Main Regulator Research Articles

Obesity as the main factor of metabolic syndrome influencing on respiratory function

In the review, a generalized analysis of current scientific data explaining the physiological mechanisms of the influence of obesity on respiratory system is carried out. The multifactorial nature of the respiratory effect of obesity, including mechanical and inflammatory effects, is emphasized. The consequences of restrictive and obstructive changes in the biomechanics of respiration, changes in the topographic distribution of lung ventilation, mismatch of ventilation and perfusion, and a decrease in the efficiency of the respiratory muscles are considered. Elucidation of the central mechanisms of the respiratory action of proinflammatory mediators expressed by adipose tissue cells is recognized as a promising area of research. Special attention is paid to the action of leptin, which is the main regulator of metabolism and respiratory control in obesity. Its ability to modulate the central respiratory chemosensitive is discussed. It is assumed that an increase in pulmonary ventilation due to an increase in leptin production in obesity has a compensatory character and allows obese patients to maintain normocapnia despite an increase in mechanical load on respiration system. Whereas leptin resistance and suppressed hypercapnic ventilation response play a key role in the development of obesity–hypoventilation syndrome. It is concluded that it is necessary to further study the physiological mechanisms of the influence of obesity on the respiratory function in order to find new effective therapeutic methods for the treatment of diseases associated with obesity, which is the main factor in the development of metabolic syndrome.

TXN promotes tumorigenesis by activating the ERK1/2 and ERK5 signaling pathways regulating c-Myc in non-small cell lung cancer

Lung cancer is the primary cause of cancer-related deaths worldwide, particularly for non-small cell lung cancer (NSCLC). However, the exact mechanism underlying tumor formation remains unclear. It is widely acknowledged that inflammation and oxidative stress occur in the tumor microenvironment, promoting cell malignant growth and metastasis. Thioredoxin-1 (TXN), the main regulator of oxidative stress, plays a significant role in the development of NSCLC. However, the specific tumor-promoting mechanism is still being investigated. This study aimed to examine the function and mechanism of TXN in NSCLC. The effects of knockdown or overexpression TXN on cell proliferation, invasion and apoptosis were evaluated by Cell Counting Kit-8, colony formation, wound healing, transwell, TUNEL staining, and flow cytometric assays. Western blotting was performed to analyze the regulation of TXN and downstream proteins suppressed by genes and pharmacology. TXN knockdown significantly suppressed cell proliferation, invasion and promoted apoptosis both in vitro and in vivo, whereas TXN overexpression reversed these malignant phenotypes. We found that TXN regulated c-Myc expression through ERK1/2 and ERK5 signaling pathways. Suppressing ERK1/2 led to the compensatory activation of ERK5, and simultaneously inhibiting ERK1/2 and ERK5 synergistically reduced c-Myc expression, further attenuating cell proliferation, invasion and enhanced apoptosis. Our results indicated tumor promotion of TXN in NSCLC and TXN regulated c-Myc in the interest of tumorigenesis through ERK1/2 and ERK5 signaling pathways. Targeting TXN and blocking the ERK1/2 and ERK5 pathways could potentially offer new therapeutic strategies for NSCLC.

Abstract A013: Microenvironment driven dynamic deposition of histone H3.3 controls entry and exit from dormancy in disseminated cancer cells

Abstract Breast cancer metastases can go undetected for long periods of time, are highly resistant to therapy, and are the cause for more than 90% of breast cancer mortality. Dormancy is a critical step in the metastatic cascade, whereby disseminated cancer cells (DCCs) enter a non-proliferative state at distal sites until they reactivate and give rise to overt metastasis, often years after disease remission. As such, dormancy represents a major clinical obstacle in breast cancer patient care. Chromatin remodeling serves as a foundation for the cellular reprogramming required to drive cell fate transitions. However, little is known about the nature of this remodeling in dormancy. Here, we hypothesize that differential deposition of histone H3.3 determines whether DCCs enter and remain dormant in secondary organs or proliferate and form overt metastases. We used a combination of H3.3 deposition sensors and genetic tools to manipulate H3.3 and HIRA in vitro and in vivo to track H3.3 deposition through the metastatic cascade and evaluate its importance for dormancy. To evaluate relevance to patient disease we developed a dormancy score to implement from publicly available RNA seq. To define mechanism of regulation, we used a combination of whole genome sequencing (ChIP-seq, ATAC-seq and RNA-seq) followed by in-depth analysis and functional validation. Our data demonstrates that H3.3 incorporation at secondary sites is heterogenous, revealing a subset of DCCs with low H3.3 in chromatin. Blocking H3.3 deposition onto chromatin leads to a reversible arrest in G0/G1 which tracks with the activation of canonical dormancy signaling. Mechanistically, our data supports a model where blocking H3.3 incorporation into chromatin directly represses the expression of SKP2, the main regulator of progression through the restriction point of cell cycle via regulation of p27 degradation. SKP2 suppression in turn leads to the accumulation of p27 thereby causing cell cycle arrest in G0/G1 and dormancy establishment. We traced this H3.3-mediated regulation of dormancy through the dynamic regulation of H3.3 histone chaperone, HIRA, whose levels inversely correlate with dormancy in experimental models as well as in patient samples. We found that HIRA levels respond to the metastatic microenvironment where fibrotic conditions induce HIRA expression and trigger metastatic outgrowth in an H3.3-dependent manner. Together, our work unveils for the first time a patient-relevant epigenetic mechanism, impinging on H3.3 deposition, as a major regulatory point of DCCs’ cell fate at secondary organs controlling both entry of DCC’s into dormant state as well as their reactivation and thriving as overt metastasis. Citation Format: Stanislav Drapela, Cristina Megino-Luque, Rojan Chimeh Rad, Devesh Raizada, Nadir Sarigul, Didem Ilter, Brian J. Czerniecki, Jose Javier Bravo-Cordero, Ana P. Gomes. Microenvironment driven dynamic deposition of histone H3.3 controls entry and exit from dormancy in disseminated cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A013.

Exploring the Updated Roles of Ferroptosis in Liver Diseases: Mechanisms, Regulators, and Therapeutic Implications.

Ferroptosis, a newly discovered mode of cell death, is a type of iron-dependent regulated cell death characterized by intracellular excessive lipid peroxidation and imbalanced redox. As the liver is susceptible to oxidative damage and the abnormal iron accumulation is a major feature of most liver diseases, studies on ferroptosis in the field of liver diseases are of great interest. Studies show that targeting the key regulators of ferroptosis can effectively alleviate or even reverse the deterioration process of liver diseases. System Xc- and glutathione peroxidase 4 are the main defense regulators of ferroptosis, while acyl-CoA synthetase long chain family member 4 is a key enzyme causing peroxidation in ferroptosis. Generally speaking, ferroptosis should be suppressed in alcoholic liver disease, non-alcoholic fatty liver disease, and drug-induced liver injury, while it should be induced in liver fibrosis and hepatocellular carcinoma. In this review, we summarize the main regulators involved in ferroptosis and then the mechanisms of ferroptosis in different liver diseases. Treatment options of drugs targeting ferroptosis are further concluded. Determining different triggers of ferroptosis can clarify the mechanism of ferroptosis occurs at both physiological and pathological levels.

Anti-Obesity Effects of Calcium Fructoborate by Inhibiting Adipogenesis and Increasing SIRT's Expression in 3T3-L1 Cells.

Obesity is a global public health problem that can lead to mortality and morbidity. Studies on the pathophysiology of obesity for effective and safe treatments are focused on the mechanisms of adipogenesis. The association between boron treatment and weight loss has been reported, but its anti-adipogenic mechanisms and effects on preadipocytes remain unclear. This study aims to investigate the effects of boron compounds boric acid (BA) and calcium fructoborate (CaFB) on adipogenesis using the most widely used in vitro 3T3-L1 cellular model. In our study, cytotoxicity, Oil Red O (ORO), gene and protein expression analyses and cellular NAD measurements of boron compounds were performed. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) transcription factors are the main regulators of adipogenesis, and boron compounds affect them at gene and protein levels by showing anti-obesity effects. This is the first study to show that CaFB has anti-obesity properties in mouse adipocytes. Sirtuins, known as the longevity genes, were also activated from boron treatment. Results of this research provide new basic knowledge and insights into the effect of boron-based compounds on obesity. It also offers potential prospects for the development of effective treatment and/or supportive treatment methods.

Modifying the Glycocalyx of Melanoma Cells via Metabolic Glycoengineering Using N-Acetyl-d-glucosamine Analogues.

Tumor cells are decorated with aberrant glycan structures on cell surfaces. It is well known that the glycocalyx serves as a main cellular regulator, although its role in cancer is still not completely understood. Over recent decades, several non-natural monosaccharides carrying clickable groups have been introduced in melanoma cells. This technique, called Metabolic Glycoengineering (MGE), opens up the possibility of altering the cell's glycocalyx via click chemistry using a two-step approach. This study expands the field of MGE by showing the successful metabolic incorporation of novel alternative artificial glucosamine derivatives. The latter were either deoxygenated or blocked by methyl ether in position 4 to generate deficient glycosylation patterns, while being extended by an alkyne to enable click chemistry as a one-step approach. As a result, we observed a reduced proliferation rate of melanoma cells. Furthermore, using a lectin array, the decrease in high mannose epitopes was observed. In summary, the successful use of alternative artificial glucosamine derivatives enabled a significant alteration in the glycocalyx, consequently influencing cell behavior.

The role of leptin in endometrium disorders: literature review

Leptin is not only the main regulator of energy balance, but also it affects the reproductive and immune systems. Leptin and its receptors are expressed in the endometrium and are actively involved in the embryo implantation. According to numerous studies, expression and level changes of leptin are associated with the inflammatory and autoimmune diseases, including endometriosis and chronic endometritis. Hyperplastic and inflammatory diseases of the uterus are accompanied by a violation of the receptivity of the endometrium due to the dysregulation of many factors involved in proliferation, vascularization and decidualization of cells. Activity of most of these factors is due to the leptin action, however, there are no studies of the direct effect of leptin in the pathogenesis of disorders of the endometrium in hyperplastic and inflammatory diseases.Thus, the purpose of this literature review was to describe the putative molecular mechanisms of the effect of leptin on the development of endometrial pathology.Literature search was carried out from 03/20/2023 to 05/11/2023 using scientific literature databases: NCBI PubMed, Google Scholar (foreign sources), Cyberleninka, Elibrary (domestic sources): references for the period 1995-2023 were analyzed. The following keywords were used for the search: leptin, endometrial dysfunction, endometrial receptivity, inflammation, pelvic inflammatory disease.

Demyelination in cuprizone mice is ameliorated by calycosin mediated through astrocyte Nrf2 signaling pathway

Oxidative stress plays a pivotal role in multiple sclerosis (MS), triggering demyelination predominantly through excessive peroxide production and the depletion of antioxidants. The accumulation of oxidative damage can be caused by dysregulation of astrocytes, which are the brain's main regulators of oxidative homeostasis. Calycosin, an essential bioactive component extracted from Astragalus, is recognized for its neuroprotective properties. Although recent research has highlighted calycosin's neuroprotective capabilities, its role in demyelinating conditions like MS remains unclear. In this work, we examined the possible molecular mechanism of calycosin's neuroprotective effect on cuprizone (CPZ)-induced demylination in mice. According to our research, calycosin successfully reduced demyelination and behavioral dysfuction in CPZ mice. Calycosin also decreased the production of oxidative stress and enhanced the expression of antioxidants in CPZ mice and in astrocytes induced by hydrogen peroxide (H2O2). Furthermore, both in vivo and in vitro experiments demonstrated that calycosin promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) along with the upregulation of heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase (SOD). Importantly, the application of all-trans retinoic acid (ATRA), a specific inhibitor of Nrf2, effectively reversed the myelin-protective and antioxidant effects conferred by calycosin. This study suggested that calycosin might exert neuroprotection by inhibiting oxidative stress and reducing demyelination via the activation of astrocyte Nrf2 signaling. These findings indicated that calycosin might be a potential candidate for treating MS.

The efflux system CdfX exports zinc that cannot be transported by ZntA in Cupriavidus metallidurans.

Cupriavidus metallidurans is able to survive exposure to high concentrations of transition metals, but is also able to grow under metal starvation conditions. A prerequisite of cellular zinc homeostasis is a flow equilibrium combining zinc uptake and efflux processes. The mutant strain ∆e4 of the parental plasmid-free strain AE104 with a deletion of all four chromosomally encoded genes of previously known efflux systems ZntA, CadA, DmeF, and FieF was still able to efflux zinc in a pulse-chase experiment, indicating the existence of a fifth efflux system. The gene cdfX, encoding a protein of the cation diffusion facilitator (CDF) family, is located in proximity to the cadA gene, encoding a P-type ATPase. Deletion of cdfX in the ∆e4 mutant resulted in a further decrease in zinc resistance. Pulse-chase experiments with radioactive 65Zn(II) and stable-isotope-enriched 67Zn(II) provided evidence that CdfX was responsible for the residual zinc efflux activity of the mutant strain ∆e4. Reporter gene fusions with cdfX-lacZ indicated that the MerR-type regulator ZntR, the main regulator of zntA expression, was responsible for zinc- and cadmium-dependent upregulation of cdfX expression, especially in mutant cells lacking one or both of the previously characterized efflux systems, ZntA and CadA. Expression of zntR also proved to be controlled by ZntR itself as well as by zinc and cadmium availability. These data indicate that the cdfX-cadA region provides C. metallidurans with a backup system for the zinc-cadmium-exporting P-type ATPase ZntA, with CdfX exporting zinc and CadA cadmium.IMPORTANCEBacteria have evolved the ability to supply the important trace element zinc to zinc-dependent proteins, despite external zinc concentrations varying over a wide range. Zinc homeostasis can be understood as adaptive layering of homeostatic systems, allowing coverage from extreme starvation to extreme resistance. Central to zinc homeostasis is a flow equilibrium of zinc comprising uptake and efflux reactions, which adjusts the cytoplasmic zinc content. This report describes what happens when an imbalance in zinc and cadmium concentrations impairs the central inner-membrane zinc efflux system for zinc by competitive inhibition for this exporter. The problem is solved by activation of Cd-exporting CadA or Zn-exporting CdfX as additional efflux systems.

Activation, regulation, and effects of the mitochondrial unfolded protein response in endothelial cells

Abstract Introduction Endoplasmic reticulum stress and the resulting unfolded protein response (UPRER) play significant roles in endothelial dysfunction and atherosclerosis. However, the understanding of the mitochondrial unfolded protein response (UPRMito) in endothelial dysfunction is limited. This study aims to characterize the activation, regulation, and effects of the UPRMito to provide a comprehensive understanding of endothelial UPRMito. Methods and Results RNA sequencing and quantitative proteomic analysis were employed to investigate the impact of stressors on mitochondrial (MitoBlock-6, CDDO) and endoplasmic (Tunicamycin) protein homeostasis on the transcriptome and proteome of Human Coronary Artery Endothelial cells (HCAEC, Fig.1). The analysis identified 12,745 unique transcripts and 7,891 unique proteins. Mitocarta annotation highlighted the regulatory effect induced by these stressors, particularly on mitochondrial proteins and transcripts (Fig. 2). Geneset enrichment analyses revealed upregulation of pathways regulated by activating transcription factors 4/5 (ATF4/5) and the integrated stress response marker CHOP. Analysis of protein expression changes unveiled that 21 proteins were upregulated by both CDDO and MitoBlock-6, including essential mitochondrial chaperones (HSP10 and HSP60). In contrast, the UPRER stressor Tunicamycin did not induce canonical UPRMito activation. Additionally, 142 proteins were commonly downregulated, affecting crucial biological processes such as mitochondrial translation, mitochondrial gene expression, and ribosome biogenesis. Importantly, NAD+ADP-ribosyltransferase activity was highly downregulated, involving PARP proteins with roles in DNA damage repair. On a functional level, mitochondrial stressors led to dose-dependent alterations in HCAEC viability, apoptosis, and reactive oxygen species formation (ROS) measured by resazurin-based assays, caspase 3/7 activity, and MitoSox staining, respectively. Low doses and short incubations with mitochondrial stressors promoted endothelial cell health through cytoprotective UPRMito pathways, while prolonged incubation led to unresolvable mitochondrial stress (exemplarily shown in Fig. 3). SiRNA-mediated gene silencing of the UPRMito regulators ATF4 and ATF5 promoted mitochondrial ROS formation in cells treated with mitochondrial stressors. These effects were particularly more pronounced after ATF5-Knockdown, suggesting that ATF5 is the main regulator of the UPRMito in HCAEC (Fig. 4). Moreover, high-dose UPRMito stressors led to an impairment in mitochondrial membrane integrity with cytosolic release of pro-inflammatory mtDNA. Conclusion UPRMito and UPRER lead to a different stress response. Depending on the underlying conditions, both stress responses can exert both cytoprotective and cytotoxic effects. Further studies are required to analyze the in vivo relevance and possible therapeutic exploitation of the UPRMito in endothelial dysfunction and atherosclerosis.Figures

Lineage‐specific duplication of a CYCLOIDEA2 gene and the diversification of capitula in the tribe Anthemideae (Asteraceae)

AbstractGene duplications have contributed to the innovation of morphological traits during plant evolution. An outstanding example is the role of CYCLOIDEA2 (CYC2) gene duplications in the formation of the complex structure of Asteraceae capitula. Previous studies have shown that Chrysanthemum lavandulifolium (Fischer ex Trautv.) Makino and a few other species of the Asteraceae harbor two copies of CYC2e. Here, we identified a lineage‐specific CYC2e duplication event at the root of the phylogeny of the tribe Anthemideae by analyzing the evolutionary history of CYC2 genes across the Asteraceae. Although the gene expression patterns of CYC2e1 and CYC2e2 were similar in most of floral organs, CYC2e1 promoted ligule elongation by promoting cell expansion, whereas CYC2e2 showed a weak inhibiting effect on the ray‐floret development, and overexpression of CYC2e2 resulted in the maldevelopment of stamens in disc florets. These results indicated differentiated functions of CYC2e1 and CYC2e2 in the capitulum development of the Anthemideae. Given the previous data that identified CYC2g and CYC2d as main regulators in ligule morphogenesis, we comparatively analyzed 10 Asteraceae genomic data, and identified tandem arrays of CYC2g, CYC2d, and CYC2e. We further investigated the regulatory relationships between CYC2g/CYC2d and CYC2e genes, and found that CYC2g can activate the expression of CYC2e1. The findings of this study elucidate the synergistic roles of CYC2 genes in regulating the formation of the Asteraceae capitula and enhance understanding of the mechanism of duplicated gene retention during plant evolution.

Alternative sulphur metabolism in the fungal pathogen Candida parapsilosis

Candida parapsilosis is an opportunistic fungal pathogen commonly isolated from the environment and associated with nosocomial infection outbreaks worldwide. We describe here the construction of a large collection of gene disruptions, greatly increasing the molecular tools available for probing gene function in C. parapsilosis. We use these to identify transcription factors associated with multiple metabolic pathways, and in particular to dissect the network regulating the assimilation of sulphur. We find that, unlike in other yeasts and filamentous fungi, the transcription factor Met4 is not the main regulator of methionine synthesis. In C. parapsilosis, assimilation of inorganic sulphur (sulphate) and synthesis of cysteine and methionine is regulated by Met28, a paralog of Met4, whereas Met4 regulates expression of a wide array of transporters and enzymes involved in the assimilation of organosulfur compounds. Analysis of transcription factor binding sites suggests that Met4 is recruited by the DNA-binding protein Met32, and Met28 is recruited by Cbf1. Despite having different target genes, Met4 and Met28 have partial functional overlap, possibly because Met4 can contribute to assimilation of inorganic sulphur in the absence of Met28.

The ketone body β-hydroxybutyrate ameliorates neurodevelopmental deficits in the GABAergic system of daf-18/PTEN Caenorhabditis elegans mutants.

A finely tuned balance between excitation and inhibition (E/I) is essential for proper brain function. Disruptions in the GABAergic system, which alter this equilibrium, are a common feature in various types of neurological disorders, including autism spectrum disorders (ASDs). Mutations in Phosphatase and Tensin Homolog (PTEN), the main negative regulator of the phosphatidylinositol 3-phosphate kinase/Akt pathway, are strongly associated with ASD. However, it is unclear whether PTEN deficiencies can differentially affect inhibitory and excitatory signaling. Using the Caenorhabditis elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) inputs regulate muscle activity, we found that daf-18/PTEN mutations impact GABAergic (but not cholinergic) neurodevelopment and function. This selective impact results in a deficiency in inhibitory signaling. The defects observed in the GABAergic system in daf-18/PTEN mutants are due to reduced activity of DAF-16/FOXO during development. Ketogenic diets (KGDs) have proven effective for disorders associated with E/I imbalances. However, the mechanisms underlying their action remain largely elusive. We found that a diet enriched with the ketone body β-hydroxybutyrate during early development induces DAF-16/FOXO activity, therefore improving GABAergic neurodevelopment and function in daf-18/PTEN mutants. Our study provides valuable insights into the link between PTEN mutations and neurodevelopmental defects and delves into the mechanisms underlying the potential therapeutic effects of KGDs.

The ketone body β-hydroxybutyrate ameliorates neurodevelopmental deficits in the GABAergic system of daf-18/PTEN Caenorhabditis elegans mutants

A finely tuned balance between excitation and inhibition (E/I) is essential for proper brain function. Disruptions in the GABAergic system, which alter this equilibrium, are a common feature in various types of neurological disorders, including autism spectrum disorders (ASDs). Mutations in Phosphatase and Tensin Homolog (PTEN), the main negative regulator of the phosphatidylinositol 3-phosphate kinase/Akt pathway, are strongly associated with ASD. However, it is unclear whether PTEN deficiencies can differentially affect inhibitory and excitatory signaling. Using the Caenorhabditis elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) inputs regulate muscle activity, we found that daf-18/PTEN mutations impact GABAergic (but not cholinergic) neurodevelopment and function. This selective impact results in a deficiency in inhibitory signaling. The defects observed in the GABAergic system in daf-18/PTEN mutants are due to reduced activity of DAF-16/FOXO during development. Ketogenic diets (KGDs) have proven effective for disorders associated with E/I imbalances. However, the mechanisms underlying their action remain largely elusive. We found that a diet enriched with the ketone body β-hydroxybutyrate during early development induces DAF-16/FOXO activity, therefore improving GABAergic neurodevelopment and function in daf-18/PTEN mutants. Our study provides valuable insights into the link between PTEN mutations and neurodevelopmental defects and delves into the mechanisms underlying the potential therapeutic effects of KGDs.

Novel, soluble 3-heteroaryl-substituted tanshinone mimics attenuate the inflammatory response in murine macrophages

The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can lead to beneficial anti-inflammatory effects. To this aim, we previously synthesized a novel class of small molecules named Tanshinone Mimics (TMs) able to interfere with HuR-RNA binding, and that dampen the LPS-induced immune response. Herein, we present a novel series of TMs, encompassing thiophene 3/TM9 and 4/TM10, furan 5/TM11 and 6/TM12, pyrrole 7b/TM13, and pyrazole 8. The furan-containing 5(TM11) showed the greatest inhibitory effect of the series on HuR-RNA complex formation, as suggested by RNA Electromobility Shift Assay and Time-Resolved FRET. Molecular Dynamics Calculation of HuR − 5/TM11 interaction, quantum mechanics approaches and Surface Plasmon Resonance data, all indicates that, within the novel heteroaryl substituents, the furan ring better recapitulates the chemical features of the RNA bound to HuR. Compound 5/TM11 also showed improved aqueous solubility compared to previously reported TMs. Real-time monitoring of cell growth and flow cytometry analyses showed that 5/TM11 preferentially reduced cell proliferation rather than apoptosis in murine macrophages at immunomodulatory doses. We observed its effects on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages, showing that 5/TM11 significantly reduced the expression of proinflammatory cytokines as Cxcl10 and Il1b.

Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (PG1) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. PG1 and its improved derivatives PG1-3, D-PGS4 and D-PGS5, proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, PG13-controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the PG1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes.

The novel selective inhibitors of cyclin-dependent kinase 4/6: in vitro and in silico study.

One of the main regulators in the cell cycle is cyclin-dependent kinase 4 and 6 (CDK4/6). FDA has approved CDK4/6 inhibitors for the treatment of patients with metastatic breast cancer. However, the development of selective agents remains problematic due to the conservation of their ATP binding sites. In the previous in silico study, ZINC585292724, ZINC585292587, ZINC585291674, and ZINC585291474 have been identified as potential inhibitors. Therefore, the present study aimed to analyze the selectivity and inhibitory activity of the four compounds against CDK4/6 in vitro as well as determine the potential for their further development in silico. The in vitro results showed that the four compounds had good selectivity towards both kinases, due to their similar structure. In agreement with the in silico results, ZINC585291674 produced the best inhibitory activity against CDK4 and CDK6, with IC50 of 184.14nM and 111.78nM, respectively. Their ADMET profile were also similar to reference compound of Palbociclib. Based on this, ZINC585291674 can be used as a lead compound for further inhibitor development.

Metabolic regulation of mitochondrial morphologies in pancreatic beta cells: coupling of bioenergetics and mitochondrial dynamics

Cellular bioenergetics and mitochondrial dynamics are crucial for the secretion of insulin by pancreatic beta cells in response to elevated levels of blood glucose. To elucidate the interactions between energy production and mitochondrial fission/fusion dynamics, we combine live-cell mitochondria imaging with biophysical-based modeling and graph-based network analysis. The aim is to determine the mechanism that regulates mitochondrial morphology and balances metabolic demands in pancreatic beta cells. A minimalistic differential equation-based model for beta cells is constructed that includes glycolysis, oxidative phosphorylation, calcium dynamics, and fission/fusion dynamics, with ATP synthase flux and proton leak flux as main regulators of mitochondrial dynamics. The model shows that mitochondrial fission occurs in response to hyperglycemia, starvation, ATP synthase inhibition, uncoupling, and diabetic conditions, in which the rate of proton leakage exceeds the rate of mitochondrial ATP synthesis. Under these metabolic challenges, the propensities of tip-to-tip fusion events simulated from the microscopy images of the mitochondrial networks are lower than those in the control group and prevent the formation of mitochondrial networks. The study provides a quantitative framework that couples bioenergetic regulation with mitochondrial dynamics, offering insights into how mitochondria adapt to metabolic challenges.

Gas Chromatography Tandem Mass Spectrometry (GC-MS/MS) for High-Throughput Screening of Pesticides in Rice Samples Collected in Bangladesh and Saudi Arabia

Gas Chromatography Tandem Mass Spectrometry (GC-MS/MS) with modified QuEChERS sample preparation has been applied to the high-throughput screening of pesticide residuals in rice collected from Bangladesh and Saudi Arabia markets. Both countries consume high volumes of rice, which is a fundamental food for their populations. We report optimized sample preparation and mass spectrometry analysis protocols, which can be rapidly deployed in analytical laboratories. The screening of four groups (organophosphorus, synthetic pyrethroid, organonitrogen, and organochlorine) of a total of 115 pesticides can be performed within ~10 min using a matrix-matched calibration. For most compounds, the limits of detection and quantification (LOD/LOQ) are well below the maximum residue levels (MRLs) of the main regulators. The method generally demonstrates acceptable recovery values (91 compounds 75–125% and 10 compounds 30–75%). Out of 55 rice samples analyzed, 16 samples (29%) contained pesticide residues above LOQ. Four samples contained chlorpyrifos with concentrations ranging from 21.3 to 71.9 µg/kg, ten samples contained tebuconazole (34.7–69.0 µg/kg), and three samples contained pirimiphos methyl (10.7–20.7 µg/kg). The concentrations of the pesticide residues detected in these samples are well below MRL of FAO/WHO (chlorpyrifos, 500 µg/kg; tebuconazole, 1500 µg/kg; pirimiphos methyl, 7000 µg/kg).

7424 Measurements of Key Regulators of Energy Intake in Young and Midlife Women with Overweight or Obesity

Abstract Disclosure: E. Tama: None. R. Rivera: None. S. Fansa: None. D. Anazco: None. W. Ghusn: None. S. Faubion: None. A. Acosta: Consulting Fee; Self; Amgen Inc. Grant Recipient; Self; Vivus USA, Novo Nordisk. C. Shufelt: None. M.D. Hurtado: None. Introduction: Midlife women (ages 40 to 65) experience age-related weight gain (0.4-0.7 kg/year) due to physiologic and psychosocial changes. Evidence supports that age-related decrease in lean mass and its subsequent effect on energy expenditure are key physiologic factors. To date, the are no data describing if changes in the main regulators of energy intake (i.e., hunger, satiation, and satiety) across women’s lifespan play a role in weight gain during midlife. The aim of this study is to characterize measurements of satiation, satiety, and appetite sensations through early adulthood and midlife in women with obesity. Methods: This is a cross sectional study of women aged 19-65 with overweight or obesity who have undergone measurements of (i) body composition by DXA scan; (ii) resting energy expenditure (REE) by indirect calorimetry; (iii) satiation by calories consumed to satiation (CTS) during an ad libitum meal test; (iv) satiety by time needed to empty 50% of the stomach content (GEt1/2) in a gastric emptying study and plasma satiety hormone levels (PYY, GLP-1, and CCK); and (v) appetite sensations by visual analog scales (VAS) for hunger and fullness. We present data as mean ± standard deviation. We performed simple correlations between these variables and age. We performed multiple regression analyses to adjust for body composition and REE. Results: Our cohort included 552 mostly white (93%) women, with a mean age of 41±11 years and a mean BMI of 37±7 kg/m2. Increased age significantly correlated with higher waist circumference (r=0.1, p=0.006), waist/hip ratio (r=0.1, p=0.01), and total fat mass % (r=0.1, p=0.04); and with lower lean mass % (r=-0.1, p=0.04) and REE (r=-0.3, p<0.0001). Increased age was associated with decreased hunger sensation (i.e., lower preprandial VAShunger after an overnight fast, r=-0.1, p=0.04; and lower postprandial VAShunger 4hr after a standardized 320-kcal breakfast, r=-0.1, p=0.001), stronger satiation (i.e., lower CTS, r=-0.1, p=0.001), and more prolonged satiety (i.e., higher GEt1/2, r=0.1, p=0.02; higher postprandial GLP-1 levels, r=0.1, p=0.03; and higher postprandial VASfullness after and ad libitum meal, p=0.1, p=0.008). After adjusting for lean mass % and REE, the correlations between age and hunger sensations and satiation measurement, but not satiety measurements, were maintained. Conclusion: Our results show that older women have lower lean mass % and REE. After adjusting for these variables, older women experience decreased hunger sensation and achieve satiation with a lower number of calories. As these changes in theory would protect against midlife weight gain, the differences in body composition and REE remain the most likely culprit. Further research is necessary to investigate if there are other key factors that may contribute to weight gain such as the hedonic or emotional components of food intake. Presentation: 6/2/2024