Adjustment of the main biosynthesis modules to enhance the production of l-homoserine in Escherichia coli W3110.

l-homoserine is an important platform compound of many valuable products. Construction of microbial cell factory for l-homoserine production from glucose has attracted a great deal of attention. In this study, l-homoserine biosynthesis pathway was divided into three modules, the glucose uptake and upstream pathway, the downstream pathway, and the energy supply module. Metabolomics of the chassis strain HS indicated that the supply of ATP was inadequate, therefore, the energy supply module was firstly modified. By balancing the ATP supply module, the l-homoserine production increased by 66% to 12.55 g/L. Further, the results indicated that the upstream pathway was blocked, and increasing the culture temperature to 37°C could solve this problem and the l-homoserine production reached 21.38 g/L. Then, the downstream synthesis pathways were further strengthened to balance the fluxes, and the l-homoserine production reached the highest reported level of 32.55 g/L in shake flasks. Finally, fed-batch fermentation in a 5-L bioreactor was conducted, and l-homoserine production could reach to 119.96 g/L after 92 h cultivation, with the yield of 0.41 g/g glucose and productivity of 1.31 g/L/h. The study provides a well research foundation for l-homoserine production by microbial fermentation with the capacity for industrial application.

USF1 Silencing Reduces Ferroptosis Resistance in Prostate Cancer Cells.

BACKGROUND: Ferroptosis is an iron-dependent cell death mode. Ferroptosis resistance is related to prostate cancer (PCa) invasion; However, there is vague understanding with regard to the underlying mechanism. This study was undertaken to clarify the role and mechanism of upstream stimulatory factor 1 (USF1) in ferroptosis resistance in invasive PCa. METHODS: USF1 was silenced in the human PCa cell lines C4-2B and PC-3. After these cells were treated with a ferroptosis inhibitor, cell viability and invasion and the expression of glutathione peroxidase 4 (GPX4) were evaluated. Chromatin immunoprecipitation and Dual-luciferase reporter assay suggested an interaction between USF1 and brain-expressed X-linked protein 1 (BEX1). Consequently, BEX1 was overexpressed in USF1-silenced C4-2B and PC-3 cells and its effects on cell viability and invasion and GPX4 expression were examined. RESULTS: USF1 silencing mitigated PCa cell viability and invasion. Treatment with a ferroptosis inhibitor counteracted the inhibitory roles of USF1 silencing in cell invasion and GPX4 expression. Additionally, USF1 silencing decreased BEX1 expression and USF1 was found to bind to the BEX1 promoter. BEX1 overexpression reversed the influences of USF1 silencing on the viability, BEX1 protein expression, invasive ability and ferroptosis of PCa cells. CONCLUSIONS: USF1 activates the transcription of BEX1, preventing PCa cell ferroptosis and promoting cell invasion. Therefore, USF1 silencing may inhibit the progression of PCa by reducing ferroptosis resistance.

Combined effects of flood, drought and land use dominate water quality and nutrient exports in Jialing River basin, SW China.

Climate change and the associated increase in hydroclimatic extremes necessitate a deeper understanding of the resulting water quality responses. This study investigates the combined impacts of hydroclimatic extremes and land uses on water quality of the Chinese Jialing River, of which the middle and downstream areas experienced a flood in 2021 and a severe drought in 2022. Water Quality Index (WQI) and nutrient loads were assessed using daily data from 22 monitoring stations across the Jialing River and its two tributaries, the Qujiang River and Fujiang River, over 2021-2022. The results indicate a slight upward trend in water quality, as reflected by the WQI, for the tributaries from 2021 to 2022, while a declining trend was observed in the mainstream. Floods had a more pronounced impact on water quality than droughts, particularly on nutrient concentrations, and both dilution and flushing effects were observed as discharge increased in the Jialing River and its tributaries. Notably, water quality deterioration was most pronounced in the downstream areas with land uses dominated by cropland and built-up area, where intensified rainfall distributed and exacerbated nutrient losses in the rainy seasons. Nutrient fluxes, including Chemical Oxygen Demand (COD), Total Phosphorus (TP), and Total Nitrogen (TN), were closely linked to discharge, with hydroclimatic extremes therefore significantly affecting nutrient exports. This study elucidates the complex interactions between land use, extreme weather and water quality in the Jialing River Basin. Our findings underscore the need to strengthen the management of non-point source pollutants in the downstream areas of the Jialing River to address the challenges posed by anticipated increases in extreme rainfall in the near future.

Facilitation of diabetic wound healing by far upstream element binding protein 1 through augmentation of dermal fibroblast activity.

AIMS: Diabetes mellitus (DM) often leads to wound healing complications, partly attributed to the accumulation of advanced glycosylation end products (AGEs) that impair fibroblast function. Far Upstream Element Binding Protein 1 (FUBP1) regulates cell proliferation, migration, and collagen synthesis. However, the impact of FUBP1 on diabetic wound healing remains unknown. This study is designed to explore the function and mechanisms of FUBP1 in diabetic wound healing. METHODS: Eighteen Sprague-Dawley rats (weighing 220-240 g) were randomly assigned to three groups (n = 6): a control group (NC) of healthy rats, a model group (DM) of untreated diabetic rats, and a treatment group (DM + FUBP1) of diabetic rats accepting FUBP1 treatment. A 10 mm diameter circular full-thickness skin defect was created on the back of each rat. On days 1 and 7, rats in the treatment group received local injections of 5 µg FUBP1 protein at the wound site, whereas the control group and model group were administered saline. Wound healing was documented on days 0, 3, 7, 10, and 14, with tissue samples from the wound areas collected on day 14 for histological analysis, including H&E staining, Masson's trichrome staining, and immunohistochemistry. Western blot analysis was utilized to assess the expression of GSK-3ß, Wnt3a, and ß-catenin. In vitro, the effects of various concentrations of AGEs on cell viability and FUBP1 expression were examined in human dermal fibroblasts (HDF). Cells were genetically modified to overexpress FUBP1 using lentiviral vectors and were cultured for 48 h in media with or without AGEs. The impacts on fibroblast proliferation, migration, and Wnt/ß-catenin signaling were evaluated using CCK-8, scratch assays, and Western blot analysis. RESULTS: Animal investigation revealed that from day 7 onwards, the wound healing rate of the treatment group was higher than that of the model group but lower than the control group. On day 14, the wound healing rates were as follows: control group (0.97 ± 0.01), model group (0.84 ± 0.03), and treatment group (0.93 ± 0.01). These differences were statistically significant. Histological analysis indicates that FUBP1 promotes granulation tissue formation, re-epithelialization, and collagen deposition in treatment group. Additionally, FUBP1 protein expression decreased in dermal fibroblasts when exposed to AGEs. Overexpression of FUBP1 significantly enhanced fibroblast proliferation and migration, activating the Wnt/ß-catenin pathway and mitigating the inhibitory effects of AGEs. CONCLUSIONS: Our results suggest that FUBP1 can be a promising therapeutic target for diabetic wound healing, potentially counteracting the detrimental effects of AGEs on dermal fibroblasts through the Wnt/ß-catenin pathway.

Embedding Physical Therapy in the Pediatric Primary Care Setting: Qualitative Analysis of Pediatricians' Insights on Potential Collaborative Roles and Benefits.

The growing need for collaborative healthcare teams to meet complex health challenges has led to physical therapists (PTs) being embedded in adult primary care settings for many years now. However, this model of care has not been found in pediatrics. This qualitative study sought to gain insights from pediatricians on the potential of embedding pediatric PTs in primary care. Participants were nine pediatricians practicing in both urban and rural, hospital-based and private settings. Semi-structured interviews were recorded, transcribed, and analyzed via thematic analysis per published methods, assuring trustworthiness. Three overarching themes emerged: pediatricians' priorities aligned with the Quadruple Aim of Healthcare, embedded PTs could fill multiple roles in pediatrics, and they could see a wide variety of patients, highlighting real potential benefits in primary care. Participants endorsed in-office focused treatments, screening to determine optimal care pathways, and ongoing patient follow-up as potential PT roles in this setting. Providers thought that PTs could help manage care for musculoskeletal complaints, high-risk infants, medically complex children, autism, and obesity. An advanced-trained PT having attributes of confidence, adaptability, and open-mindedness was desired. All participants endorsed pediatric primary care PTs as having potentially high value in their practice. This is the first known study on the potential of embedding a PT in the pediatric primary care setting, offering valuable insights from pediatricians to be leveraged in implementation planning.

The fed-batch production of mannosylerythritol lipids by Ustilago maydis DSM 4500 from hydrophilic carbon sources.

Glycolipids are a class of widely studied biosurfactants with excellent applicability in cosmetic and pharmaceutical formulations. This class of biosurfactants includes mannosylerythritol lipids (MELs), which have gained particular interest due to their moisturizing and healing activity for dry and damaged human skin, arising from conditions such as eczema. Traditionally, MELs have been produced by growing certain basidiomycetous yeasts on vegetable oils. However, oils are a comparatively expensive substrate, which negatively affects the economic performance of MEL production. In addition to this, vegetable oils significantly complicate the downstream processing required to produce a product with the required purity for most applications. To address these challenges, this study investigated MEL-A production exclusively from hydrophilic carbon sources by Ustilago maydis DSM 4500. By implementing a fed-batch production strategy, maximum MEL-A concentration of 0.87 g/L was achieved from glucose exclusively. Also, adding micronutrients (such as MnSO4) to MEL-A production showed a 24.1% increase in the product titer, implying other metabolites are formed, favoring MEL production.

Uncovering the functions and mechanisms of regulatory elements-associated non-coding RNAs.

Over the past decade, regulatory non-coding RNAs (ncRNAs) produced by RNA Pol II have been revealed as meaningful players in various essential cellular functions. In particular, thousands of ncRNAs are produced at transcriptional regulatory elements such as enhancers and promoters, where they may exert multiple functions to regulate proper development, cellular programming, transcription or genomic stability. Here, we review the mechanisms involving these regulatory element-associated ncRNAs, and particularly enhancer RNAs (eRNAs) and PROMoter uPstream Transcripts (PROMPTs). We contextualize the mechanisms described to the processing and degradation of these short lived RNAs. We summarize recent findings explaining how ncRNAs operate locally at promoters and enhancers, or further away, either shortly after their production by RNA Pol II, or through post-transcriptional stabilization. Such discoveries lead to a converging model accounting for how ncRNAs influence cellular fate, by acting on transcription and chromatin structure, which may further involve factors participating to 3D nuclear organization.

Physics-informed neural networks for biopharmaceutical cultivation processes: Consideration of varying process parameter settings.

We present a new modeling approach for the study and prediction of important process outcomes of biotechnological cultivation processes under the influence of process parameter variations. Our model is based on physics-informed neural networks (PINNs) in combination with kinetic growth equations. Using Taylor series, multivariate external process parameter variations for important variables such as temperature, seeding cell density and feeding rates can be integrated into the corresponding kinetic rates and the governing growth equations. In addition to previous approaches, PINNs also allow continuous and differentiable functions as predictions for the process outcomes. Accordingly, our results show that PINNs in combination with Taylor-series expansions for kinetic growth equations provide a very high prediction accuracy for important process variables such as cell densities and concentrations as well as a detailed study of individual and combined parameter influences. Furthermore, the proposed approach can also be used to evaluate the outcomes of new parameter variations and combinations, which enables a saving of experiments in combination with a model-driven optimization study of the design space.

Auto-transduction in lentiviral vector bioprocessing: A quantitative assessment and a novel inhibition strategy.

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7-6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.

Confinement controls the directional cell responses to fluid forces.

Our understanding of how fluid forces influence cell migration in confining environments remains limited. By integrating microfluidics with live-cell imaging, we demonstrate that cells in tightly-but not moderately-confined spaces reverse direction and move upstream upon exposure to fluid forces. This fluid force-induced directional change occurs less frequently when cells display diminished mechanosensitivity, experience elevated hydraulic resistance, or sense a chemical gradient. Cell reversal requires actin polymerization to the new cell front, as shown mathematically and experimentally. Actin polymerization is necessary for the fluid force-induced activation of NHE1, which cooperates with calcium to induce upstream migration. Calcium levels increase downstream, mirroring the subcellular distribution of myosin IIA, whose activation enhances upstream migration. Reduced lamin A/C levels promote downstream migration of metastatic tumor cells by preventing cell polarity establishment and intracellular calcium rise. This mechanism could allow cancer cells to evade high-pressure environments, such as the primary tumor.

LRRK2 in Parkinson's disease: upstream regulation and therapeutic targeting.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common causes of Parkinson's disease (PD) to date. Dysfunction in LRRK2 enzymatic activities and elevated protein levels are associated with the disease. How is LRRK2 activated, and what downstream molecular and cellular processes does LRRK2 regulate? Addressing these questions is crucial to decipher the disease mechanisms. In this review we focus on the upstream regulations and briefly discuss downstream substrates of LRRK2 as well as the cellular consequences caused by these regulations. Building on these basic findings, we discuss therapeutic strategies targeting LRRK2 and highlight the challenges in clinical trials. We further highlight the important questions that remains to be answered in the LRRK2 field.

"I Was Treated Differently": Reproductive Health Care Experiences Among Women With Recent Experiences of Incarceration, Homelessness, And/Or Substance Use in a Medically Underserved Area in the Southwestern US.

Background: Women with upstream social determinants of health, particularly those with recent experiences of incarceration, homelessness, and/or substance use, encounter a series of barriers in accessing health care services and consequently face poor sexual and reproductive health outcomes. Driven by a community concern for increasing rates of syphilis and congenital syphilis among women who are structurally disadvantaged, this study focuses on their experiences with reproductive healthcare access across healthcare settings.Research Design and Study Sample: This community-based pláticas (conversational) research project gathered 12 in-depth interviews and testimonios (testimonies) with women who reported a criminalized upstream barrier (incarceration, homelessness, and/or substance use) in a small city in the southwestern U.S. - most of whom identified as Latina/Hispanic.Analysis and Results: Using a grounded analysis and drawing upon Chicana feminist methodologies, this study identifies four major themes: (1) homelessness and economic vulnerabilities, (2) incarceration and health care, (3) drug use, provider stigma, and motherhood, and (4) desired changes to the healthcare experience.Conclusion: Results highlight the need for economic and transportation supports, community-based preventive services as alternatives to incarcerated healthcare, along with more compassionate and structurally competent provider-patient dialogue.

Ribosome Profiling and RNA Sequencing Reveal Translation and Transcription Regulation under Acute Heat Stress in Rainbow Trout (Oncorhynchus mykiss, Walbaum, 1792) Liver.

Rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) is an important economic cold-water fish that is susceptible to heat stress. To date, the heat stress response in rainbow trout is more widely understood at the transcriptional level, while little research has been conducted at the translational level. To reveal the translational regulation of heat stress in rainbow trout, in this study, we performed a ribosome profiling assay of rainbow trout liver under normal and heat stress conditions. Comparative analysis of the RNA-seq data with the ribosome profiling data showed that the folding changes in gene expression at the transcriptional level are moderately correlated with those at the translational level. In total, 1213 genes were significantly altered at the translational level. However, only 32.8% of the genes were common between both levels, demonstrating that heat stress is coordinated across both transcriptional and translational levels. Moreover, 809 genes exhibited significant differences in translational efficiency (TE), with the TE of these genes being considerably affected by factors such as the GC content, coding sequence length, and upstream open reading frame (uORF) presence. In addition, 3468 potential uORFs in 2676 genes were identified, which can potentially affect the TE of the main open reading frames. In this study, Ribo-seq and RNA-seq were used for the first time to elucidate the coordinated regulation of transcription and translation in rainbow trout under heat stress. These findings are expected to contribute novel data and theoretical insights to the international literature on the thermal stress response in fish.

Reverse Chromatin Immunoprecipitation (R-ChIP).

DNA-protein interactions play fundamental roles in diverse biological functions. The gene-centered method is used to identify the upstream regulators of defined genes. In this study, we developed a novel method for capturing the proteins that bind to certain chromatin fragments or DNA sequences, which is called reverse chromatin immunoprecipitation (R-ChIP). This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin or DNA fragments, and the DNA-associated proteins are then analyzed using mass spectrometry. This method can capture DNA-associated proteins with sufficient quantity and purity for identification.

Phosphate deficiency alters transcript isoforms via alternative transcription start sites.

Alternative transcription start sites (TSS) are widespread in eukaryotes and can alter the 5' UTR length and coding potential of transcripts. Here we show that inorganic phosphate (Pi) availability regulates the usage of several alternative TSS in Arabidopsis (Arabidopsis thaliana). In comparison to phytohormone treatment, Pi had a pronounced and specific effect on the usage of many alternative TSS. By combining short-read RNA sequencing with long-read sequencing of full-length mRNAs, we identified a set of 45 genes showing alternative TSS under Pi deficiency. Alternative TSS affected several processes, such as translation via the exclusion of upstream open reading frames present in the 5' UTR of RETICULAN LIKE PROTEIN B1 mRNA, and subcellular localization via removal of the plastid transit peptide coding region from the mRNAs of HEME OXYGENASE 1 and SULFOQUINOVOSYLDIACYLGLYCEROL 2. Several alternative TSS also generated shorter transcripts lacking the coding potential for important domains. For example, the EVOLUTIONARILY CONSERVED C-TERMINAL REGION 4 (ECT4) locus, which encodes an N6-methyladenosine (m6A) reader, strongly expressed under Pi deficiency a short noncoding transcript (named ALTECT4) ~550 nt long with a TSS in the penultimate intron. The specific and robust induction of ALTECT4 production by Pi deficiency led to the identification of a role for m6A readers in primary root growth in response to low phosphate that is dependent on iron and is involved in modulating cell division in the root meristem. Our results identify alternative TSS usage as an important process in the plant response to Pi deficiency.

Hybrid Synthetic Promoters in Saccharomyces cerevisiae Built on Foreign Promoter Sequences.

Traditionally, hybrid promoters are constructed, in Saccharomyces cerevisiae, by joining the core region and the upstream activating sequences from different native promoters. Here, we describe a new design that makes use of the core promoters from foreign organisms: viruses, humans, and the yeast Schizosaccharomyces pombe. With this approach, we realized a library of 59 new constitutive promoters that span over nine folds in gene expression.

Translational regulation enhances distinction of cell types in the nervous system.

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Regulatory factors of Nrf2 in age-related macular degeneration pathogenesis.

Age-related macular degeneration (AMD) is a complicated disease that causes irreversible visual impairment. Increasing evidences pointed retinal pigment epithelia (RPE) cells as the decisive cell involved in the progress of AMD, and the function of anti-oxidant capacity of PRE plays a fundamental physiological role. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a significant transcription factor in the cellular anti-oxidant system as it regulates the expression of multiple anti-oxidative genes. Its functions of protecting RPE cells against oxidative stress (OS) and ensuing physiological changes, including inflammation, mitochondrial damage and autophagy dysregulation, have already been elucidated. Understanding the roles of upstream regulators of Nrf2 could provide further insight to the OS-mediated AMD pathogenesis. For the first time, this review summarized the reported upstream regulators of Nrf2 in AMD pathogenesis, including proteins and miRNAs, and their underlying molecular mechanisms, which may help to find potential targets via regulating the Nrf2 pathway in the future research and further discuss the existing Nrf2 regulators proved to be beneficial in preventing AMD.

Modulation of prion protein expression through cryptic splice site manipulation.

Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP's sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5' untranslated region. This exon is homologous to exon 2 in nonprimate species but contains a start codon that would yield an upstream open reading frame with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.

Hospice Administrators' and Providers' Perspectives on Providing Upstream Palliative Care: Facilitators, Barriers, and Policy Prescriptions.

Background: Among patients with serious illness, palliative care before hospice enrollment is associated with improved quality of life, reduced symptom burden, and earlier transitions to hospice. However, fewer than half of eligible patients receive specialty palliative care referrals. As most hospice clinicians and administrators have experience in specialty palliative care, several emerging programs propose engaging hospice clinicians to provide early palliative care. Objective: We sought to identify barriers and facilitators to upstream palliative care. Design: We conducted a key informant qualitative study among hospice administrators and clinicians. Setting/Subjects: We conducted semi-structured interviews with 23 hospice administrators and clinicians in eight states from March to August 2022. We identified participants using snowball and purposive sampling using states that participate in Medicare Advantage's value-based insurance design Model. Results: Respondents indicated that barriers to early palliative care included inadequate staffing and reimbursement. Hospice clinicians providing community-based palliative care can address access barriers and improve transitions to hospice. Respondents expressed desire for payer guidance in identifying eligible patients but were cautious about payers acting as direct palliative care providers. However, payers could facilitate uptake by broadening and specifying coverage of services to include goals of care conversations and symptom management. Routine referrals initiated by objective measures could potentially increase access. Conclusions: Utilizing hospice providers to provide upstream palliative care can increase access, improve outcomes, and ease the transition to hospice.