Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.

γ-Glutamylation of Isopropylamine by Fermentation.

Glutamylation yields N-functionalized amino acids in several natural pathways. γ-Glutamylated amino acids may exhibit improved properties for their industrial application, e. g., as taste enhancers or in peptide drugs. γ-Glutamyl-isopropylamide (GIPA) can be synthesized from isopropylamine (IPA) and l-glutamate. In Pseudomonas sp. strain KIE171, GIPA is an intermediate in the biosynthesis of l-alaninol (2-amino-1-propanol), a precursor of the fluorochinolone antibiotic levofloxacin and of the chloroacetanilide herbicide metolachlor. In this study, fermentative production of GIPA with metabolically engineered Pseudomonas putida KT2440 using γ-glutamylmethylamide synthetase (GMAS) from Methylorubrum extorquens was established. Upon addition of IPA during growth with glycerol as carbon source in shake flasks, the recombinant strain produced up to 21.8 mM GIPA. In fed-batch bioreactor cultivations, GIPA accumulated to a titer of 11 g L-1 with a product yield of 0.11 g g-1 glycerol and a volumetric productivity of 0.24 g L-1 h-1 . To the best of our knowledge, this is the first fermentative production of GIPA.

Graph-based sequential beamforming.

This paper presents a Bayesian estimation method for sequential direction finding. The proposed method estimates the number of directions of arrivals (DOAs) and their DOAs performing operations on the factor graph. The graph represents a statistical model for sequential beamforming. At each time step, belief propagation predicts the number of DOAs and their DOAs using posterior probability density functions (pdfs) from the previous time and a different Bernoulli-von Mises state transition model. Variational Bayesian inference then updates the number of DOAs and their DOAs. The method promotes sparse solutions through a Bernoulli-Gaussian amplitude model, is gridless, and provides marginal posterior pdfs from which DOA estimates and their uncertainties can be extracted. Compared to nonsequential approaches, the method can reduce DOA estimation errors in scenarios involving multiple time steps and time-varying DOAs. Simulation results demonstrate performance improvements compared to state-of-the-art methods. The proposed method is evaluated using ocean acoustic experimental data.

Bayesian detection and tracking of odontocetes in 3-D from their echolocation clicks.

Localization and tracking of marine animals can reveal key insights into their behaviors underwater that would otherwise remain unexplored. A promising nonintrusive approach to obtaining location information of marine animals is to process their bioacoustic signals, which are passively recorded using multiple hydrophones. In this paper, a data processing chain that automatically detects and tracks multiple odontocetes (toothed whales) in three dimensions (3-D) from their echolocation clicks recorded with volumetric hydrophone arrays is proposed. First, the time-difference-of-arrival (TDOA) measurements are extracted with a generalized cross-correlation that whitens the received acoustic signals based on the instrument noise statistics. Subsequently, odontocetes are tracked in the TDOA domain using a graph-based multi-target tracking (MTT) method to reject false TDOA measurements and close gaps of missed detections. The resulting TDOA estimates are then used by another graph-based MTT stage that estimates odontocete tracks in 3-D. The tracking capability of the proposed data processing chain is demonstrated on real acoustic data provided by two volumetric hydrophone arrays that recorded echolocation clicks from Cuvier's beaked whales (Ziphius cavirostris). Simulation results show that the presented MTT method using 3-D can outperform an existing approach that relies on manual annotation.

Automating multi-target tracking of singing humpback whales recorded with vector sensors.

Passive acoustic monitoring is widely used for detection and localization of marine mammals. Typically, pressure sensors are used, although several studies utilized acoustic vector sensors (AVSs), that measure acoustic pressure and particle velocity and can estimate azimuths to acoustic sources. The AVSs can localize sources using a reduced number of sensors and do not require precise time synchronization between sensors. However, when multiple animals are calling concurrently, automated tracking of individual sources still poses a challenge, and manual methods are typically employed to link together sequences of measurements from a given source. This paper extends the method previously reported by Tenorio-Hallé, Thode, Lammers, Conrad, and Kim [J. Acoust. Soc. Am. 151(1), 126-137 (2022)] by employing and comparing two fully-automated approaches for azimuthal tracking based on the AVS data. One approach is based on random finite set statistics and the other on message passing algorithms, but both approaches utilize the underlying Bayesian statistical framework. The proposed methods are tested on several days of AVS data obtained off the coast of Maui and results show that both approaches successfully and efficiently track multiple singing humpback whales. The proposed methods thus made it possible to develop a fully-automated AVS tracking approach applicable to all species of baleen whales.

From Aquaculture to Aquaculture: Production of the Fish Feed Additive Astaxanthin by Corynebacterium glutamicum Using Aquaculture Sidestream.

Circular economy holds great potential to minimize the use of finite resources, and reduce waste formation by the creation of closed-loop systems. This also pertains to the utilization of sidestreams in large-scale biotechnological processes. A flexible feedstock concept has been established for the industrially relevant Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin. In this study, we aimed to use a preprocessed aquaculture sidestream for production of carotenoids, including the fish feed ingredient astaxanthin by C. glutamicum. The addition of a preprocessed aquaculture sidestream to the culture medium did not inhibit growth, obviated the need for addition of several components of the mineral salt's medium, and notably enhanced production of astaxanthin by an engineered C. glutamicum producer strain. Improved astaxanthin production was scaled to 2 L bioreactor fermentations. This strategy to improve astaxanthin production was shown to be transferable to production of several native and non-native carotenoids. Thus, this study provides a proof-of-principle for improving carotenoid production by C. glutamicum upon supplementation of a preprocessed aquaculture sidestream. Moreover, in the case of astaxanthin production it may be a potential component of a circular economy in aquaculture.

DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-ß-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-ß-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

Probabilistic focalization for shallow water localization.

Localizing and tracking an underwater acoustic source is a key task for maritime situational awareness. This paper presents a sequential Bayesian estimation method for passive acoustic source localization in shallow water. The proposed probabilistic focalization approach associates detected directions of arrival (DOAs) to modeled DOAs and jointly estimates the time-varying source location. Embedded ray tracing makes it possible to incorporate environmental parameters that characterize the acoustic waveguide. Due to its statistical model, the proposed method can provide robustness in scenarios with severe environmental uncertainty. We demonstrate performance advantages compared to matched field processing using data collected during the SWellEx-96 experiment.

Sequential sparse Bayesian learning for time-varying direction of arrival.

This paper presents methods for the estimation of the time-varying directions of arrival (DOAs) of signals emitted by moving sources. Following the sparse Bayesian learning (SBL) framework, prior information of unknown source amplitudes is modeled as a multi-variate Gaussian distribution with zero-mean and time-varying variance parameters. For sequential estimation of the unknown variance, we present two sequential SBL-based methods that propagate statistical information across time to improve DOA estimation performance. The first method heuristically calculates the parameters of an inverse-gamma hyperprior based on the source signal estimate from the previous time step. In addition, a second sequential SBL method is proposed, which performs a prediction step to calculate the prior distribution of the current variance parameter from the variance parameter estimated at the previous time step. The SBL-based sequential processing provides high-resolution DOA tracking capabilities. Performance improvements are demonstrated by using simulated data as well as real data from the SWellEx-96 experiment.

Simultaneous Production of Psilocybin and a Cocktail of ß-Carboline Monoamine Oxidase Inhibitors in "Magic" Mushrooms.

The psychotropic effects of Psilocybe "magic" mushrooms are caused by the l-tryptophan-derived alkaloid psilocybin. Despite their significance, the secondary metabolome of these fungi is poorly understood in general. Our analysis of four Psilocybe species identified harmane, harmine, and a range of other l-tryptophan-derived ß-carbolines as their natural products, which was confirmed by 1D and 2D NMR spectroscopy. Stable-isotope labeling with 13 C11 -l-tryptophan verified the ß-carbolines as biosynthetic products of these fungi. In addition, MALDI-MS imaging showed that ß-carbolines accumulate toward the hyphal apices. As potent inhibitors of monoamine oxidases, ß-carbolines are neuroactive compounds and interfere with psilocybin degradation. Therefore, our findings represent an unprecedented scenario of natural product pathways that diverge from the same building block and produce dissimilar compounds, yet contribute directly or indirectly to the same pharmacological effects.

Tracking of multiple surface vessels based on passive acoustic underwater arrays.

This paper introduces an approach to localize and track an unknown number of non-cooperative surface vessels based on passive acoustic sensing of their noise radiated underwater. Time-Difference of Arrival (TDOA) measurements are extracted from pairs of hydrophones, and a recently introduced Bayesian framework for multi-object tracking is employed to detect and track vessels from TDOA measurements. Results based on data from a three-dimensional compact hydrophone array towed by an autonomous vehicle confirm that non-cooperative surface vessels can be detected and tracked.

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre-Fertilization Processes by Quantitative Proteomics.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable-isotope dimethyl labeling prior to nanoLC-MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N-glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

A proteomic analysis of ferulic acid metabolism in Amycolatopsis sp. ATCC 39116.

The pseudonocardiate Amycolatopsis sp. ATCC 39116 is used for the biotechnical production of natural vanillin from ferulic acid. Our laboratory has performed genetic modifications of this strain previously, but there are still many gaps in our knowledge regarding its vanillin tolerance and the general metabolism. We performed cultivations with this bacterium and compared the proteomes of stationary phase cells before ferulic acid feeding with those during ferulic acid feeding. Thereby, we identified 143 differently expressed proteins. Deletion mutants were constructed and characterized to analyze the function of nine corresponding genes. Using these mutants, we identified an active ferulic acid ß-oxidation pathway and the enzymes which constitute this pathway. A combined deletion mutant in which the ß-oxidation as well as non-ß-oxidation pathways of ferulic acid degradation were deleted was unable to grow on ferulic acid as the sole source of carbon and energy. This mutant differs from the single deletion mutants and was unable to grow on ferulic acid. Furthermore, we showed that the non-ß-oxidation pathway is involved in caffeic acid degradation; however, its deletion is complemented even in the double deletion mutant. This shows that both pathways can complement each other. The ß-oxidation deletion mutant produced significantly reduced amounts of vanillic acid (0.12 instead of 0.35 g/l). Therefore, the resulting mutant could be used as an improved production strain. The quinone oxidoreductase deletion mutant (ΔytfG) degraded ferulic acid slower at first but produced comparable amounts of vanillin and significantly less vanillyl alcohol when compared to the parent strain.

Development of an Improved System for the Generation of Knockout Mutants of Amycolatopsis sp. Strain ATCC 39116.

The Gram-positive actinomycete Amycolatopsis sp. strain ATCC 39116 is used for the industrial production of natural vanillin. Previously, the only gene deletion performed in this strain targeted the gene vdh, coding for a vanillin dehydrogenase. The generation of this mutant suffered from a high number of illegitimate recombinations and a low rate of homologous recombination. To alleviate this, we constructed an optimized deletion system based on a modified suicide vector. Thereby, we were able to increase the rate of homologous integration from less than 1% of the analyzed clones to 20% or 50%, depending on the targeted gene. We were furthermore able to reduce the screening effort needed to identify homogenotes through the use of the rpsL gene from Saccharopolyspora erythraea, which confers streptomycin sensitivity on clones still carrying the suicide vector. The new suicide vector is p6SUI5ERPSL, and its applicability was demonstrated by the deletion of three Amycolatopsis gene clusters. The deletion of the first of the gene clusters, coding for an aldehyde oxidase (yagRST), led to no altered phenotype compared to the parent strain; deletion of the second, coding for a vanillic acid decarboxylase (vdcBCD), led to a phenotype that was strongly impaired in its growth with vanillic acid as the sole carbon source and also unable to form guaiacol; and deletion of the third, coding for a vanillate demethylase (vanAB), led to only a negligible impact in comparison. Therefore, we showed that decarboxylation of vanillic acid is the main degradation pathway in Amycolatopsis sp. ATCC 39116 while the demethylation plays only a minor role and does not compensate the deletion of vdcBCD IMPORTANCE: Amycolatopsis sp. ATCC 39116 is an important microorganism used for the production of natural vanillin from ferulic acid. In contrast to this importance, it has previously been shown that this strain is hard to manipulate on a genetic level. We therefore generated an optimized system to facilitate the deletion of genes in this strain. This allowed us to greatly reduce the time and work requirements for generating deletions. This could allow the improvement of vanillin production in the future and also the elucidation of metabolic pathways. To test our deletion system, we deleted three gene clusters in Amycolatopsis sp. ATCC 39116. One showed no involvement in the metabolism of vanillin, while the second proved to be the main pathway of vanillic acid degradation and completely stopped the formation of the off-flavor guaiacol. The third appeared to have only a negligible impact on the degradation of vanillic acid.

Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin.

UNLABELLED: The Gram-positive bacterium Amycolatopsis sp. ATCC 39116 is used for the fermentative production of natural vanillin from ferulic acid on an industrial scale. The strain is known for its outstanding tolerance to this toxic product. In order to improve the productivity of the fermentation process, the strain's metabolism was engineered for higher final concentrations and molar yields. Degradation of vanillin could be decreased by more than 90% through deletion of the vdh gene, which codes for the central vanillin catabolism enzyme, vanillin dehydrogenase. This mutation resulted in improvement of the final concentration of vanillin by more than 2.2 g/liter, with a molar yield of 80.9%. Further improvement was achieved with constitutive expression of the vanillin anabolism genes ech and fcs, coding for the enzymes feruloyl-coenzyme A (CoA) synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech). The transcription of both genes was shown to be induced by ferulic acid, which explains the unwanted adaptation phase in the fermentation process before vanillin was efficiently produced by the wild-type cells. Through the constitutive and enhanced expression of the two genes, the adaptation phase was eliminated and a final vanillin concentration of 19.3 g/liter, with a molar yield of 94.9%, was obtained. Moreover, an even higher final vanillin concentration of 22.3 g/liter was achieved, at the expense of a lower molar yield, by using an improved feeding strategy. This is the highest reported vanillin concentration reached in microbial fermentation processes without extraction of the product. Furthermore, the vanillin was produced almost without by-products, with a molar yield that nearly approached the theoretical maximum. IMPORTANCE: Much effort has been put into optimization of the biotechnological production of natural vanillin. The demand for this compound is growing due to increased consumer concerns regarding chemically produced food additives. Since this compound is toxic to most organisms, it has proven quite difficult to reach high concentrations and molar yields. This study shows that improvements in the final vanillin concentrations and molar yields can be made through a combination of modification of the fermentation parameters and molecular strain engineering, without the need for methods such as continuous extraction from the fermentation broth. Using this approach, we were able to reach a final vanillin concentration of 22.3 g/liter, which is the highest vanillin concentration reported to date that was generated with Amycolatopsis sp. ATCC 39116 without additional extraction of the toxic product.

Regioselective Dichlorination of a Non-Activated Aliphatic Carbon Atom and Phenolic Bismethylation by a Multifunctional Fungal Flavoenzyme.

The regioselective functionalization of non-activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono- and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O-methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH2 -dependent enzyme is uniquely capable of the stepwise gem-dichlorination of a non-activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.

Self-change from problems with alcohol and drugs: A scoping review of the literature since 2010.

ISSUES: Self-change from alcohol and drug use problems is increasingly acknowledged in research. Despite the growing number of published studies, the most recent broad review of this dispersed field dates back to 2010. The present review narratively synthesises key findings from empirical studies and critically identifies research gaps and directions for further research. APPROACH: Following the PRISMA guidelines for scoping reviews, a systematic search was conducted in multiple scientific databases, resulting in the identification of 56 relevant articles with explicit empirical results on self-change. KEY FINDINGS: The scoping review presents findings related to: (i) methods and definitions used; (ii) the prevalence of self-change; (iii) indicators of self-change; (iv) the process of self-change; and (v) population views on self-change. CONCLUSION: The review highlights the significant growth in research on self-change considering key themes as well as the need for a relational and time-bound approach to self-change in research and practice.

Exploring indicators of natural recovery from alcohol and drug use problems: findings from the life in recovery survey in Flanders.

INTRODUCTION: Research has established natural recovery (NR) as an important pathway to substance use recovery. Studies investigating correlates of NR have mainly focused on demographic and substance use variables rather than life circumstances. This study seeks to better understand the phenomenon of natural recovery by (i) validating the international scientific literature concerning demographic and substance use indicators of NR in Flanders and (ii) assessing the additional explanatory power of recovery strengths and barriers during active addiction, controlling for demographic and substance use covariates. METHODS: A total of 343 persons in recovery from alcohol or drug use problems (≥ 3 months) completed an online cross-sectional survey in Flanders. Participants in NR and in recovery after following treatment were compared using multivariate linear regression models. Reasons for not following treatment were analyzed using inductive thematic analysis. RESULTS: Higher education level, lower severity of dependence, and cannabis use as the main problem substance (vs. alcohol) were statistically significant (p < 0.05) correlates of NR. When scores for the number of barriers and strengths associated with active addiction were added, barriers (but not strengths) were significantly associated with NR. When barrier items were individually tested, having untreated emotional or mental health problems, having a driver's license revoked and damaging property were statistically significant correlates. The most reported reason for not entering treatment was not experiencing any need to do so. CONCLUSION: The results highlight the importance of a holistic approach to recovery support across multiple life domains. Limitations and opportunities for further research are discussed.

Enhancing astaxanthin biosynthesis and pathway expansion towards glycosylated C40 carotenoids by Corynebacterium glutamicum.

Astaxanthin, a versatile C40 carotenoid prized for its applications in food, cosmetics, and health, is a bright red pigment with powerful antioxidant properties. To enhance astaxanthin production in Corynebacterium glutamicum, we employed rational pathway engineering strategies, focused on improving precursor availability and optimizing terminal oxy-functionalized C40 carotenoid biosynthesis. Our efforts resulted in an increased astaxanthin precursor supply with 1.5-fold higher ß-carotene production with strain BETA6 (18 mg g-1 CDW). Further advancements in astaxanthin production were made by fine-tuning the expression of the ß-carotene hydroxylase gene crtZ and ß-carotene ketolase gene crtW, yielding a nearly fivefold increase in astaxanthin (strain ASTA**), with astaxanthin constituting 72% of total carotenoids. ASTA** was successfully transferred to a 2 L fed-batch fermentation with an enhanced titer of 103 mg L-1 astaxanthin with a volumetric productivity of 1.5 mg L-1 h-1. Based on this strain a pathway expansion was achieved towards glycosylated C40 carotenoids under heterologous expression of the glycosyltransferase gene crtX. To the best of our knowledge, this is the first time astaxanthin-ß-D-diglucoside was produced with C. glutamicum achieving high titers of microbial C40 glucosides of 39 mg L-1. This study showcases the potential of pathway engineering to unlock novel C40 carotenoid variants for diverse industrial applications.