Spatial sorting caused by downstream dispersal: implication for morphological evolution in isolated populations of fat minnow inhabiting small streams flowing through terraced rice paddies.

The evolutionary forces arising from differential dispersal are known as "spatial sorting," distinguishing them from natural selection arising from differential survival or differential reproductive success. Spatial sorting is often considered to be transient, because it is offset by the return of dispersers in many cases. However, in riverine systems, spatial sorting by downstream dispersal can be cumulative in habitats upstream of migration barriers such as weirs or falls, which can block the return of the dispersers. Terraced rice paddies are often found on steep mountain slopes in Japan and often incorporate small streams with numerous migration barriers. This study investigated the morphological features of fat minnow, Rhynchocypris oxycephalus jouyi (Cyprinidae), inhabiting above-barrier habitats of the small streams flowing through flood-prone terraced rice paddies and examined their function via a mark-recapture experiment. Although this study did not reveal a consistent pattern across all local populations, some above-barrier populations were characterised by individuals with a thinner caudal peduncle, thinner body, and longer ventral caudal fin lobes than those in neighbouring mainstream populations. A mark-recapture experiment during flooding showed that a thinner caudal peduncle and deeper body helped fat minnow avoid downstream dispersal and ascend a small step, and suggested that a longer ventral caudal fin lobe was important for ascending. These results suggest that the caudal morphologies of some above-barrier populations avoid or reduce the risk of downstream dispersal, supporting the idea that spatial sorting shapes functional traits, enhancing the spatial persistence of individuals in upstream habitats.

Synthetic biology approaches and bioseparations in syngas fermentation.

Fossil fuel use drives greenhouse gas emissions and climate change, highlighting the need for alternatives like biomass-derived syngas. Syngas, mainly H2 and CO, is produced via biomass gasification and offers a solution to environmental challenges. Syngas fermentation through the Wood-Ljungdahl pathway yields valuable chemicals under mild conditions. However, challenges in scaling up persist due to issues like unpredictable syngas composition and microbial fermentation contamination. This review covers advancements in genetic tools and metabolic engineering to expand product range, highlighting crucial enabling technologies that expedite strain development for acetogens and other non-model organisms. This review paper provides an in-depth exploration of syngas fermentation, covering microorganisms, gas composition effects, separation techniques, techno economic analysis, and commercialization efforts.

Escherichia coli-based biorefining process yields optically pure lactic acid from fermented second-generation feedstocks.

Within the circular bioeconomy the production of optically pure LA from 2nd generation feedstocks would be ideal but it is very challenging. In this paper genetically engineered Escherichia coli strains were created to resolve racemic LA solutions synthesised and produced from the fermentation of organic waste or ensiled grass. Refining LA racemic mixtures into either a D- or L-LA was achieved by cells being able to consume one LA isomer as a sole carbon and energy source while not being able to consume the other. A D-LA refining strain JSP0005 was grown on fermented source-sorted organic household waste and different grass silage leachates, which are 2nd generation feedstocks containing up to 33 g/L lactic acid racemate. In all growth experiments, L-LA was completely removed leaving D-LA as the only LA stereoisomer, i.e. resulting in optically pure D-LA, which also increased by as much as 248.6 % from its starting concentration, corresponding to 38 g/L. The strains resulting from this study are a promising first step towards a microbial based LA biorefining process.

Comparative Evaluation of Local and Downstream Responses in Two Commercially Available Paclitaxel-Coated Balloons in Healthy Peripheral Arteries of a Swine Model.

OBJECTIVE: To investigate the local, downstream, and systemic effects of two different paclitaxel-coated balloons. DESIGN: Preclinical study in healthy peripheral arteries of a swine model, with randomized allocation of the distribution of the devices: the test paclitaxel-coated balloon (PCB) (Luminor®), a control PCB (IN.PACT®), and a plain angioplasty balloon (Oceanus®), considering single (1×) and overlapping (3×) doses with simple blind histologic analysis. METHODS: Twenty animals underwent balloon angioplasty at 1× or 3× doses in the external and internal branches of both femoral arteries and were followed-up for 28 days. Post-procedural and follow-up angiography were carried out. Comprehensive necropsy and histology were used to evaluate the local, downstream and systemic effects. RESULTS: Angioplasty was successfully carried out in all animals. Significant protocol deviations appeared in three arteries (treated with Oceanus®) without clinical relevance. Those samples were excluded from the analysis. All the animals survived the follow-up period without major clinical issues. Local signs of drug toxicity were less marked with Luminor® than IN.PACT® at 1× dose, including endothelial loss (p=0.0828), intima/media inflammation (p=0.0004), transmural medial smooth muscle cell (SMC) loss (p=0.0016), wall thickness loss (p=0.0141), presence of fibrin in the vascular wall (p=0.0054), and adventitial inflammation (p=0.0080). A similar pattern was observed at the 3× dose for endothelial loss (p=0.0011), intima/media inflammation (p< 0.0001), circumferential SMC loss (p=0.0004), medial SMC replacement with proteoglycans (p=0.0014), fibrin (p=0.0034), and collagen content (p=0.0205). Downstream vascular histologic changes were mild although more prevalent in the IN.PACT® 3× group (p=0.006). No systemic effects of toxicity were detected in any of the samples analyzed. CONCLUSION: Luminor® showed better healing pattern (lower inflammation, and endothelial and muscular loss) than IN.PACT® balloon. The effect was evident at single and triple doses. The prevalence of downstream lesions, albeit low, was higher with the triple dose of IN.PACT® compared with Luminor®.

DOE-based process optimization for development of efficient methods for purification of recombinant hepatitis B surface antigen from Pichia pastoris feedstock using Capto adhere resin.

Background: The multimodal chromatography resins, such as Capto adhere, are considered good candidates to be utilized in downstream processing due to their high capacity and selectivity; however, their multimodal interactions lead to an intricacy in the adsorption-desorption patterns and systematic characterization of conditions for process steps is necessary. Methods: Capto adhere, a strong ion exchanger with multimodal functionality, was used in this study for the final aim of recombinant hepatitis B surface antigen (rHBsAg) purification from Pichia pastoris (P. pastoris) industrial feedstock. Optimization of various parameters was done using the design of experiments (DOE) approach to determine the best binding and non-binding conditions. Results: Maximum rHBsAg binding on Capto adhere occurred in 20 mM sodium acetate, pH 4.5, and a binding capacity of about 0.75 mg/ml was achieved, which was much higher than rHBsAg binding capacity of other resins reported so far. In elution optimization investigations, it was revealed that 1 M arginine (buffered in 50 mM sodium phosphate, pH 6.5) was the most efficient eluting agent. The binding and elution optimal conditions were utilized for further purification of rHBsAg from P. pastoris industrial feedstock in bind-elute mode, and the recovery and purity of the obtained rHBsAg were about 60% and 100%, respectively. Following optimization in the flow-through purification mode, the target protein recovery was significantly increased (up to 97%) and the target protein purity of more than 95% was achievable. SEC-HPLC analysis showed that the obtained retention times for the purified rHBsAg were similar to those reported previously. Conclusions: These results suggest that Capto adhere under such optimized conditions can be considered as a good candidate for efficient purification of rHBsAg from P. pastoris industrial feedstock in downstream processing.

The Caenorhabditis elegans protein SOC-3 permits an alternative mode of signal transduction by the EGL-15 FGF receptor.

Fibroblast Growth Factors and their receptors (FGFRs) comprise a cell signaling module that can stimulate signaling by Ras and the kinases Raf, MEK, and ERK to regulate animal development and homeostatic functions. In Caenorhabditis elegans, the sole FGFR ortholog EGL-15 acts with the GRB2 ortholog SEM-5 to promote chemoattraction and migration by the sex myoblasts (SMs) and fluid homeostasis by the hypodermis (Hyp7). Cell-specific differences in EGL-15 signaling were suggested by the phenotypes caused by egl-15(n1457), an allele that removes a region of its C-terminal domain (CTD) known to bind SEM-5. To determine how mutations altered EGL-15 activity in the SMs and Hyp7, we used the kinase reporter ERK-KTR to measure activation of the ERK ortholog MPK-1. Consequences of egl-15(n1457) were cell-specific, resulting in loss of MPK-1 activity in the SMs and elevated activity in Hyp7. Previous studies of Hyp7 showed that loss of the CLR-1 phosphatase causes a fluid homeostasis defect termed "Clear" that is suppressed by reduction of EGL-15 signaling, a phenotype termed "Suppressor of Clear" (Soc). To identify mechanisms that permit EGL-15 signaling in Hyp7, we conducted a genetic screen for Soc mutants in the clr-1; egl-15(n1457) genotype. We report the identification of SOC-3, a protein with putative SEM-5-binding motifs and PH and PTB domains similar to DOK and IRS proteins. In combination with the egl-15(n1457) mutation, loss of either soc-3, the GAB1 ortholog soc-1, or the SHP2 ortholog ptp-2, reduced MPK-1 activation. We generated alleles of soc-3 to test the requirement for the SEM-5-binding motifs, finding that residue Tyr356 is required for function. We propose that EGL-15-mediated SM chemoattraction relies solely on the direct interaction between SEM-5 and the EGL-15 CTD. In Hyp7, EGL-15 signaling uses two mechanisms: the direct SEM-5 binding mechanism; and an alternative, CTD-independent mechanism involving SOC-3, SOC-1, and PTP-2. This work demonstrates that FGF signaling uses distinct, tissue-specific mechanisms in development, and identifies SOC-3 as a potential adaptor that facilitates Ras pathway activation by FGFR.

Vitamin B-6 Prevents Heart Failure with Preserved Ejection Fraction Through Downstream of Kinase 3 in a Mouse Model.

BACKGROUND: There is an urgent need to develop an efficient therapeutic strategy for heart failure with preserved ejection fraction (HFpEF), which is mediated by phenotypic changes in cardiac macrophages. We previously reported that vitamin B-6 inhibits macrophage-mediated inflammasome activation. OBJECTIVES: We sought to examine whether the prophylactic use of vitamin B-6 prevents HFpEF. METHODS: HFpEF model was elicited by a combination of high-fat diet and Nω-nitro-l-arginine methyl ester supplement in mice. Cardiac function was assessed using conventional echocardiography and Doppler imaging. Immunohistochemistry and immunoblotting were used to detect changes in the macrophage phenotype and myocardial remodeling-related molecules. RESULTS: Co-administration of vitamin B-6 with HFpEF mice mitigated HFpEF phenotypes, including diastolic dysfunction, cardiac macrophage phenotypic shifts, fibrosis, and hypertrophy. Echocardiographic improvements were observed, with the E/E' ratio decreasing from 42.0 to 21.6 and the E/A ratio improving from 2.13 to 1.17. The exercise capacity also increased from 295.3 to 657.7 min. However, these beneficial effects were negated in downstream of kinase (DOK) 3-deficient mice. Mechanistically, vitamin B-6 increased DOK3 protein concentrations and inhibited macrophage phenotypic changes, which were abrogated by an AMP-activated protein kinase inhibitor. CONCLUSIONS: Vitamin B-6 increases DOK3 signaling to lower risk of HFpEF by inhibiting phenotypic changes in cardiac macrophages.

Interpopulation variation in dispersal behavior of fat minnow Rhynchocypris oxycephalus jouyi juveniles inhabiting fragmented habitats.

In many organisms, including fishes, downstream dispersal is often phenotype-dependent. Phenotype-dependent downstream dispersal can generate evolutionary pressure via spatial sorting, which non-randomly removes phenotypes enhancing downstream dispersal from upstream populations. Spatial sorting due to downstream dispersal could accumulate in fish populations in above-barrier habitats to which dispersed individuals cannot return, resulting in functional traits that reduce downstream dispersal. This evolutionary mechanism may be more important in smaller above-barrier habitats where downstream emigration occurs over shorter dispersal distances. This study observed the dispersal behavior of fat minnow Rhynchocypris oxycephalus jouyi juveniles in an experimental tank to examine whether juveniles from small above-barrier habitats show more behaviors favorable for reducing downstream dispersal than those from large above-barrier habitats. Juveniles from small above-barrier habitats avoided downstream dispersal for longer durations than those from large above-barrier habitats, but there was no difference in the frequency of ascending attempts. These results support the notion that behavioral traits of juveniles in small above-barrier populations have been refined by spatial sorting to reduce downstream dispersal. The finding that interpopulation variation in dispersal behavior occurred only for certain dispersal directions implies that the behavioral consequences of habitat fragmentation are more complex than previously assumed.

A practical handbook on single-cell RNA sequencing data quality control and downstream analysis.

Advancements in single-cell analysis have facilitated high-resolution observation of the transcriptome in individual cells. However, standards for obtaining high-quality cells and data analysis pipelines remain variable. Here, we provide the groundwork for improving the quality of single-cell analysis by delineating guidelines for selecting high-quality cells and considerations throughout the analysis. This review will streamline researchers' access to single-cell analysis and serve as a valuable guide for analysis.

Balancing pH and yield: exploring itaconic acid production in Ustilago cynodontis from an economic perspective.

BACKGROUND: Itaconic acid is a promising bio-based building block for the synthesis of polymers, plastics, fibers and other materials. In recent years, Ustilago cynodontis has emerged as an additional itaconate producing non-conventional yeast, mainly due to its high acid tolerance, which significantly reduces saline waste coproduction during fermentation and downstream processing. As a result, this could likely improve the economic viability of the itaconic acid production process with Ustilaginaceae. RESULTS: In this study, we characterized a previously engineered itaconate hyper-producing Ustilago cynodontis strain in controlled fed-batch fermentations to determine the minimal and optimal pH for itaconate production. Under optimal fermentation conditions, the hyper-producing strain can achieve the theoretical maximal itaconate yield during the production phase in a fermentation at pH 3.6, but at the expense of considerable base addition. Base consumption is strongly reduced at the pH of 2.8, but at cost of production yield, titer, and rate. A techno-economic analysis based on the entire process demonstrated that savings due to an additional decrease in pH control reagents and saline waste costs cannot compensate the yield loss observed at the highly acidic pH value 2.8. CONCLUSIONS: Overall, this work provides novel data regarding the balancing of yield, titer, and rate in the context of pH, thereby contributing to a better understanding of the itaconic acid production process with Ustilago cynodontis, especially from an economic perspective.

Proteomics-based method to comprehensively model the removal of host cell protein impurities.

Mechanistic models mostly focus on the target protein and some selected process- or product-related impurities. For a better process understanding, however, it is advantageous to describe also reoccurring host cell protein impurities. Within the purification of biopharmaceuticals, the binding of host cell proteins to a chromatographic resin is far from being described comprehensively. For a broader coverage of the binding characteristics, large-scale proteomic data and systems level knowledge on protein interactions are key. However, a method for determining binding parameters of the entire host cell proteome to selected chromatography resins is still lacking. In this work, we have developed a method to determine binding parameters of all detected individual host cell proteins in an Escherichia coli harvest sample from large-scale proteomics experiments. The developed method was demonstrated to model abundant and problematic proteins, which are crucial impurities to be removed. For these 15 proteins covering varying concentration ranges, the model predicts the independently measured retention time during the validation gradient well. Finally, we optimized the anion exchange chromatography capture step in silico using the determined isotherm parameters of the persistent host cell protein contaminants. From these results, strategies can be developed to separate abundant and problematic impurities from the target antigen.

A review on microbes mediated resource recovery and bioplastic (polyhydroxyalkanoates) production from wastewater.

BACKGROUND: Plastic is widely utilized in packaging, frameworks, and as coverings material. Its overconsumption and slow degradation, pose threats to ecosystems due to its toxic effects. While polyhydroxyalkanoates (PHA) offer a sustainable alternative to petroleum-based plastics, their production costs present significant obstacles to global adoption. On the other side, a multitude of household and industrial activities generate substantial volumes of wastewater containing both organic and inorganic contaminants. This not only poses a threat to ecosystems but also presents opportunities to get benefits from the circular economy. Production of bioplastics may be improved by using the nutrients and minerals in wastewater as a feedstock for microbial fermentation. Strategies like feast-famine culture, mixed-consortia culture, and integrated processes have been developed for PHA production from highly polluted wastewater with high organic loads. Various process parameters like organic loading rate, organic content (volatile fatty acids), dissolved oxygen, operating pH, and temperature also have critical roles in PHA accumulation in microbial biomass. Research advances are also going on in downstream and recovery of PHA utilizing a combination of physical and chemical (halogenated solvents, surfactants, green solvents) methods. This review highlights recent developments in upcycling wastewater resources into PHA, encompassing various production strategies, downstream processing methodologies, and techno-economic analyses. SHORT CONCLUSION: Organic carbon and nitrogen present in wastewater offer a promising, cost-effective source for producing bioplastic. Previous attempts have focused on enhancing productivity through optimizing culture systems and growth conditions. However, despite technological progress, significant challenges persist, such as low productivity, intricate downstream processing, scalability issues, and the properties of resulting PHA.

Recent developments on sustainable biobutanol production: a novel integrative review.

Renewable and sustainable biofuel production, such as biobutanol, is becoming increasingly popular as a substitute for non-renewable and depleted petrol fuel. Many researchers have studied how to produce butanol cheaply by considering appropriate feedstock materials and bioprocess technologies. The production of biobutanol through acetone-butanol-ethanol (ABE) is highly sought after around the world because of its sustainable supply and lack of competition with food. The purpose of this study is to present the current biobutanol production research and to analyse the biobutanol research conducted during 2006 to 2023. The keyword used in this study is "Biobutanol," and the relevant data was extracted from the Web of Science database (WoS). According to the results, institutions and scholars from the People's Republic of China, the USA, and India have the highest number of cited papers across a broad spectrum of topics including acetone-butanol-ethanol (ABE) fermentation, biobutanol, various pretreatment techniques, and pervaporation. The success of biobutanol fermentation from biomass depends on the ability of the fermentation operation to match the microbial behaviour along with the appropriate bioprocessing strategies to improve the entire process to be suitable for industrial scale. Based on the review data, we will look at the biobutanol technologies and appropriate strategies that have been developed to improve biobutanol production from renewable biomass.

Insulin evolution: A holistic view of recombinant production advancements.

The increased prevalence of diabetes and the growing popularity of non-invasive methods of recombinant human insulin uptake, such as oral insulin, have increased insulin demand, further limiting the affordability of insulin. Over 40 years have passed since the development of engineered microorganisms that replaced the animal pancreas as the primary source of insulin. To stay ahead of the need for insulin in the present and the future, a few drawbacks with the existing expression systems need to be alleviated, including the inclusion body formation, the use of toxic inducers, and high process costs. To address these bottlenecks and improve insulin production, a variety of techniques are being used in bacteria, yeasts, transgenic plants and animals, mammalian cell lines, and cell-free expression systems. Different approaches for the production of insulin, including two-chain, proinsulin or mini-proinsulin, preproinsulin coupled with fusion protein, chaperone, signal peptide, and purification tags, are explored in upstream, whereas downstream processing takes into account the recovery of intact protein in its bioactive form and purity. This article focuses on the strategies used in the upstream and downstream phases of the bioprocess to produce recombinant human insulin. This review also covers a range of analytical methods and tools employed in investigating the genuity of recombinant human insulin.

Recovery and encapsulation of Dunaliella salina ß-carotene through a novel sustainable approach: Sequential application of an ionic liquid as naturally-derived solvent and emulsifier.

Dunaliella salina is a promising source of ß-carotene, widely employed in the food industry. This study aimed to evaluate the sequential application of the Ionic Liquid (IL) cholinium oleate as an extraction solvent for D. salina ß-carotene recovery and, sequentially, as emulsifier for emulsion-based products obtained therefrom. The IL was evaluated regarding its ability to permeabilize the cells and recover ß-carotene at different temperatures (25-65 °C) and IL concentrations (0-46%). The use of the IL as solvent greatly improved ß-carotene recovery (>84%). The IL already present in the obtained extracts loaded with recovered ß-carotene was sequentially used as emulsifier in the production of nanoemulsions (NE). NE presented a ß-carotene entrapment efficiency of 100% and were kinetically stable for 30 days and presented droplet size, size distribution, and ζ-potential of 220 nm, 0.21, and -67 mV, respectively. These results indicate that using IL sequential as solvent and emulsifier has potential applications in the food industry.

Scalable microfluidic method for tunable liposomal production by a design of experiment approach.

Liposomes constitute a widespread drug delivery platform, gaining more and more attention from the pharmaceutical industry and process development scientists. Their large-scale production as medicinal products for human use is all but trivial, especially when parenteral administration is required. In this study an off-the-shelf microfluidic system and a methodological approach are presented for the optimization, validation and scale-up of highly monodisperse liposomes manufacturing. Starting from a Doxil®-like formulation (HSPC, MPEG-DSPE and cholesterol), a rational approach (Design of Experiments, DoE) was applied for the screening of the process parameters affecting the quality attributes of the product (mainly size and polydispersity). Additional DoEs were conducted to determine the effect of critical process parameters "CPPs" (cholesterol concentration, total flow rate "TFR" and flow rate ratio "FRR"), thus assessing the formulation and process robustness. A scale-up was then successfully accomplished. The procedure was applied to a Marqibo®-like formulation as well (sphingomyelin and cholesterol) to show the generality of the proposed formulation, process development and scale-up approach. The application of the system and method herein presented enables the large-scale manufacturing of liposomes, in compliance with the internationally recognized regulatory standards for pharmaceutical development (Quality by Design).

Structural study of a light chain mispaired bispecific predicts mechanism of downstream separation.

Bispecific antibodies expressed and assembled from a single upstream culture require the correct balance and pairing of four different heavy and light chains (HC and LC). The increased potential for chain-mispaired species challenges the downstream purification of this new format. While clearance of HC-mispaired species, including homodimers and half-antibodies, has been assessed, removal of LC mispairs requires a more stringent approach. Here, we report two case studies in which separation is achieved, as well as the structural basis of these separations: (A) In the first case, a main species with a positively charged patch in the correctly formed variable fragment (Fv) is disrupted when paired with the wrong LC. This LC-mispaired variant binds more weakly to a cation exchange resin and can be washed off in a chromatography step. (B) A second molecule whose LC mispair introduces a negative-charge patch and hydrophobic patch in close proximity, presenting increased binding to a multimodal anion exchange resin. This LC-mispaired variant can be retained on the column under conditions in which the bispecific is recovered. In both case studies, the molecular structural analysis by protein surface properties models correlated well with the chromatography experiments. The comprehensive interpretation of experimental and computational results has provided a better understanding of strategies and potential applications for predicting the downstream purification of complex molecules.

Process analytical technology in Downstream-Processing of Drug Substances- A review.

Process Analytical Technology (PAT) has revolutionized pharmaceutical manufacturing by providing real-time monitoring and control capabilities throughout the production process. This review paper comprehensively examines the application of PAT methodologies specifically in the production of solid active pharmaceutical ingredients (APIs). Beginning with an overview of PAT principles and objectives, the paper explores the integration of advanced analytical techniques such as spectroscopy, imaging modalities and others into solid API substance production processes. Novel developments in in-line monitoring at academic level are also discussed. Emphasis is placed on the role of PAT in ensuring product quality, consistency, and compliance with regulatory requirements. Examples from existing literature illustrate the practical implementation of PAT in solid API substance production, including work-up, crystallization, filtration, and drying processes. The review addresses the quality and reliability of the measurement technologies, aspects of process implementation and handling, the integration of data treatment algorithms and current challenges. Overall, this review provides valuable insights into the transformative impact of PAT on enhancing pharmaceutical manufacturing processes for solid API substances.

Efficacy and Safety of Dual Paclitaxel and Sirolimus Nanoparticle-Coated Balloon.

We evaluated a novel dual active pharmaceutical ingredient (API) drug-coated balloon (DCB), which consists of a coating of nanoparticles encapsulating low-dose paclitaxel (PTX) in combination with sirolimus in a synergistic ratio. Compared to the PTX DCB, the dual API DCB demonstrated similar inhibition of cell proliferation in vitro but at a significantly lower total drug dose (over 13 times lower than sirolimus nanoparticles). Animal experiments demonstrated that the dual API DCB is more effective in inhibiting intimal cell proliferation with insignificant downstream embolic effects and myocardial damage compared to the PTX DCB. These findings indicate that dual API DCBs have a high potential to demonstrate improved clinical outcomes and a greater safety profile than the PTX DCBs.