Cellular Production Research Articles

ADP-Ribosylation Factor-Interacting Protein 2 Acts as a Novel Regulator of Mitophagy and Autophagy in Podocytes in Diabetic Nephropathy.

(1) Background: Differentiated podocytes are particularly vulnerable to oxidative stress and cellular waste products. The disease-related loss of postmitotic podocytes is a direct indicator of renal disease progression and aging. Podocytes use highly specific regulated networks of autophagy and endocytosis that counteract the increasing number of damaged protein aggregates and help maintain cellular homeostasis. Here, we demonstrate that ARFIP2 is a regulator of autophagy and mitophagy in podocytes both in vitro and in vivo. (2) Methods: In a recent molecular regulatory network analysis of mouse glomeruli, we identified ADP-ribosylation factor-interacting protein 2 (Arfip2), a cytoskeletal regulator and cofactor of ATG9-mediated autophagosome formation, to be differentially expressed with age. We generated an Arfip2-deficient immortalized podocyte cell line using the CRISPR/Cas technique to investigate the significance of Arfip2 for renal homeostasis in vitro. For the in vivo analyses of Arfip2 deficiency, we used a mouse model of Streptozotozin-induced type I diabetes and investigated physiological data and (patho)histological (ultra)structural modifications. (3) Results: ARFIP2 deficiency in immortalized human podocytes impedes autophagy. Beyond this, ARFIP2 deficiency in human podocytes interferes with ATG9A trafficking and the PINK1-Parkin pathway, leading to the compromised fission of mitochondria and short-term increase in mitochondrial respiration and induction of mitophagy. In diabetic mice, Arfip2 deficiency deteriorates autophagy and leads to foot process effacement, histopathological changes, and early albuminuria. (4) Conclusions: In summary, we show that ARFIP2 is a novel regulator of autophagy and mitochondrial homeostasis in podocytes by facilitating ATG9A trafficking during PINK1/Parkin-regulated mitophagy.

Stabilization of myoglobin from different species (produced by cellular agriculture) using food-grade natural and synthetic antioxidants

Cellular agriculture products, like myoglobin, are increasingly used by the food industry to provide desirable sensory properties to plant-based meat substitutes. This study elucidated the physicochemical properties and redox stability of myoglobin from both natural (equine) and cellular agriculture (bovine, sperm whale, and leopard) sources. The electrical characteristics and water-solubility of the different myoglobin samples were measured from pH 2.5 to 8.5. The isoelectric point of the myoglobin samples depended on the species, being pH 5.5 for equine, pH 4.5 for leopard and bovine, and pH 6.5 for sperm whale. All myoglobin samples had a solubility greater than 80% across the entire pH range studied. All myoglobin solutions appeared red and had two peaks in their UV–visible absorbance spectra after one day, which is consistent with oxymyoglobin formation. Equine myoglobin at pH 8 was selected to study its redox and color stability over time, where the oxymyoglobin oxidative status closely paralleled with the redness of the solutions. The effects of antioxidants (ascorbic acid, caffeic acid, catechin, gallic acid, quercetin, taxifolin, Trolox, and 4-methylcatechol) on the redox and color stability (redness) of the equine myoglobin (pH 8.0) was also studied. Antioxidants with low reduction potential values (ascorbic acid and quercetin) were particularly effective at enhancing the color stability of oxymyoglobin. The computational modeling study showed that amino acids on the myoglobin interacted with antioxidants through hydrogen bonds. The insights obtained may have important implications for the use of cellular agriculture to produce myoglobin for food applications.

Structural insights into respiratory complex I deficiency and assembly from the mitochondrial disease-related ndufs4−/− mouse

Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4−/− mouse, to define the structural, biochemical, and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of the complex I from ndufs4−/− mouse hearts revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12, which replaces its paralogue NDUFAF2 in mature complex I, is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free enzyme. We propose that NDUFAF2 recruits the NADH-dehydrogenase module during assembly of the complex. Taken together, the findings provide new molecular-level understanding of the ndufs4−/− mouse model and complex I-linked mitochondrial disease.

Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.

Short communication: ROS production and mitochondrial membrane potential in hemocytes of marine bivalves, Mytilus galloprovincialis and Magallana gigas, under hypoosmotic stress.

Bivalve mollusks that inhabit low-depth coastal and estuarine areas frequently experience osmotic stress that may be also associated with alterations of antioxidant enzyme activities and markers of oxidative stress. Mitochondria are a major source of reactive oxygen species (ROS) in eucaryotic cells. Overpoduction of ROS induces oxidative stress leading to a damage of intracellular compartments and cell death. In euryhaline bivalves, information concerning cellular ROS production upon osmotic stress and changes in mitochondrial membrane potential is scarce. The present study investigates osmotic stability and hemocytes` regulatory volume decrease (RVD) of Mediterranean mussel (Mytilus galloprovincialis) and the Pacific oyster (Magallana gigas). We also studied dynamic changes in intracellular ROS levels and mitochondrial membrane potential in hemocytes undergoing the RVD response following hypoosmotic swelling. Our data revealed that osmotic stability of mussel and oyster hemocytes did not significantly differ. Loss of environmental osmolarity from 460.0±2.0mOsml-1 to 216.0±4.0mOsml-1 resulted in an increase of hemocyte volume by 60% of the initial cellular volume in mussels and by 28% in oysters. After rapid hypoosmotic swelling hemocytes of both species demonstrated the RVD response. At the end of 60min exposure to hypoosmotic environment, hemocyte volume significantly decreased in both species by 10-12% compared to the maximal hemocyte volume. Hypoosmotic shock induced an increase of mitochondrial membrane potential in hemocytes of mussels and oysters. In mussels, increased mitochondrial membrane potential was accompanied with decreased ROS levels in hemocytes, whereas oyster hemocytes showed enhanced ROS production.

Spatial and Single-Cell Investigations Illuminate Theragnostic value and Immune Landscape of Mitochondrial Dynamin-Like GTPase in Breast Cancer.

Background: As we delve into the intricate world of mitochondrial inner membrane proteins, particularly Optic Atrophy types 1 and 3 (OPA1/3), we uncover their pivotal role in maintaining mitochondrial dynamic equilibrium and fusion, crucial for cellular energy production and synthesis. Despite extensive scrutiny, the significance of OPA1/3 in breast cancer (BRCA) and its interplay with the immune microenvironment remain elusive. Materials and Methods: We meticulously sourced BRCA data from renowned repositories such as The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Gene Expression Omnibus (GEO), and the Human Protein Atlas (HPA), leveraging cutting-edge techniques including single-cell RNA-sequencing (scRNA-seq), spatial transcriptomics, and pharmacogenomics. Through multifaceted data analysis, we endeavored to unravel the intricate role and potential value of OPA1/3 in BRCA tumorigenesis and progression. Results: Our investigation reveals a conspicuous upregulation of OPA1/3 expression in BRCA, correlating with dismal prognoses. Kaplan-Meier plot analysis underscores that heightened OPA1/3 levels are associated with poor survival rates. Both clinical specimens and biobank biopsies corroborate the elevated expression of OPA1/3 in breast cancer patients. Moreover, scRNA-seq unveils a strong correlation between OPA1/3 and macrophage infiltration in the BRCA immune milieu, alongside its association with the cellular communication network involving CXCL, TGFb, VEGF, and IL16. Conclusion: In light of these findings, OPA1/3 emerges as a promising contender for therapeutic targeting and as a potential diagnostic, prognostic, and survival biomarker in BRCA. The implications of our study underscore the pressing need to explore these novel biomarkers to enhance patient outcomes.

Environmental endocrine disruptor-induced mitochondrial dysfunction: a potential mechanism underlying diabetes and its complications.

Diabetes and its complications significantly affect individuals' quality of life. The etiology of diabetes mellitus and its associated complications is complex and not yet fully understood. There is an increasing emphasis on investigating the effects of endocrine disruptors on diabetes, as these substances can impact cellular processes, energy production, and utilization, ultimately leading to disturbances in energy homeostasis. Mitochondria play a crucial role in cellular energy generation, and any impairment in these organelles can increase susceptibility to diabetes. This review examines the most recent epidemiological and pathogenic evidence concerning the link between endocrine disruptors and diabetes, including its complications. The analysis suggests that endocrine disruptor-induced mitochondrial dysfunction-characterized by disruptions in the mitochondrial electron transport chain, dysregulation of calcium ions (Ca2+), overproduction of reactive oxygen species (ROS), and initiation of signaling pathways related to mitochondrial apoptosis-may be key mechanisms connecting endocrine disruptors to the development of diabetes and its complications.

Research Progress of Coenzyme Q in Diabetes Mellitus and Its Common Complications.

Coenzyme Q has garnered significant attention due to its potential role in enhancing cellular energy production and its antioxidant properties. We delve into the therapeutic potential of coenzyme Q in managing diabetes mellitus and its complications, highlighting its capacity to improve mitochondrial function, reduce inflammation and oxidative stress, and correct lipid profiles. Coenzyme Q has shown promise in ameliorating insulin resistance and alleviating complications such as diabetic peripheral neuropathy, kidney disease, retinopathy, and cardiomyopathy. However, its clinical application is limited by poor bio-availability. This review also provides a comprehensive overview of current therapeutic strategies for diabetes complications involving coenzyme Q, including stimulating endogenous synthesis and utilizing carrier transport systems, offering insights into mechanisms for enhancing coenzyme Q bio-availability. These findings suggest that, with improved delivery methods, coenzyme Q could become a valuable adjunct therapy in the management of diabetes mellitus.

Mechanisms of the NAD+ salvage pathway in enhancing skeletal muscle function.

Nicotinamide adenine dinucleotide (NAD+) is crucial for cellular energy production, serving as a coenzyme in oxidation-reduction reactions. It also supports enzymes involved in processes such as DNA repair, aging, and immune responses. Lower NAD+ levels have been associated with various diseases, highlighting the importance of replenishing NAD+. Nicotinamide phosphoribosyltransferase (NAMPT) plays a critical role in the NAD+ salvage pathway, which helps sustain NAD+ levels, particularly in high-energy tissues like skeletal muscle.This review explores how the NAMPT-driven NAD+ salvage pathway influences skeletal muscle health and functionality in aging, type 2 diabetes mellitus (T2DM), and skeletal muscle injury. The review offers insights into enhancing the salvage pathway through exercise and NAD+ boosters as strategies to improve muscle performance. The findings suggest significant potential for using this pathway in the diagnosis, monitoring, and treatment of skeletal muscle conditions.

Long-term culture of patient-derived mammary organoids in non-biogenic electrospun scaffolds for identifying metalloprotein and motor protein activities in aging and senescence.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotypein tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactonescaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids andpatient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

Role of Lipoic Acid in Testosterone Production in Males.

Testosterone extends its impact beyond sexual function, playing a crucial role in shaping overall male health, including aspects such as muscle mass, bone density, mood regulation, and energy levels. Lipoic acid, a cofactor for specific enzymes, particularly dehydrogenases involved in cellular energy production, has been studied for its impact on testosterone. This comprehensive review systematically scoured PubMed and Scopus databases using the keywords "lipoic acid" and "testosterone." It encompassed all relevant English papers published from November 1971 to the present, including full texts and abstracts, along with research elucidating the biochemical mechanisms linking lipoic acid to testosterone. In summary, lipoic acid consistently restores testosterone levels, offering promise as an intervention in testicular health, especially in cases of testicular toxicity caused by various harmful agents. Its mechanisms encompass nitric oxide enhancement, fortification of testicular antioxidants, elevation of luteinizing hormone, enhancement of steroidogenesis, and the maintenance of energy production. These mechanisms underscore the therapeutic potential of lipoic acid for testicular health.

IPSC-derived NK cells expressing high-affinity IgG Fc receptor fusion CD64/16A to mediate flexible, multi-tumor antigen targeting for lymphoma.

NK cells can mediate tumor cell killing by natural cytotoxicity and by antibody-dependent cell-mediated cytotoxicity (ADCC), an anti-tumor mechanism mediated through the IgG Fc receptor CD16A (FcγRIIIA). CD16A polymorphisms conferring increased affinity for IgG positively correlate with clinical outcomes during monoclonal antibody therapy for lymphoma, linking increased binding affinity with increased therapeutic potential via ADCC. We have previously reported on the FcγR fusion CD64/16A consisting of the extracellular region of CD64 (FcγRI), a high-affinity Fc receptor normally expressed by myeloid cells, and the transmembrane/cytoplasmic regions of CD16A, to create a highly potent and novel activating fusion receptor. Here, we evaluate the therapeutic potential of engineered induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as an "off-the-shelf", antibody-armed cellular therapy product with multi-antigen targeting potential. iNK cells were generated from iPSCs engineered to express CD64/16A and an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein for cytokine independence. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells to examine in in vitro and in vivo assays using the Raji lymphoma cell line. ADCC was evaluated in real-time by IncuCyte assays and using a xenograft mouse model with high circulating levels of human IgG. Our data show that CD64/16A expressing iNK cells can mediate potent anti-tumor activity against human B cell lymphoma. In particular, (i) under suboptimal conditions, including low antibody concentrations and low effector-to-target ratios, iNK-CD64/16A cells mediate ADCC, (ii) iNK-CD64/16A cells can be pre-loaded with tumor-targeting antibodies (arming) to elicit ADCC, (iii) armed iNK-CD64/16A cells can be repurposed with additional antibodies to target new tumor antigens, and (iv) cryopreserved, armed iNK-CD64/16A are capable of sustained ADCC in a tumor xenograft model under saturating levels of human IgG. iNK-CD64/16A cells allow for a flexible use of antibodies (antibody arming and antibody targeting), and an "off-the-shelf" platform for multi-antigen recognition to overcome limitations of adoptive cell therapies expressing fixed antigen receptors leading to cancer relapse due to antigen escape variants.

Insights into the role of legionella effectors on host metabolic perturbations.

Legionella infection, the causative agent of Legionnaires' disease, represents a significant threat to human health. The pathogenesis of this infection is intricately linked to the complex interactions between the bacterium and its host, resulting in profound metabolic perturbations. Central to these metabolic shifts is the bacterium's modulation of lipid metabolism, with changes in lipid synthesis and breakdown modifying membrane composition and function. These alterations can influence cellular signaling and immune responses, further contributing to disease progression. It also disrupts glucose utilization and lipid metabolism, altering cellular energy production and immune responses. Additionally, Legionella infection perturbs amino acid and protein metabolism, affecting protein synthesis and degradation, leading to changes in cellular functions and immune responses. This mini-review underscores the complexity of metabolic perturbations in Legionella infection and their significance in host-pathogen interactions. Understanding these metabolic shifts provides valuable insights into the pathogenesis of Legionnaires' disease and could lead to the development of novel therapeutic strategies.

Isolation, Identification and Screening of Potential Marine Actinomycete for Lipase Production under Submerged Fermentation by using Tributyrin substrate

Ninety-six isolates were collected at various depths and sea shores of the Bay of Bengal near Visakhapatnam Coastal of Andhra Pradesh. These isolates were screened for the isolation of lipase activity by tributyrin agar clearing method and cultured under submerged fermentation and assayed for extra cellular lipase production by using tributyrin as substrate. Cultural characteristics of the selected strain was done by incorporating different media. Various parameters namely morphological, biochemical, physiological and molecular were used for the characterization and identification of actinomycetes isolates. Results indicated that all the isolates showed lipolytic activity after primary screening. The marine isolate T9 was the most active one thus the same was selected for production of lipase. The 16S rDNA amplification for phylogenetic study revealed that the isolate was highly related to Nocardiopsis dassonvillei. In conclusion, these results increased the scope of finding industrially important marine actinomycetes from Visakhapatnam Coastal of Andhra Pradesh.

Evaluating the Number of Cellular and/or Tissue-Based Product Applications Required to Treat Diabetic Foot Ulcers and Venous Leg Ulcers in Non-Hospital Outpatient Department Settings

Evaluating the Number of Cellular and/or Tissue-Based Product Applications Required to Treat Diabetic Foot Ulcers and Venous Leg Ulcers in Non-Hospital Outpatient Department Settings

Construction of Mn-N-C nanoparticles with multienzyme-like properties and photothermal performance for the effective treatment of bacterial infections.

In this work, we successfully constructed Mn-coordinated nitrogen-carbon nanoparticles (Mn-N-C NPs) exhibiting multienzyme-like activities. In a bacterial infectious microenvironment, the POD-like and OXD-like activities of Mn-N-C NPs could synergistically trigger the generation of ROS (˙OH and O2˙-), causing oxidative damage to the bacterial cell membrane for killing bacteria. Alternatively, in neutral or weak alkaline normal tissues, the excessive O2˙- could be converted into O2 and H2O2via the SOD-like ability of Mn-N-C NPs, and subsequently their CAT-like activity catalyzed excess H2O2 into H2O and O2 for protecting normal cells through the antioxidant defense. Mn-N-C NPs also possessed a good NIR-photothermal performance, which could enhance their POD-like and OXD-like activities. Furthermore, Mn-N-C NPs could facilitate the GSH oxidation process and disrupt the intrinsic balance in the bacterial protection microenvironment with the assistance of H2O2, which is beneficial for rapid bacterial death. Undoubtedly, the Mn-N-C NPs + H2O2 system showed the highest antibacterial activity when irradiated with an 808 nm laser, destroying the bacterial membrane and causing the efflux of proteins. Moreover, the Mn-N-C NPs + H2O2 system was immune to the development of bacterial resistance and could efficiently disrupt the formation of a bacterial biofilm with negligible cytotoxicity and low hemolysis ratio. Finally, Mn-N-C NPs exhibited an excellent antibacterial performance in vivo and could accelerate wound healing without cellular inflammation production. Therefore, due to their significant therapeutic effects, Mn-N-C NPs show great potential in fighting antibiotic-resistant bacteria.

A Multicenter Retrospective Study Comparing a Polylactic Acid CAMP With Intact Fish Skin Graft or a Collagen Dressing in the Management of Diabetic Foot Ulcers and Venous Leg Ulcers

Background. Venous leg ulcers (VLUs) and diabetic foot ulcers (DFUs) are examples of chronic wounds that pose an ongoing health care challenge. Despite significant progress in managing such wounds, challenges persist that require innovative solutions. Cellular, acellular, and matrix-like products (CAMPs) are advanced therapies designed to facilitate chronic wound healing. Polylactic acid (PLA) is a synthetic material used in alloplastic CAMPs that has shown promise in the management of burns and traumatic injuries. Objective. To retrospectively assess the effect of PLA in promoting chronic wound healing compared with 2 other well-established CAMPs. Materials and Methods. This retrospective chart review, which was conducted in 3 high-volume wound care clinics, aimed to compare the efficacy of 2 advanced wound care products vs a PLA alloplastic CAMP in promoting DFU and VLU closure. The study included 131 wounds treated with a non-CAMP collagen dressing, fish skin grafts (FSGs), or PLA matrices. Inclusion criteria included single Wagner grade 1 or 2 DFUs or Clinical-Etiology-Anatomy-Pathophysiology classification score C6 VLUs, present for at least 12 to 52 weeks, and measuring between 1 cm² and 20 cm². Patients received weekly treatments with 1 of 3 CAMPs, along with standard care. The primary outcome was time to achieve full healing, and the secondary outcome was the proportion of ulcers healed at 12 weeks. Results. The PLA alloplastic CAMP demonstrated superior outcomes, with a 50% and 20% reduction in time to heal DFUs compared with collagen dressings and FSG, respectively (P < .001). For VLUs, PLA exhibited even more impressive results, achieving 95% and 40% reduction compared with collagen and FSG, respectively (P < .001). PLA allografts displayed a 55% higher rate of full healing by 12 weeks compared with collagen dressings, and a 26% higher rate compared with FSG (P < .001). Conclusion. This study highlights the unique attributes of PLA for achieving wound closure. PLA-based alloplastic CAMPs are promising treatments, offering rapid healing, increased closure rates, and multifaceted benefits for wound healing.

A viable hypomorphic mutation in the mitochondrial ribosome subunit, MRPS-31, exhibits mitochondrial dysfunction in C. elegans.

The mitochondrial ribosome (mitoribosome) translates mitochondrial genome encoded proteins essential for cellular energy production. Given this critical role, defects in the mitoribosome can cause mitochondrial stress and manifest as multisystemic diseases. In a screen for unique activators of the mitochondrial unfolded protein response (UPR mt ) in Caenorhabditis elegans , we recovered a strain harboring a missense mutation in the gene encoding mitochondrial ribosome protein S31 ( MRPS-31 )-a component of the mitoribosome small subunit. Herein, we confirm causality of the mrps-31 allele and characterize its induction of UPR mt and impact on organismal development, providing a valuable model for further study of the mitoribosome.

Using Small Molecules for Targeting Heavy Metals in Neurotoxicity and Neuroinflammation.

Pharmaceutical drugs, natural toxins, industrial chemicals, and various environmental toxins negatively impact the nervous system. A significant cause of many neurodegenerative diseases is neurotoxicity. Although trace amounts of heavy metals are required for the proper functioning of several metabolic pathways, their dysregulation can cause many cellular and molecular alterations, which can enhance the risks associated with several neurodegenerative diseases. For example, high levels of heavy metals like manganese (Mn) affect the central nervous system with implications in both higher-order cognitive and motor functions. In addition, the buildup of amyloid aggregates and metal ions in the brain of patients with Alzheimer's disease is associated with disease pathogenesis. Small molecules capable of targeting neuroinflammation and neuroprotection pathways would be valuable to elucidate the pathological pathways associated with metal toxicity in neurogenerative disease. This chapter will summarize the necessary steps involved in (1) culturing of cell lines and maintenance of animal models, (2) design and preparation of samples of small molecules and treatment methodologies, (3) RNA and protein isolation and preparation of tissue and cell culture samples for quantitative studies, and (4) quantitative estimation of cellular products.

Guarana (Paullinia cupana) as a potential tool for mesenchymal stromal cells priming in regenerative medicine.

Mesenchymal stromal cells (MSCs) have therapeutic potential due to their abilities of differentiation, immunomodulation, and migration to injured tissues, potentiating such effects when cells are activated. Guarana (Paullinia cupana) is a tropical plant species found in South America that is known for its antioxidant, stimulant, and cicatricial effects. The guarana extract is composed of many substances and caffeine is the main component. The objective was to evaluate the effects of guarana and caffeine on MSCs. After the initial characterization, MSCs were treated with Paullinia cupana (10, 100, and 1000 μg/mL) or caffeine (0.4, 4, and 40 μg/mL) for 24 h. MSCs treatment with 1000 μg/mL guarana increased cell polarity, viability, cell migration to chemoattractant, antioxidant potential, and liberation of extracellular vesicles (EVs), while it reduced the levels of autophagy. MSCs treated with 100 and 1000 μg/mL guarana or 40 μg/mL caffeine showed a decrease of cell proliferation. No treatment affected the cellular area and cell cycle of MSCs. The study shows in vitro evidence that guarana could be a promising alternative for activating MSCs to promote better cellular products for future clinical therapies.