatio-temporal characterization of drought variability in data-scarce regions using global precipitation data: a case study in Cauto river basin, Cuba.

Drought is considered the most severe water-related disaster in the Cauto river basin, which is the longest river and the main agricultural producer in Cuba. Better understanding of drought characteristics is crucial to drought management. Given the sparsity of ground-based precipitation observations in the Cauto, this study aims at using gridded global precipitation to analyze the spatio-temporal variations of drought in this river basin. Firstly, the monthly Climate Hazards Group InfraRed Precipitation with Station data (CHIRPS) was calibrated with the gauged precipitation using the Thiessen polygon-based method and linear least squares regression equations. Then, the gridded standardized precipitation index (SPI) with time scales of 3, 6, 9 months and drought characteristics, namely, drought frequency, duration and intensity were calculated using the calibrated CHIRPS. Finally, the spatio-temporal analysis was performed to investigate the variations of drought in the Cauto river basin in time and space. The obtained results show that the calibrated CHIRPS is highly consistent with the gauged observations and is capable of determining the magnitude, time, and spatial extent of drought events in the Cauto river basin. The trend analysis by the Mann-Kendall test reveals that although the trend is not statistically significant, the SPI tends to decrease with time in the dry season, which indicates the more severe drought. The spatial analysis indicates that the lower altitude area of the Cauto river basin is suffered from longer drought duration and higher drought intensity than the upper one. This study expresses the importance of open global precipitation data sources in monitoring and quantifying drought characteristics in data-scarce regions.

Understanding and tackling meat reduction in different cultural contexts: a segmentation study of Swiss and Vietnamese consumers.

Objective: This study aims to disclose and compare meat consumer segments in Switzerland and Vietnam, which differ in terms of their socioeconomic and cultural settings (the former is a developed country, and the latter is an emerging one) to develop a set of segment-specific recommendations that might be applied to consumption in comparable contexts, that is, in other developed countries and other emerging economies. Methods: Data were collected through two online surveys: one for Swiss residents from randomly selected households and one for Vietnamese urban residents recruited via snowball sampling. The final sample size was N = 643 for Switzerland and N = 616 for Vietnam. Hierarchical cluster analyses followed by K-means cluster analyses revealed five distinct clusters in both countries. Results: Three clusters were common to both countries: meat lovers (21% in Switzerland and 19% in Vietnam), proactive consumers (22% in Switzerland and 14% in Vietnam) and suggestible consumers (19% in Switzerland and 25% in Vietnam). Two were specific to each country, namely traditional (19%) and basic (21%) consumers in Switzerland and confident (16%) and anxious (26%) consumers in Vietnam. Conclusion: Relying on voluntary actions, nudging techniques, private initiatives and consumers' sense of responsibility will certainly be useful but will nevertheless be insufficient to achieve a planetary health diet within the given timeframe (the 2030 Agenda for Sustainable Development). Governments will have no choice but to activate all levers within their sphere of influence - including regulatory measures - and oblige private sector actors to commit to the measures imposed on them. A binding international agenda with common objectives and measures is a judicious approach. Unlike most previous studies, which focused on meat consumption intensity and frequency or diet type to segment consumers, our approach, based on psychographic profiles, allows the identification of segments that share common drivers and barriers and thus the development of better-targeted measures to reduce meat consumption.

Socio-behavioural factors influencing meat intake and meat reduction intention in Vietnam and Switzerland.

Meat consumption is declining in developed countries but increasing in emerging countries. This study, for the first time, compares the socio-behavioural factors influencing individuals' meat consumption level and meat reduction intention between Vietnam, an emerging economy and Switzerland, a developed country. Online consumer surveys were conducted in late 2022, yielding 552 usable replies from Switzerland and 592 from Vietnam for this study. Drawing upon an extended Protection Motivation Theory and using structural equation modelling, we found similarities as well as differences in the determinants of meat consumption behaviour. Perceived health risks of meat overconsumption, self-efficacy of meat reduction, attitude toward ethical and environmental issues, and pressure from family members' reluctance to change diet drove the intention to reduce meat in both countries. Meat attachment emerges as the most important determinant of meat consumption level in not only Switzerland but also Vietnam and thus presents the largest barrier to meat reduction. The association between response cost of eating less meat and intention to reduce meat was negative in Switzerland but positive in Vietnam. Self-efficacy of meat consumption reduction influenced meat consumption level solely in Switzerland. Ethical and environmental attitudes significantly facilitated meat reduction intention of Swiss respondents only, reflecting cultural differences. Policy implications were discussed.

Surgical outcomes of premeatal and retromeatal cerebellopontine angle meningioma in Vietnam: a single-center prospective cross-sectional study.

Cerebellopontine angle (CPA) meningiomas are the second most common tumor of the CPA. Depending on the site of dural attachment, the relationship between the tumor and critical neurovascular structures of the CPA is variable. This study aims to evaluate the influence of CPA meningioma location in relation to the internal auditory canal (IAC) on clinical symptoms, radiological presentations, and surgical treatments and outcomes which has been rarely reported in Vietnam. Patients and methods: A prospective study on 33 patients treated with microsurgery from August 2020 to May 2022 at the Neurosurgery Center, Viet Duc University Hospital. Results: The mean age of 27 females (85%) and 6 (15%) males was 54±12 years. Based on their location to the IAC, there were 16 premeatal cases (49%) (anterior to the IAC) and 17 retromeatal cases (15%) (posterior to the IAC). The time of diagnosis of the retromeatal group was later (16.5 vs. 9.7 months), the average tumor size of the 2 groups was not different, but when there was brainstem compression, the average tumor size of retromeatal group was larger (49 vs. 44 mm). The clinical presentations of the retromeatal group were related to the cerebellar symptoms, while trigeminal neuropathy symptoms all came from the premeatal group. Gross total resection of the premeatal group was 31% and of the retrometal group was 71%. The results of preserving the facial nerve function of the premeatal group were lower (44 vs. 82%). Postoperative Karnofsky score of the retromeatal group improved, while the premeatal group did not change. Conclusions: Classification of CPA meningiomas according to their location to the IAC plays an important role in diagnosis and treatment, affecting clinical symptoms, surgical strategy as well as surgical outcomes.

The Interaction of Two Widely Used Endodontic Irrigants, Chlorhexidine and Sodium Hypochlorite, and Its Impact on the Disinfection Protocol during Root Canal Treatment.

In recent years, sodium hypochlorite and chlorhexidine digluconate have been the gold standard of irrigation solutions utilized within the disinfection protocol during root canal treatments. Nowadays, it is known that, during chemical disinfection of the root canal, consecutive application of sodium hypochlorite and chlorhexidine digluconate leads to the formation of an orange-brown precipitate. This precipitate is described as being chemically similar to para-chloroaniline, which is suspected to have cytotoxic and carcinogenic effects. Concerns also exist regarding its influence on the leakage of root canal fillings, coronal restorations, and tooth discoloration. The purpose of this article is to review the literature on the interaction of sodium hypochlorite and chlorhexidine digluconate on the tooth and its surrounding tissues, and to discuss the effect of the precipitate formed during root canal treatment. We further address options to avoid the formation of the precipitate and describe alternative irrigation solutions that should not interact with sodium hypochlorite or chlorhexidine digluconate.

Influence of Ascorbic Acid as a Growth and Differentiation Factor on Dental Stem Cells Used in Regenerative Endodontic Therapies.

BACKGROUND: Vitamin C is one of the major extracellular nonenzymatic antioxidants involved in the biosynthesis of collagen. It promotes the growth of fibroblasts, wound healing processes, and enhances the survival and differentiation of osteoblasts. The potential effects of ascorbic acid on human dental pulp cells (DPC) and the cells of the apical papilla (CAP) used in actual regenerative endodontic procedures remain largely unknown. In this study, we investigated the possible employment of ascorbic acid in the differentiation and regenerative therapies of DPC and CAP. METHODS: Nine extracted human wisdom teeth were selected for this study. Subpopulations of stem cells within DPC and CAP were sorted with the mesenchymal stem cell marker STRO-1, followed by treatments with different concentrations (0 mM, 0.1 mM, 0.5 mM, and 1.0 mM) of ascorbic acid (AA), RT-PCR, and Western blot analysis. RESULTS: FACS analysis revealed the presence of cell subpopulations characterized by a strong expression of mesenchymal stem cell marker STRO-1 and dental stem cell markers CD105, CD44, CD146, CD90, and CD29. Treatment of the cells with defined amounts of AA revealed a markedly increased expression of proliferation marker Ki-67, especially in the concentration range between 0.1 mM and 0.5 mM. Further investigations demonstrated that treatment with AA led to significantly increased expression of common stem cell markers OCT4, Nanog, and Sox2. The most potent proliferative and expressional effects of AA were observed in the concentration of 0.1 mM. CONCLUSIONS: AA might be a novel and potent growth promoter of human dental cells. Increasing the properties of human dental pulp cells and the cells of the apical papilla using AA could be a useful factor for further clinical developments of regenerative endodontic procedures.

Boosting the acetol production in methanotrophic biocatalyst Methylomonas sp. DH-1 by the coupling activity of heteroexpressed novel protein PmoD with endogenous particulate methane monooxygenase.

BACKGROUND: Methylacidiphilum sp. IT6 has been validated its C3 substrate assimilation pathway via acetol as a key intermediate using the PmoCAB3, a homolog of the particulate methane monooxygenase (pMMO). From the transcriptomic data, the contribution of PmoD of strain IT6 in acetone oxidation was questioned. Methylomonas sp. DH-1, a type I methanotroph containing pmo operon without the existence of its pmoD, has been deployed as a biocatalyst for the gas-to-liquid bioconversion of methane and propane to methanol and acetone. Thus, Methylomonas sp. DH-1 is a suitable host for investigation. The PmoD-expressed Methylomonas sp. DH-1 can also be deployed for acetol production, a well-known intermediate for various industrial applications. Microbial production of acetol is a sustainable approach attracted attention so far. RESULTS: In this study, bioinformatics analyses elucidated that novel protein PmoD is a C-terminal transmembrane-helix membrane with the proposed function as a transport protein. Furthermore, the whole-cell biocatalyst was constructed in Methylomonas sp. DH-1 by co-expression the PmoD of Methylacidiphilum sp. IT6 with the endogenous pMMO to enable acetone oxidation. Under optimal conditions, the maximum accumulation, and specific productivity of acetol were 18.291 mM (1.35 g/L) and 0.317 mmol/g cell/h, respectively. The results showed the first coupling activity of pMMO with a heterologous protein PmoD, validated the involvement of PmoD in acetone oxidation, and demonstrated an unprecedented production of acetol from acetone in type I methanotrophic biocatalyst. From the data achieved in batch cultivation conditions, an assimilation pathway of acetone via acetol as the key intermediate was also proposed. CONCLUSION: Using bioinformatics tools, the protein PmoD has been elucidated as the membrane protein with the proposed function as a transport protein. Furthermore, results from the assays of PmoD-heteroexpressed Methylomonas sp. DH-1 as a whole-cell biocatalyst validated the coupling activity of PmoD with pMMO to convert acetone to acetol, which also unlocks the potential of this recombinant biocatalyst for acetol production. The proposed acetone-assimilated pathway in the recombinant Methylomonas sp. DH-1, once validated, can extend the metabolic flexibility of Methylomonas sp. DH-1.

Single-Image 3-D Reconstruction: Rethinking Point Cloud Deformation.

Single-image 3-D reconstruction has long been a challenging problem. Recent deep learning approaches have been introduced to this 3-D area, but the ability to generate point clouds still remains limited due to inefficient and expensive 3-D representations, the dependency between the output and the number of model parameters, or the lack of a suitable computing operation. In this article, we present a novel deep-learning-based method to reconstruct a point cloud of an object from a single still image. The proposed method can be decomposed into two steps: feature fusion and deformation. The first step extracts both global and point-specific shape features from a 2-D object image, and then injects them into a randomly generated point cloud. In the second step, which is deformation, we introduce a new layer termed as GraphX that considers the interrelationship between points like common graph convolutions but operates on unordered sets. The framework can be applicable to realistic image data with background as we optionally learn a mask branch to segment objects from input images. To complement the quality of point clouds, we further propose an objective function to control the point uniformity. In addition, we introduce different variants of GraphX that cover from best performance to best memory budget. Moreover, the proposed model can generate an arbitrary-sized point cloud, which is the first deep method to do so. Extensive experiments demonstrate that we outperform the existing models and set a new height for different performance metrics in single-image 3-D reconstruction.

Ultrafast proton-coupled isomerization in the phototransformation of phytochrome.

The biological function of phytochromes is triggered by an ultrafast photoisomerization of the tetrapyrrole chromophore biliverdin between two rings denoted C and D. The mechanism by which this process induces extended structural changes of the protein is unclear. Here we report ultrafast proton-coupled photoisomerization upon excitation of the parent state (Pfr) of bacteriophytochrome Agp2. Transient deprotonation of the chromophore's pyrrole ring D or ring C into a hydrogen-bonded water cluster, revealed by a broad continuum infrared band, is triggered by electronic excitation, coherent oscillations and the sudden electric-field change in the excited state. Subsequently, a dominant fraction of the excited population relaxes back to the Pfr state, while ~35% follows the forward reaction to the photoproduct. A combination of quantum mechanics/molecular mechanics calculations and ultrafast visible and infrared spectroscopies demonstrates how proton-coupled dynamics in the excited state of Pfr leads to a restructured hydrogen-bond environment of early Lumi-F, which is interpreted as a trigger for downstream protein structural changes.