In mammals, CMs during development are diploid and mononucleated. In response to cardiac maturation, CMs undergo polyploidization and binucleation associated with CM functional changes. The transition from mononucleation to binucleation coincides with unique metabolic changes and shift in energy generation. Recent studies provide evidence that metabolic reprogramming promotes CM cell cycle reentry and changes in ploidy and nucleation state in the heart that together enhances cardiac structure and function after injury. This review summarizes current literature regarding changes in CM ploidy and nucleation during development, maturation and in response to cardiac injury. Importantly, how metabolism affects CM fate transition between mononucleation and binucleation and its impact on cell cycle progression, proliferation and ability to regenerate the heart will be discussed. The adult heart is made up of cardiomyocytes (CMs) that maintain pump function but are unable to divide and form new myocytes in response to myocardial injury. In contrast, the developmental cardiac tissue is made up of proliferative CMs that regenerate injured myocardium.  The adult mammalian heart is characterized by limited capacity to repair and regenerate ,2,3. GraCMs possess ability to proliferate during early postnatal stages but lose it by postnatal day 7. Before permanent cell cycle withdrawal, postnatal CMs go through a last wave of incomplete cell cycle, characterized by the uncoupling of karyokinesis from cytokinesis, thus triggering CM binucleation ,38. FielSeveral studies on cardiac development show that the yield of DNA replication in the adult murine heart is a topic still debated. However, immunofluorescence analysis with tritiated thymidine or BrdU staining performed in murine heart suggest that the number of CMs entering the cell cycle in an adult heart is very low ,50. NotaCurrently, the biggest challenge in basic cardiovascular science is to eliminate the cardiomyocytes loss, found during several cardiovascular disorders and improve heart regeneration. As largely described, the CMs maintain proliferation ability during the pre-natal phase thus favoring reparative and regenerative capability of the heart following injuries. Conversely, the adult myocardium shows a very low power of regeneration unable to ensure an effective replacement of the dead cells upon damage . The mamEvidence from literature suggests changes in polyploidy and nucleation impact postnatal CMs transition between proliferation and terminal differentiation .However, the trigger signals of this endo-nuclear mechanism are still debated. Interestingly, Fink and coworkers have observed significant changes occurring in early postnatal development in murine hearts . After bBesides morphology and structural adaptations, the postnatal CMs show specific electric modifications in response to physiological stimuli that occur during fate transition: from proliferation to terminal differentiation. Interestingly, Chen et al. have described a different electrical phenotype exhibited by mononucleated and binucleated CMs resident in the left atrium and pulmonary vein. Notably, the binucleated CMs showed significant arrhythmogenic activity with increased expression in gap junctions and intercalary discs thus allowing a fast propagation of the electrical impulse through the entire organ.Additionally, ploidy also influenced calcium dynamics in CMs, increasing the expression of genes involved in calcium handling/excitation-contraction coupling pathways . Lastly,Multinucleation and polyploidy represent typical characteristics in mammalian post- natal CMs, coinciding with terminal differentiation when most CMs become acytokinetic . AdditioCardiac hypertrophy and polyploidization are closely connected and essential for maintaining CM function . SpecifiHowever, besides the pathological and deleterious hypertrophic phenotype found in post-myocardial injuries, the exercise training also may promote a progressive increase in cardiac cell size thus resulting in a new physiological phenotype of heart hypertrophy, especially found in athletes .Interestingly, a current investigation has described a significant enhancement in murine mature CM proliferation after exercise training accompanied by a slight increase in cardiac polyploidization . TherefoAlthough, aging is characterized by a significant decrease in cardiac mass this is not accompanied by a parallel polyploidy reduction . ConversSeveral reports have elucidated the possibility to regulate polyploidization mechanism in cardiac tissue, through manipulation of specific modulators involved in cell replication, thus promoting heart cell proliferation  105,106106. CellAnother specific cardiac cell-cycle target to regulate polyploidization is YAP1 (Yes-associated protein). This transcriptional effector belonging to the Hippo signaling pathway is involved in heart cell proliferation, improving cardiac function  and cellTherefore, it’s reasonable to hypothesize that Hippo/YAP-1 signaling axis might represent a novel promising target to treat cardiometabolic disorders, through the modulation of the cardiac cell cycle and regeneration mechanism . In thisMNDCMs formation, thus supporting heart regeneration after injury [Defects in chromosome segregation might induce cytokinesis dysfunction, which in turn can trigger the apoptotic responses to promote carcinogenesis and can impact polyploidization in the heart. Notably, the impaired mitotic microtubule distribution is accompanied by aberrant actomyosin ring anchorage and can result in significant increase of binucleated CMs. Interestingly, a recent study described severe alterations in CM cytokinesis during development after deletion of growth arrest-specific 2 like 3 (Gas2l3) protein. Gas2l3 is a crucial cytoskeletal linker protein, involved in stabilizing and formation of the actin and microtubule network. Gas2l3 knock-out mice displayed multinucleated cardiac cells, thus suggesting its pivotal role in the heart polyploidization process . Similarr injury ,132. AccYekelchyk recently described a similar transcriptome profiling exhibited by mono- and multi-nucleated mouse adult ventricular myocytes. Notably, through single-cell RNA-sequencing the investigators evaluated the heterogeneity among CMs both in the physiological and pathological conditions, observing identical sets of genes expressed by individual mono- and multi-nucleated CMs [Conversely, significant phenotypic differences emerged in the dynamics of cell renewal and turnover in the human heart. Interestingly, ated CMs . TherefoIn mammals during development, CMs are all mononucleated and diploid (MNDCMs) with strong inherent replicative potential. In fact, MNDCMs retain the embryonic/fetal gene imprinting, essential for the regeneration of the cardiac tissue, even after injury ,100,134.Over the years, studies have shown that the energetic state of cells can impact ploidy, cell size and function that can then be utilized by cell signaling cascades to implement specific growth and functional endpoints ,148. In Dysregulated adenosine 5′-diphosphate: adenosine 5′-triphosphate (ADP:ATP) homeostasis is linked to development of hypertrophic cardiomyopathy (HCM) . CardiacStudies recently have implicated metabolic mechanisms to significantly participate in the polyploidization process and cell cycle regulation in the heart .The main metabolic feature of proliferating CMs is enhanced dependence on glycolysis for energy generation . HoweverTherefore, based on this evidence there is a greater need to explore how CM metabolic processes regulate polyploidization and cell cycle in the heart. This may lead to identification of a novel potential molecular target that regulates both cell proliferation and polyploidization in the heart post injury. Most likely, the study of cardiac mitochondria might represent a future cellular target to manipulate cardiac regeneration. Are the responses to control the CM cell cycle hidden in the mitochondria?Based on a long body of evidence and convincing data, we think that innovative therapeutic approaches are needed to favor reparative mechanisms in the mature heart after injury, promoting the CM cell cycle and DNA replication. In this regard, it would be interesting to dissect the pathophysiological role played by the polyploidization process. Indeed, this mechanism could represent the future key solution to aid the biggest challenge in cardiovascular field: reprogramming CM cell cycle to promote cardiac regeneration post injury. Nevertheless, the specific biologic consequence of the increased CM polyploidization in the adult heart is still debated. Although, a large amount of experimental evidence demonstrates pro-regenerative effects of mononucleated diploid cardiac cells, in parallel other data shows that the polyploidization induces the CM cell cycle exit thus limiting their proliferation and cardiac regeneration. Interestingly, the progressive maturation of CMs is accompanied by a gradual increase in oxidative stress and ROS production with significant mitochondria deterioration and chromatin remodeling, resulting in polyploidization and cell cycle arrest. Thus, we speculate that cardiac mitochondria may modulate the chromatin architecture in CMs thus driving heart proliferation. Moreover, literature regarding hepatic cells polyploidization regulatory processes may represent another promising therapeutic strategy to identify key modulators possibly active in the CMs. Therefore, future approaches that focus on stimulation of cardiac regeneration post injury should scrupulously evaluate the impact of both polyploidization and proliferation to avoid any imbalance in the CM regeneration processes.


According to Globocan 2020 data, approximately 308,102 new cases of brain cancer and central nervous system (CNS) cancers were reported worldwide, with 251,329 reported deaths. Reactive oxygen and nitrogen species (RONS) alter various signaling pathways to regulate cell stability. In this review, we will discuss the altered metabolism in brain cancer and emphasize the role of reactive oxygen species (ROS), reactive nitrogen species (RNS), and the tumor microenvironment in controlling metabolic shifts in brain cancer. We will also highlight potential therapies for targeting ROS, inhibiting cancer cell proliferation, and addressing gliomal stem-like cells.Brain cancer is known as one of the deadliest cancers globally. One of the causative factors is the imbalance between oxidative and antioxidant activities in the body, which is referred to as oxidative stress (OS). As part of regular metabolism, oxygen is reduced by electrons, resulting in the creation of numerous reactive oxygen species (ROS). Inflammation is intricately associated with the generation of OS, leading to the increased production and accumulation of reactive oxygen and nitrogen species (RONS). Glioma stands out as one of the most common malignant tumors affecting the central nervous system (CNS), characterized by changes in the redox balance. Brain cancer cells exhibit inherent resistance to most conventional treatments, primarily due to the distinctive tumor microenvironment. Oxidative stress (OS) plays a crucial role in the development of various brain-related malignancies, such as glioblastoma multiforme (GBM) and medulloblastoma, where OS significantly disrupts the normal homeostasis of the brain. In this review, we provide in-depth descriptions of prospective targets and therapeutics, along with an assessment of OS and its impact on brain cancer metabolism. We also discuss targeted therapies. Brain tumors are the prevalent malignancies of the CNS. Many of these malignancies are difficult to treat and have a high fatality rate . Brain t2O2) with the involvement of metal ions such as iron (Fe) and copper (Cu). This process results in the creation of hydroxyl radicals (•OH) and hydroxyl anions (OH−)  to regulate hypoxia, the link among the species-specific beneficial and detrimental biochemistries is delicate, which makes the context of the brain bittersweet, where excellent redox signaling functionality readily results in OS when electrons go off track. With the help of targeting ROS or targeting glioma–stem cells, there is great potential for future advances, which supports the recent idea of integrative treatments focused intensively on the pathophysiology of OS in brain cancer.The numerous complicated linked factors that make the brain vulnerable to oxidative stress are unsaturated lipid accumulation, excessive glycolysis, mitochondrial glycolysis, calcium and glutamate production, limited antioxidant defense, redox active transition metals, RNA oxidation, and the auto-oxidation of neurotransmitters. Because it uses chemically different reactive species to carry out heterogeneous signaling processes, the brain is vulnerable to OS. By employing nitric oxide to adjust the synaptic elasticity, lipid radicals for ferroptosis signaling when lipid signaling stops working, or mitochondria [O


The study of well-preserved organic matter (OM) within mineral concretions has provided key insights into depositional and environmental conditions in deep time. Concretions of varied compositions, including carbonate, phosphate, and iron-based minerals, have been found to host exceptionally preserved fossils. Organic geochemical characterization of concretion-encapsulated OM promises valuable new information of fossil preservation, paleoenvironments, and even direct taxonomic information to further illuminate the evolutionary dynamics of our planet and its biota. Full exploitation of this largely untapped geochemical archive, however, requires a sophisticated understanding of the prevalence, formation controls and OM sequestration properties of mineral concretions. Past research has led to the proposal of different models of concretion formation and OM preservation. Nevertheless, the formation mechanisms and controls on OM preservation in concretions remain poorly understood. Here we provide a detailed review of the main types of concretions and formation pathways with a focus on the role of microbes and their metabolic activities. In addition, we provide a comprehensive account of organic geochemical, and complimentary inorganic geochemical, morphological, microbial and paleontological, analytical methods, including recent advancements, relevant to the characterization of concretions and sequestered OM. The application and outcome of several early organic geochemical studies of concretion-impregnated OM are included to demonstrate how this underexploited geo-biological record can provide new insights into the Earth’s evolutionary record. This paper also attempts to shed light on the current status of this research and major challenges that lie ahead in the further application of geo-paleo-microbial and organic geochemical research of concretions and their host fossils. Recent efforts to bridge the knowledge and communication gaps in this multidisciplinary research area are also discussed, with particular emphasis on research with significance for interpreting the molecular record in extraordinarily preserved fossils. The fossil record is fundamental to our understanding of major events in the evolution of life and our planet. However, the fossil record suffers from numerous biases, and not all fossils are created equally . The pro‘normal’ fossil preservation, the remains of dead organisms are subject to a range of complex biogeochemical processes encompassing degradation via eogenesis and diagenesis (within the water column and surface sediments), burial, microbial activity, authigenic mineralization and metagenesis (in sediments), weathering and exhumation. These processes destroy nearly all OM. In ‘selective’ fossil preservation, certain extremely resistant or recalcitrant biopolymers are fossilized. These include, for example, leaf cuticles of higher plants , which retain important information about extinct organisms and are vital for establishing paleodiets and reconstructing paleoenvironments at the microbial level  and isotopic studies and the enormous potential for the molecular record to reveal insights about extinct organisms. Challenges and knowledge gaps in the field are presented, along with recent efforts to bridge them.Fossiliferous concretions have been identified in strata ranging in age from Mesoproterozoic to Holocene  as seen δ13C of cholestane (−30.5 ‰), short-chain C17/C19n-alkanes (−34.8 ‰) and phytane (−34.0 ‰) support a source from phytoplankton consumed by the crustacean; the different values for these compounds reflect differences in biosynthetic pathways for different compound classes within phytoplankton cells  indicate a source from autolithified sulfate reducing bacteria (SRB) involved in concretion formation  has produced countless exceptionally preserved fossils in concretions, including an ichthyosaur bone that preserves intact cholesterol , 2017. ISeveral Mesozoic deposits in the Americas are known to host fossiliferous carbonate concretions in shales [e.g., the Upper Jurassic Cordillera de Domeyko Fromation in Chile ; the Lowδ13C of n-alkanes and fatty acids (C19-30) varied between −28 ‰ to −32 ‰, which was attributed to input from C3 plants. Short-chain fatty acids, probably of a bacterial origin (C14-18) showed lower δ values (−24 ‰ to −30 ‰).Among Cenozoic deposits with fossiliferous concretions, the Eocene London Clay Formation of the UK is perhaps one of the best known and studied . ConcretBacillus strains isolated from decaying shrimp (Artemia sinica) larvae and sea urchin (Heliocidaris erythrogramma) embryos were recorded to destroy the soft tissue. Chlorobi) via biomarker analysis. In some instances, initial, rapid heterotrophic decay leads to the release of high concentrations of cations  to the immediate environment; this may result in the stabilization and preservation of the remaining tissues, provided that the rates of the mineralization or stabilization processes remain higher than tissue decomposition  with organic matter (OM) is the most relevant process for fossilization and has been extensively studied by researchers over the last century . BIM is The process of concretion formation is in essence a two-step process: (1) microbial communities adhere to the substrate, leading to the formation of a biofilm; and (2) the biofilms act as a nucleation site which, in the presence of soluble calcium ions under a high pH environment, results in biomineralization of calcium carbonate .Pseudomonas aeruginosa accumulates minerals on the inside and outside of the biofilm by trapping fine abiotic calcite particles as well as granules of calcite through biomineralization  produced by biofilms influence precipitation and regulate patterns of mineralization ; these EPSs then become encapsulated within the biofilm . Biominelization . Biofilmlization . Li et alization . Biofilmlization , 2016.Mineralization requires supersaturation of specific ions in the surrounding environment which can be governed by microbial activity in and around the cadaver immediately upon death. Depending upon the availability of organic substrates and cations, different mineralization reactions can occur, including silicification, phosphatization, pyritization or carbonate precipitation . Among tMicrobial function and metabolic activity in biomineral precipitation  can be grouped into three categories :Precipitation of microbial metabolic pathway by-products. Metabolic pathways like photosynthesis, sulfate reduction, denitrification, anaerobic sulfide oxidation, ureolysis and methanogenesis might circumstantially lead to mineralization . The sig2+, Mg2+) when available  and remove free cations from solution. Once EPS degrade, the captured cations remain, and so the local concentration of cations increases and promotes calcium carbonate precipitation.Entrapment by extracellular polymeric substances (EPS). EPS mainly comprise polysaccharides and proteins along with nucleic acids, lipids, uronic acids . As EPS In the molecular fossil record, metabolic pathways influencing soft tissue preservation have been recorded via biomarker analysis. For example,  reportedNumerous physicochemical parameters influence the formation and composition of microbially mediated concretions, a fact attested to by the variable size (both absolute and relative to the concretion nucleus) and geochemistry of concretions (and the fossils they host) in the fossil record. Several of these physicochemical factors are explored below.Pseudomonas sp.), sulfate reduction to H2S, and anaerobic methane oxidation (coupled with sulfate reduction) are known to increase the alkalinity and change the chemistry around cells leading to carbonate precipitation , forms during early diagenesis in marine sediments and under relatively low temperatures ranging from-2°C to 10°C. This biomineral has been widely used as a low-temperature indicator to reconstruct paleoclimate (Other factors including dissolved oxygen (DO), conductivity and temperature have been demonstrated to be contributing variables in concretion formation e.g., . Glendonoclimate . AccordiCarbonate mineral precipitation is controlled by a number of complex, interrelated chemical processes. This includes alkalinity changes induced by microbiological processes, which can be dictated by the local environment and pore water chemistry e.g., , or infl3) precipitation is a consequence of BSR, wherein sulfate reducing bacteria oxidize OM to CO2 to obtain energy and reduce sulfate to H2S , which has been demonstrated to facilitate exceptional OM preservation 3F) and occur most in deposits of Jurassic–Cretaceous age, when phosphogenic-favoring environments were particularly prevalent , Ca and CO2 . Phospharevalent . Phospharevalent  and Funarevalent . Numerourevalent , the Namrevalent  and the revalent . A famourevalent . Skin, grevalent . Phospharevalent . In bonerevalent . The crerevalent . One loc, Brazil .A range of sophisticated and multidisciplinary analytical approaches can be utilized in the robust interrogation of fossiliferous concretions and the fossils they contain ; Table 2An understanding of the metabolic activities that took place at the time of fossilization can be obtained through various analytical techniques, using either direct  or indirect (microlaminations with OM or micropeloids) microscopic and elemental evidence . This unThis analysis provides a useful first screening to assess microbial mineral interactions, investigate the role of microbes and biofilms in different fossiliferous concretions and the physical nature of fossils. A range of microscopic and other microanalytical techniques have been utilized in the field of paleomicrobiology, including scanning electron microscopy, optical microscopy, fluorescence and confocal microscopy, atomic force microscopy and various analysis using synchrotron radiation.Lagerstätte (part of the Santana Group of Brazil), SEM imaging revealed direct evidence of microbial morphotypes and textural features of mineralized EPS, thereby providing strong evidence of the influence of microbial mats in the fossilization process (Scanning electron microscopy (SEM) and petrographic analysis with coupled X-ray spectroscopy (SEM/EDS) reveals morphological features along with the chemical constituents, thereby providing a comprehensive picture of microenvironments during fossilization . To date process .Optical (light) Microscopy helps in visualizing the fine details of an object by creating a magnified image through a series of glass lenses. Optical microscopy has been widely used in the study of concretions e.g., .in situ, thus providing supplementary information about chemical properties and the distribution of components of EPS.Confocal Scanning laser Microscopy (CSLM) creates sharp and distinctive images of an exact plane of a specimen . It alloFluorescence Microscopy is based on the emission of luminescence because of absorption of photons, when the samples treated with special fluorescent reagents exhibit Stokes shift. This technique can be used to identify structures in fixed and living biological samples , and helAtomic Force Microscopy (AFM) is a form of scanning probe microscopy, which uses a probe or tip to map the contours of the sample . AFM is Itrax X-Ray Fluorescence (XRF) core scanning extends traditional XRF characterization of the elemental composition of bulk sediment material by providing high-resolution elemental distributions. These have important implications for understanding paleoenvironmental and geochemical records e.g., , and canOmic techniques are a set of high-throughput methods used to study the structure, function, and interactions of microbial communities in different environments. These techniques include DNA metabarcoding, metagenomics, metatranscriptomics, proteomics, and metabolomics, which analyze the genetic material, RNA transcripts, proteins, and metabolites present in a microbial community, respectively  provides researchers with innovative ways to study the past . The stuMicrobial culturing, enzymatic analysis, physico-chemical parameters and viability analysis – Microbial culturing and enrichment is a simple and efficient method to enhance the multiplication of microbes (associated with fossil/sediment samples) by supplementation of nutrients under controlled laboratory conditions  followed by characterization of EPS, biofilm properties and any extracellular enzymes  produced by the grown cultures as demonstrated in prior studies . Microbiin situ characterization techniques to identify those with the most promising impregnations  for more detailed analytical attention. Given the typically limited amounts of fossil sample available and their precious nature, a range of appropriate micro characterization methods are often applied prior to non-destructive chemical analysis.It is advantageous to screen concretion collections with Morphological, molecular and microbiological characterization of concretions) can also be applied to the sequestered microfossils. Typically, concretions are opened so that the fossils can be accessed when exposed, but some techniques also allow in situ interrogation of the enclosed fossils. For instance, SEM imaging methods can provide ultrastructural information of fossilized tissues. However, SEM is limited by low electron penetration depth, meaning that it is often more effective on exposed structures, and especially on carefully prepared ultrathin (<100 nm) sections; one drawback, therefore is that these are representative of only a small fraction of the specimen. 3D images of embedded fossils can be achieved through focused ion beam (FIB)-SEM or by combining sequential milling with concurrent Energy Dispersive Spectroscopy (SEM EDS) where despite the destructive milling approach, nanoscale fossil structures can be successfully imaged to provide insights into their chemistry, ultrastructure, taphonomy and biogenicity  is now widely used for fossil observations and imaging in paleontological research . AFM stuComputed tomography (CT) extends the centuries long use of X-Rays to help visualization of geological fossils . X-ray cMicro-CT (μCT) scanning, or high resolution XCT, is also now being popularly applied in paleontology. Recent concretion fossil studies include the reinterpretation of a purported Cambrian jellyfish fossil as a pseudofossil ; the visSynchrotron exploration of the X-ray and matter interactions on a range of geological materials can provide insights on morphology, elemental composition, oxidation states, crystalline structure, magnetic properties, and others, which can measurably contribute to the investigation of biogenicity of putative biosignatures . GueriauSynchrotron propagation phase contrast micro-CT (PP-SRμCT/PPC SRμCT) identifies and maps mineral phases and their distribution at the microscale over centimeter-sized areas. Elemental information can be collected synchronously, informing on texture , crystallite size and local strain. The extremely high resolutions <100 nm;  achievabNeutron tomography (NT) is similar to CT in as much as it enables digital separation of fossils from rocks . AdvantaThe isolation of the fossil from its concretion host is critical to the success of organic geochemical analysis of fossil OM. Separation is often achieved through physical methods, although these might not fully isolate the fossil and matrix. As a result, pure matrix is commonly analyzed separately from the fossil sample to avoid contamination and accurately determine the contributions of each component.Bulk geochemical and key elemental analysis are common preliminary methods used to chemically screen concretions and their organic fossils. These methods often involve measuring the total organic and inorganic carbon contents of sediments, along with other elements commonly found in organic compounds such as nitrogen, sulphur and oxygen. Elemental analyses can be used to measure these elements in decarbonated sediments treated with hydrochloric acid, providing a total for each element.Rock-Eval pyrolysis is a widely used method in determining the thermal stability and quality of OM . It measNatural OM (NOM) samples are typically subject to various wet chemistry and other preparation techniques to provide specific fractions appropriate for separate gas chromatography mass spectrometry (GC–MS) analysis. The most important technique for organic speciation – i.e., identification of organic compounds including biomarkers – in complex samples such as the NOM that can occur in concretions is GC–MS  treatments. Thermal energy can be used to break macromolecular OM into smaller products which can pass through GC columns, in a process referred to as analytical pyrolysis. An important advantage of kerogen analyses is that its covalently bound lattice is less vulnerable to overprinting from migrating hydrocarbons or other autochthonous inputs than the free hydrocarbon fraction  is now widely considered the best practice method because of its high detection sensitivity including of ancient biochemical signatures sequestered in inorganic substrates . This pyPolar compound speciation and S-biogeochemistry: The polar fraction of solvent extracted (or HyPy liberated) NOM can be isolated by LC elution with polar solvents. Labile biomolecules, like proteins, lipids and sugars, can be in part sequestered through oxidative cross-linking, yielding N-, O-, and S- containing heterocyclic polymers e.g., . In partThe three-dimensional macromolecules produced by organic sulfurization are not amenable to GC–MS without further treatment. Raney Nickel is commonly used to selectively cleave the carbon-sulfur bonds of polar fractions, releasing a sulfur-bound organic fraction that can be analyzed by GC–MS . This apThanks to continued development, optimization and sophistication, GC–MS is being applied at increasing sensitivity and resolution. Both MS and GC detection can now be extended to multiple dimensions to support very high-resolution product detection. Two-dimensional gas chromatography (GC × GC) uses two different columns to separate compounds that co-elute in one-dimensional separations, greatly expanding resolution of complex mixtures . This alThe ability to correlate organic molecules with spatial and mineralogical sample features is an important consideration when interpreting exceptionally preserved fossils. Detailed molecular biomarker analysis, which typically requires destruction of bulk sample material, would ideally be complemented by spatially informative surface sensitive analytical methods.Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis using a laser probe can be used to create detailed maps of the elements present on the surface of a given sample. For example, Fe and S biochemical gradients measured across in concretion hosting an ichthyosaur fossil  identified much lower microbial activity in the inner part of the concretion, which likely contributed to the excellent fossil preservation .Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS) is a technique that can analyze organic and inorganic molecules simultaneously with high mass and lateral resolution. Cluster primary ion beams improve the identification of high MW organic compounds with less analyte fragmentation. This technology is useful in various fields such as material science, biology, and surface analysis . Importain situ analysis of fossil specimens can identify preserved molecular functional groups, alongside intermolecular and organo-mineral relationships  can be measured after removal of carbonate by reaction with dilute acid. Similarities in δ13Corg of Upper Cretaceous Holz Shale concretions  and host sediment was interpreted as evidence that OM was not strongly degraded during concretion formation  preserving a coprolite (see below).Stable carbon and oxygen measurements of carbonate have helped to classify the source of concretion carbon, e.g., sulfate (or iron) reduction vs. methanogenic CO2 , or biog2 . The δ34ormation , whereasormation . Stable ormation . Tripp eδ13C) and nitrogen (δ15N)  (δ34S) (δ34S CSIA).Compound-specific isotope analysis (CSIA) allows the stable isotope analysis of individual compounds in complex mixtures of sedimentary OM . The tecn (δ15N) , and hasn as δD)  and sulf) (δ34S) . The tecin situ characterization and imaging techniques able to identify concretions with promising fossil targets for more detailed geochemical analyses; and (iii) detailed geochemical characterization of the microfossils themselves.The investigation of mineral concretions and their capacity to sequester the organic remains of extant organisms is still a relatively new and immature science. The information that concretions can convey about ancient organisms and ecosystems is primarily derived from the detection of molecular biomarkers  from the well-preserved fossils sequestered within them  can be further elucidated through studies of modern mineralogy, microbes, genes, and surface physicochemistry. Detailed observation and understanding of these parameters and processes will enable a better understanding of the environmental settings that led to concretion formation in deep time.Design and develop laboratory simulations and modeling technology to evaluate field-based inferences of fossilization formation mechanisms . The intin situ interrogation of organismic fossils, both in the field and laboratory would be highly beneficial. Appropriate field application will conveniently help to lessen downstream analytical loads.Successful identification and selection of concretions that host the most promising fossils  will require the development and application of new field tools. Current evaluations of impregnated fossils with traditional imaging and other methods remain challenging. Development of new and accessible methods able to support Molecular speciation of organic sediments involves sophisticated, time-consuming procedures, restricting timely analytical processing of samples. The analytical challenges are intensified when conducted on trace amounts of organic analyses  where particularly meticulous protocols need to be implemented to distinguish sample from background signals. This can be exacerbated when baseline organic signals are high , which is the case at several famous sites with fossiliferous concretions, including the Carboniferous deposits at Mazon Creek, Illinois , the Eocene Green River Formation, Utah ; and the Cambrian Alum Shale, Sweden . New physical separation techniques able to better isolate fossils will be highly beneficial.2 rises over the last 200 years). An intensifying community focus on climate change presents an opportunity to improve societal understanding, attitudes, and behavior toward the environment  can challenge the public communication of research outcomes and the engagement of scientists in outreach initiatives. Nevertheless, scientists have an obligation  to openly communicate and share the results of their work, particularly those which tangibly impact society. The analysis of ancient organisms that are exceptionally preserved within mineral concretions will provide new information about evolutionary processes on Earth. A higher resolution of environmental and biological paleo-dynamics will add valuable context to understanding contemporary environmental and Earth science (ESS) issues, including modern climate change. Paleoenvironmental reconstructions help climate scientists assess the resilience of our planet’s biosphere, as well as its capacity to cope with and regulate widely fluctuating climate conditions. They also enable climate scientists to more accurately predict the environmental and ecological consequences of present-day climate trajectories – some of which are at close to historical highs (e.g., atmospheric temperature and COironment . Effectiironment . A basicND, PG, SP, and KG wrote the manuscript as the leading authors. KG provided


Protein-based therapeutics have revolutionized the pharmaceutical industry and become vital components in the development of future therapeutics. They offer several advantages over traditional small molecule drugs, including high affinity, potency and specificity, while demonstrating low toxicity and minimal adverse effects. However, the development and manufacturing processes of protein-based therapeutics presents challenges related to protein folding, purification, stability and immunogenicity that should be addressed. These proteins, like other biological molecules, are prone to chemical and physical instabilities. The stability of protein-based drugs throughout the entire manufacturing, storage and delivery process is essential. The occurrence of structural instability resulting from misfolding, unfolding, and modifications, as well as aggregation, poses a significant risk to the efficacy of these drugs, overshadowing their promising attributes. Gaining insight into structural alterations caused by aggregation and their impact on immunogenicity is vital for the advancement and refinement of protein therapeutics. Hence, in this review, we have discussed some features of protein aggregation during production, formulation and storage as well as stabilization strategies in protein engineering and computational methods to prevent aggregation. In this review, we have discussed some features of protein aggregation during production, formulation and storage as well as stabilization strategies in protein engineering and computational methods to prevent aggregation. They act as catalysts, scaffolds for structural integrity, signalling molecules, molecular transporters and receptors, among other functions.5,6 Their versatility enables proteins to contribute to the essential functions and maintenance of cellular and tissue integrity.7 Protein-based therapeutics provide a multitude of advantages relative to traditional small-molecule drugs. Notably, their intricate three-dimensional structures confer the ability to interact with specific receptors or molecules in the body with remarkable selectivity and affinity, with minimal side effects.8–10 The therapeutic proteins can be derived from natural sources or produced by genetic engineering.11–13 The approval and market introduction of human insulin, produced by recombinant technique in Escherichia coli (E. coli), in 1982, represented an important milestone in the field of protein therapeutics.14 Following this achievement, significant progress was made in the cloning and expression of other protein-based drugs, including human growth hormone, interferon β and α, and monoclonal antibodies.15 These advancements contributed further to the development of recombinant therapeutics and vaccines.16 The production of recombinant proteins has gradually emerged as a vital component within the biopharmaceutical industry.17,18 The application of genetic engineering techniques allows for the efficient production of large quantities of target proteins with high consistency and cost-effectiveness compared to traditional methods. This advancement facilitates enhanced availability of therapeutic proteins, thereby improving accessibility for patients requiring these treatments.19 By employing recombinant DNA technology, the production of protein-based drugs can be achieved using well-controlled and scalable systems. This allows for the generation of high-quality, consistent and purified protein products.20,21In the last three decades, protein-based therapeutics have emerged as a significant category of pharmaceuticals. Protein-based drugs encompass therapeutic agents consisting of proteins or peptides engineered to interact with specific targets within the body for the treatment or management of diseases.22 However, several challenges arise in this field. These challenges include: protein aggregation, denaturation, degradation, concomitant loss of activity, immunogenicity, non-specific distribution, rapid clearance from the body and potential toxicity.23–25 These issues raise significant concerns that need to be resolved for the successful development and application of protein-based therapeutics.26 Recent advances in protein engineering and the ability to intentionally introduce chemical and structural modifications have created a paradigm shift in the fine-tuning of protein properties.18 These advancements have enabled researchers to modify proteins at the molecular level, allowing precise control over their functional properties.27 This capability has opened new avenues for the design of protein-based therapeutics with enhanced efficacy and stability, optimized pharmacokinetics and reduced immunogenicity. The ability to manipulate and fine-tune protein properties broadens avenues for the development of next-generation biopharmaceuticals.The progress in genetic engineering technology and a deeper comprehension of diseases provide a promising opportunity in the design and development of new biomolecular entities with desired biopharmaceutical features.17 Continuous research in protein engineering, advanced drug delivery systems, and manufacturing technologies contributes to addressing these obstacles and enhancing the efficacy and safety of protein-based therapies.27Protein-based therapy faces various obstacles that can significantly impact its effectiveness and safety. Overcoming these challenges requires a combination of scientific, technological, and regulatory approaches.Our aim is to bridge the gap between the broader understanding of protein stability and its specific applications in therapeutic contexts by addressing the obstacles encountered in protein-based therapy due to protein instability.2.in vivo performance.28 Proteins are susceptible to various destabilizing factors, such as aggregation, degradation and denaturation, all of which have the potential to downgrade their intended functions.28,29 Ensuring stability is essential to preserve the efficacy and functionality of protein-based therapies. The stability of protein-based drugs is of utmost importance during the entire process of manufacturing, storage, and delivery.30 Structural instability, which can arise from misfolding, unfolding, and different non-covalent or covalent modifications as well as aggregation, may deteriorate drug efficacy and their promising curative properties.30,31 Proteins and peptides demonstrate a restricted range of stabilities, influenced by factors such as concentration, ionic strength, temperature and pH.32–36 This narrow range is commonly referred to as “marginal stability”.37,38 Moreover, many recombinant proteins are inherently unstable under the conditions in which they are expressed, leading to a loss of correct folding or increased susceptibility to proteolytic digestion. This instability poses a significant challenge to obtaining adequately folded proteins for therapeutic applications.39Stability plays a vital role in the development of protein-based therapy, encompassing aspects such as optimal storage conditions and 40,41 Strategies such as cold chain management, using temperature-controlled storage facilities and employing suitable freeze–thaw protocols are employed to ensure the integrity of therapeutic proteins.41 Maintenance of proper pH of the environment plays a critical role in preserving the protein structural integrity and prevents undesirable chemical reactions that can lead to degradation.30 Due to precise control and optimization of pH conditions, proteins can maintain their native conformations, preserving their biological activities and therapeutic efficacies throughout the entire lifecycle of the product.42 The pH significantly influences intramolecular folding and protein–protein interactions by playing a critical role. At various pH values, specific amino acid residues can undergo protonation or deprotonation, thereby impacting their capacity to form hydrogen bonds, form electrostatic and hydrophobic interactions. Alterations in pH can disturb the charge balance within the protein resulting in modifications of its conformation and stability.37,42,43 Additionally, certain proteins may interact favourably with surfaces of various vessels, leading to adsorption and decreasing the concentration of the active ingredient available for therapeutic action.30 Therefore, a proper formulation encompasses factors such as buffer systems, excipients and stabilizers, which contribute to preserving the protein native structure and preventing undesired modifications. These formulations are designed to create optimal conditions, including appropriate pH, ionic strength and osmolarity, in order to safeguard the stability of therapeutic proteins.44–46Maintaining the appropriate temperature conditions (below the midpoint of denaturation) helps to preserve protein stability and prevent undesirable changes in structure and function over time.47 Inclusion bodies are dense aggregates of misfolded or aggregated proteins that accumulate within the cytosol, nucleus, or periplasm of cells during recombinant protein production.48 Integrating chaperones into the production process can significantly improve the overall quality and functionality of the final recombinant protein product.49 Molecular chaperones, whether in the cytoplasm or the periplasm, play a crucial role in facilitating the production of properly folded recombinant proteins. In the cytoplasm, chaperones like Hsp100 (Clp), Hsp90, Hsp70 (DnaK/DnaJ/GrpE), Hsp60 (GroEL/GroES), and small heat shock proteins (sHsps) are involved in aiding the correct folding of recombinant proteins.50 Periplasmic chaperones in cells are crucial for properly folding recombinant proteins by aiding in disulfide bond formation, ensuring their soluble form.51 The efficiency of this process varies based on the protein type and is improved by proteins like FkpA and SurA, aiding in critical peptidyl–prolyl bond isomerization. Moreover, DsbA and DsbC are essential for forming disulfide bridges, preventing misfolded proteins and guiding recombinant proteins toward their functional form in the periplasmic space.50 Certain chemical additives in culture media enhance the expression of soluble recombinant proteins by inducing osmotic stress and triggering the production of osmolytes like betaine and trehalose in E. coli cells. These osmolytes serve as “chemical chaperones,” boosting the stability of native proteins and aiding in refolding unfolded polypeptides. Notably, d-sorbitol, in combination with betaine, is widely employed to reduce the formation of inclusion bodies in recombinant proteins.52Furthermore, by serving as protein folding assistants, chaperones enhance the yield of properly folded proteins, mitigating the formation of inclusion bodies during recombinant protein production.53,54 To address these stability concerns, various structural modifications such as site-specific mutations, post transcriptional modifications (PTMs) and PEGylation, have been explored to improve protein solubility and stability. These modifications aim to enhance the overall performance and longevity of protein therapeutics.55–59In the complex environment of the human body, proteins face additional challenges caused by proteases, immune responses and physiological conditions that can impact their stability. Understanding and addressing these stability concerns are critical for ensuring the effectiveness and safety of protein therapeutics.60 Pharmacokinetics encompasses absorption, distribution, metabolism and excretion of the therapeutic proteins within the body. It is important for the protein to exhibit appropriate bioavailability, distribution in the target site and elimination from the body.61 On the other hand, pharmacodynamics relates to how the proteins interact with their targets and produce desired therapeutic effects. This includes factors such as binding affinity, receptor activation and downstream signalling pathways.25,60 Achieving the desired pharmacokinetic and pharmacodynamic profiles is crucial for the efficacy, safety and dosing regimen optimization of protein-based therapeutics. It requires careful design, optimization and characterization of the protein molecules, to ensure that they properly interact well with the therapeutic target and achieve the desired therapeutic effects with none or negligible off-target effects.25,60,62–64In addition to stability, protein-based therapeutics need to possess suitable pharmacokinetic and pharmacodynamic properties to ensure optimal functionality.2.165 Aggregated proteins may be recognized by the immune system as foreign or abnormal, triggering an immune response that can result in the production of antibodies against the therapeutic protein.66 The immunogenic potential of all aggregate types remains uncertain and subject to debate within the scientific community. It is not yet fully understood whether all aggregates have the potential to elicit an immune response. Furthermore, the impact of additional clinical factors on immunogenicity is a complex area requiring further investigation.67,68 Immunogenicity refers to the development of serum anti-drug antibodies (ADAs) that specifically target and bind to the proteins of interest, such as a therapeutic proteins or drugs. This immune response can have significant implications for the efficacy and safety of the protein products and can lead to reduced their effectiveness.69,70 When ADAs bind to the therapeutic protein, they can neutralize its activity, prevent it from reaching its target or accelerate its clearance from the body. This can result in diminished therapeutic effects and reduced clinical benefits to the patients. In more severe cases, the formation of ADAs can pose serious health concerns.71,72 The immune response triggered by ADAs can lead to adverse immune reactions, including hypersensitivity reactions, allergic responses or immune-mediated diseases. Examples include the formation of ADAs leading to reduced efficacy or life-threatening conditions like pure red cell aplasia (PRCA) with Eprex.73 Thorlaksen et al.74 showed that various aggregate populations are more immunogenic compared to the native human insulin, and the degree of immune activation depends on specific aggregate features.One of the primary challenges associated with the production, distribution and storage of protein-based drugs is the risk of protein aggregation. Aggregation refers to the process in which proteins associate and form clusters or aggregates, which can negatively impact the therapeutic efficacy.75 Compact micron-sized particles with native-like structures also exhibited immunogenicity in vivo. Subvisible were more immunogenic than submicron particles or soluble oligomers76 which, is discussed in the next section. Chemical modifications of the insulin molecule did not significantly impact immunogenicity.77 The correlation between in vitro and in vivo immunogenicity assessments suggests that distinct aggregate types activate different immunological pathways, contributing to overall immunogenicity.74 A study demonstrated that changes in protein structure resulting from aggregation can alter the range of antigens targeted by the immune response. Protein aggregation increases the number of epitopes presented to major histocompatibility complex class II (MHC II) molecules.78 For example, an assay comparing non-aggregated and aggregated formulations of two human IgGs subjected to heat and shake-induced aggregation demonstrated a significant increase in the number of epitopes presented on monocyte-derived dendritic cells (MoDCs).23 This suggests that structural changes induced by aggregation may cause immune responses targeting antigens that are chemically modified or hidden in the native fold of the proteins  simulations, protein structure prediction software and simulated annealing revealed that this peptide sequence was located in a hydrophobic domain of the scFv, which tends to become exposed due to partial unfolding.80 Additionally, while native aggregates increased anti-drug antibody (ADA) levels, non-native aggregates induced ADA levels similar to, or lower than those induced by the monomer.23 This suggests that the native conformation of the protein is important for immunogenicity. Understanding the structural changes induced by aggregation and their effects on immunogenicity is crucial for the development and optimization of protein therapeutics.23,80Peptide mapping and enzyme-linked immunosorbent assay (ELISA) identified a peptide sequence, which is highly targeted by antibodies induced by protein aggregates but not by monomers in wild-type mice.81,82Analytical techniques and immunogenicity assessments are utilized to detect and evaluate protein aggregates. Monitoring and managing immunogenicity is crucial, and it involves preclinical and clinical testing, assay development, and strategies to minimize immune responses. By addressing immunogenicity, the efficacy and safety of protein products can be optimized for better therapeutic outcomes.3.83 In the folded protein, hydrophobic amino acid residues are buried in the protein avoiding contact with water.84 However, it is important to note that not all hydrophobic residues are buried within the protein structure. Water molecules can form loose structures (cages) around exposed hydrophobic regions. Consequently, folding, surface adsorption or collapse of hydrophobic patches in proteins can result in the release of structured (caged) water into the surrounding solvent, thereby increasing the overall system entropy.29,85,86 Hydrophilic residues (uncharged and charged amino acids) would prefer to be on the surface of the folded protein. In fact, burial of charged side chains is very rare.87 In contrast, buried uncharged polar side chains are very common. These buried uncharged polar side chains are involved in hydrogen bond formation; it is very rare to find unpaired hydrogen bond donors and acceptors. These buried hydrogen bonds are more stable than those exposed to polar solvent water. In addition to hydrophobic interactions and hydrogen bonds, other noncovalent interactions that contribute to the thermodynamic stability are van der Waals interactions and salt bridges.88Proteins strive to maintain their native conformations, driven by thermodynamics and the lowest energy states.85 Protein aggregation is a complex process that can occur through three main mechanisms, depending on the seeding entity involved, i.e., the native monomers, denatured proteins and pre-existing aggregates.89,90 Two or more native monomers, which exist in folded state, can self-associate and form oligomers. This self-association can be driven by complementary charge–charge interactions between different monomers or through the formation of noncovalent linkages between hydrophilic and hydrophobic residues on the protein surface. These interactions can lead to the assembly of larger aggregates.91 The size of aggregates increases over time and they become more resistant to get back to their native states. It is commonly observed that protein products contain a fraction of denatured proteins, which have a higher propensity for irreversible aggregation. While partially unfolded proteins may possess the ability to refold under specific conditions, the thermodynamics and kinetics generally favour the aggregation process over refolding.92–94 Another mechanism is that the native protein monomers can aggregate by adhering to pre-existing protein oligomers, contaminants or to the surfaces of containers. This adherence can initiate a nucleation process in which the aggregates rapidly grow in size. This expansion of aggregates further contributes to protein aggregation.30Monomeric folded proteins are marginally stable. This low thermodynamic stability may facilitate conformational changes within the protein structure, which can ultimately lead to protein instability and the subsequent aggregation.69 In biological products, a small amount of soluble aggregates (5–10%) is generally considered acceptable. Insoluble aggregates are evaluated based on the size of particles detected upon reconstitution. Particles as small as 150 μm in diameter can be visually detected in injectable products and particles below 150 μm are more likely to induce immune reactions. Subvisible particles with a hydrodynamic radius of ≤50–100 μm are deemed acceptable, while particles ≥10 μm can obstruct blood flow. The U.S. Pharmacopeia (USP) sets the limit for particulate matter in a container of ≤100 mL to be 6000 particles ≥10 μm and 600 particles ≥25 μm. However, recent regulatory scrutiny by the U.S. Food and Drug Administration (FDA) has emphasized the importance of controlling aggregates below 10 μm in biological products. Aggregates of this size pose a greater risk to product stability and immunogenicity.76,85Soluble aggregates have low molecular mass and may exhibit reversibility, while insoluble aggregates exceed the solution solubility limit and precipitate out of the solution.95 Electrostatic interactions involve the attraction or repulsion of charged protein molecules based on their electrostatic charges. These intermolecular forces play a crucial role in determining the colloidal stabilities of proteins.96 The balance between these forces can either promote or hinder protein aggregation. For example, a strong steric repulsion and electrostatic repulsion between protein molecules can prevent their close association and aggregation.97 Otherwise, if the attractive forces outweigh the repulsive forces, protein molecules may associate and form aggregates. Therefore, understanding and manipulating these intermolecular interactions are important in controlling the colloidal stability of proteins.98Colloidal stability of proteins relies on the intermolecular interactions among protein molecules. These interactions involve various forces, including short-range attractions, steric repulsions and electrostatic interactions. Short-range attractions arise from van der Waals forces between protein molecules when they come within a certain distance of each other. Steric repulsion occurs when protein molecules approach each other closely, leading to repulsive forces due to overlapping electron clouds or steric hindrance.23,78 Significantly, it has been observed that globular aggregates have a lower surface area relative to their mass/volume ratio compared to elongated filamentous aggregates. This observation implies that an increased surface area could potentially contribute to enhanced immune activation.99Aggregate morphology, influenced by the protein and stress conditions, plays a crucial role in the surface area and subsequent immunogenicity of aggregates. Comparisons between globular and filamentous aggregates of human IgG models revealed that filamentous aggregates induced higher expression of immune activation markers  on MoDCs. Filamentous aggregates were predominantly formed under heat and shaking stress, while shear stress or freeze–thawing induced more globular aggregates.100–104 The pH of a solution has been found to exert diverse effects on protein aggregation. Extreme pH conditions or significant pH shifts from the protein isoelectric point (pI) can disrupt the electrostatic interactions and hydrogen bonds within protein. This disruption often results in a decrease in protein solubility accompanied by conformational changes that ultimately lead to aggregation.105–107 Moreover, alterations in ionic conditions of solvents such as changes in the concentration of salts or divalent cations, can also influence protein solubility and facilitate aggregation.108 For instance, in the context of monoclonal antibodies (mAbs), a recent study indicated that mAbs formulated in a sodium acetate buffer demonstrated more conformational and colloidal stabilities compared to those formulated in a sodium citrate buffer.109,110In addition, oxidation induced by UV light exposure or metal contamination as well as temperature, pH, ionic conditions, mechanical agitation and freeze–thawing pressure, are among the key factors that can impact protein aggregation.111,112 Metal ions like Zn2+, Cu2+ and Pb2+ can inhibit or accelerate aggregation. Overall, understanding the precise impact of cations on protein aggregation remains a challenge, and further research is needed to establish a consensus.37,111,113,114Protein and peptide aggregation depends on protein concentration, and the presence of metal ions can affect the rate of aggregation. However, the relationship between metal ions and protein aggregation is complex and not fully understood. Under normal physiological conditions, aggregation typically does not occur, but it can be induced by certain metal ions.115,116 In a study by Alam et al.,116 it was demonstrated that methionine oxidation in antibodies leads to a significant reduction in conformational stability. However, this oxidation has a limited effect on antibody aggregation, except under extreme conditions such as low pH and elevated temperature. Conversely, tryptophan oxidation and asparagine deamidation have minimal impact on antibody conformational stability but promote aggregation over the broader range of solution conditions.116Chemical modifications, such as deamidation and oxidation, can have a significant impact on the stability of protein-based therapeutics, including antibodies. Deamidation involves the conversion of asparagine to aspartic acid or isoaspartic acid, while oxidation involves the addition of oxygen or reactive oxygen species to amino acid residues. Both modifications can lead to conformational changes, reduce solubility and increased aggregation of antibodies. These modifications can occur spontaneously or be induced by various factors.69,117 Exposure to ultraviolet (UV) light can trigger chemical reactions and oxidation, altering protein structures and promoting aggregation. Temperature plays a crucial role as elevated temperatures enhance molecular motions and unfold proteins, whereas extremely low temperatures can induce conformational changes and increase the risk of aggregation.91Furthermore, various factors contribute to protein aggregation. Mechanical agitation such as stirring, pumping or filtration, can disrupt protein structures and facilitate aggregation by inducing unfolding and exposing aggregation-prone regions. Freeze–thaw cycles encountered during storage, can exert pressure on proteins, resulting in conformational changes, unfolding and aggregation.4.27 Methionine, cysteine, histidine, tryptophan, tyrosine and phenylalanine are vulnerable to oxidation and promote aggregation.23,118 In addition, deamidation of asparagine and glutamine, disulfide bridge formation involving two cysteine residues and cross-linking due to the formation of covalent bonds between protein molecules, contribute to the aggregate formation. Consideration of these factors is, therefore, vital for maintaining the stability and effectiveness of protein therapeutics.27 Structural modifications, site-specific mutations,27 glycosylation,119 PEGylation,120 lipidation121 and protein fusion122,123 that are widely used to enhance the solubility and stability of proteins, are vital considerations in the development of protein-based therapeutics  chains to the target protein.128 During the freezing step in lyophilization, the solution is cooled to initiate ice nucleation and the growth of ice crystals. The freezing temperature is selected below the formulation glass transition temperature, and the freezing rate is chosen based on the desired ice crystal size and protein stability. Sufficient holding time at the freezing temperatures ensures thermal equilibrium. An annealing step can follow freezing to promote ice crystal growth and excipient crystallization. Annealing temperature is typically above the glass transition temperature but below the eutectic melting temperature.129,130 Optimization of these parameters is essential for achieving efficient drying and uniform freeze-dried products.131 Stabilizers play a crucial role in freeze-dried protein formulations to protect proteins during the freezing process. Cryoprotectants such as sucrose and trehalose are commonly used stabilizers. These cryoprotectants work by selectively excluding themselves from the protein surface, thereby raising the energy barrier for protein unfolding during freezing. This mechanism preserves effectively the native state of proteins, enhancing their stability throughout the freeze-drying process.132–134 Furthermore, proteins tend to adsorb at different interfaces, including the air–liquid interface during mixing or the ice–liquid or ice–air interface during freeze-drying. To counteract this phenomenon and enhance protein stability in such scenarios, surfactants are incorporated into protein formulations. Non-ionic surfactants such as Polysorbate 20, Polysorbate 80 and Poloxamer 188 are frequently employed in both liquid and freeze-dried protein formulations. These surfactants assist in preventing protein aggregation and adsorption at interfaces, thereby preserving the integrity and functionality of the proteins throughout the formulation and manufacturing processes.133,135 A study of a model protein, myoglobin, known to be susceptible to cold denaturation, emphasized the significance of employing a fast-freezing technique to mitigate aggregation and maintain the activity. The hypothesis proposed that rapid freezing reduces the exposure of the protein to cold conditions, where denaturation can occur. However, this fast freezing approach leads to the generation of small ice crystals, which, in turn, enhances the surface area available for interactions between ice and water molecules.128 Although the fast-freezing procedure proves beneficial for proteins prone to surface-induced conformational instability, it may not be suitable for all proteins. To illustrate this point, lactate dehydrogenase, another model protein, was employed as an example. The study demonstrated that the same fast-freezing method that effectively preserved myoglobin stability had adverse effects on lactate dehydrogenase. It led to protein aggregation and a subsequent loss of enzymatic activity.136 This study emphasizes the significant variation in protein stabilities and the importance of adjusting environmental parameters accordingly to prevent potentially immunogenic aggregate formation.Optimizing the freeze-drying or lyophilization process and formulation parameters is crucial for minimizing aggregation. The choice of freezing procedure should consider the specific protein stability requirements to prevent conformational changes and subsequent aggregation, ultimately reducing the risk of immunogenicity.5.5.1137,138 Furthermore, protein engineers have the ability to introduce or manipulate post-translational modifications (PTMs) in therapeutic proteins. PTMs such as glycosylation, phosphorylation, or lipidation exert substantial effects on protein stability, pharmacokinetics and immune recognition. These protein engineering strategies enhance the therapeutic potential of proteins and contribute to the development of more effective protein-based therapies.139,140Protein engineering is a crucial component of protein-based therapy, enabling the creation of tailored proteins with improved therapeutic properties. Through computational modelling and structural analysis, protein engineers modify amino acid sequences, introduce site-specific mutations and incorporate functional motifs to optimize protein stability, binding affinity and enzymatic activity.141 These mutations are designed to address issues such as protein aggregation, susceptibility to proteolytic degradation and unfavourable interactions with container surfaces. By strategically introducing mutations at precise locations, researchers can disrupt aggregation-prone regions, enhance resistance to proteolysis and mitigate undesirable adsorption, thereby improving the protein solubility and stability.27Site-specific mutations involve targeted alterations to specific amino acids within a protein sequence, exerting significant effects on its properties including solubility and stability.Here are some specific examples of insulin variants that have been developed through site-specific mutagenesis to achieve different kinetics of action.142• Rapid-acting insulin analogues such as lispro : lispro was developed through site-specific mutagenesis by exchange of proline and lysine amino acids at positions 28 and 29 in the insulin B chain. This modification enables lispro to be quickly absorbed after subcutaneous injection, leading to a faster onset of action than regular human insulin. Consequently, lispro closely imitates the rapid insulin secretion observed after meals, offering improved postprandial glucose control for individuals with diabetes.143,144• Long-acting insulin analogues : insulin glargine was engineered through site-specific mutagenesis by replacing asparagine at position 21 of the insulin A chain with glycine and adding two arginine residues at the C-terminus of the B chain. These modifications influence the solubility and stability of the insulin molecule, leading to the formation of micro-precipitates following injection. These micro-precipitates enable a gradual release of insulin over an extended duration, providing basal insulin coverage for up to 24 hours.5.2145–147 Computational methods such as molecular dynamics (MD) simulations and machine learning algorithms play a crucial role in predicting protein stability for protein-based therapy. These techniques analyse protein structures and sequences to identify regions that are prone to instability.148 Furthermore, they can predict the impact of mutations or modifications on protein stability, facilitating the design of more stable protein variants.84,149 These computational methods provide valuable insights into protein stability and contribute to the development of more robust and effective protein-based therapies. MD simulations are computational tools that simulate the movement of atoms and molecules over time. In the protein-based drugs, MD simulations provide insights into protein dynamics, stability and behaviour.150 They assess protein stability under various conditions and predict how proteins will behave. MD simulations also offer a way to study protein-ligand interactions, helping researchers to understand the binding affinities and to optimize drug candidates.151–153 Moreover, machine learning algorithms play a pivotal role in the protein-based therapy by leveraging large datasets of protein stability information to train predictive models. These models analyse protein sequences, structures and other pertinent features, enabling accurate predictions of protein stability.154–156 By harnessing the power of machine learning, researchers can streamline the screening and prioritization of protein candidates based on their predicted stabilities. This approach minimizes the need for laborious and expensive experimental screening methods, allowing researchers to allocate resources toward the most promising candidates for further development. Machine learning expedites the identification and optimization of stable protein candidates, facilitating drug discovery of protein-based therapeutics . This model was trained on the UniRef50 dataset, which consists of 24 million amino acid sequences. UniRep generates fixed-length vectors that summarize the information in protein sequences, independent of structural or evolutionary data. These vectors contain rich information and have excellent generalization ability, making them suitable for protein stability prediction. UniRep also significantly improves efficiency of protein engineering tasks, making it a versatile tool for protein engineering informatics.162Deep learning is applied to unlabelled amino acid sequences to extract essential protein features in rational protein engineering. Alley et al.163 developed an innovative computational method named BayeStab, specifically designed for predicting changes in protein thermostability caused by mutations. This method serves as a valuable tool for researchers evaluating the impact of amino acid substitutions on protein stability. By utilizing BayeStab, scientists can make accurate predictions regarding the thermodynamic stability of mutated proteins, facilitating the rational design and optimization of proteins for various applications in protein-based therapeutics.163Wang 164Furthermore, the development of DeepDDG predicts changes in protein stability caused by point mutations. By utilizing neural network-based techniques and comprehensive training on a diverse dataset, DeepDDG enhances the ability to understand and manipulate protein stability, facilitating protein engineering efforts and advancing the understanding of missense mutations in proteins. Input features of DeepDDG demonstrated that the solvent-accessible surface area (SASA) of the mutated residue is the main character for the determination of protein stability.6.165Successful prediction of protein aggregation requires the adaption of computational schemes tailored to the specific properties of the protein being studied. However, this task can be challenging for untrained users due to the requirement of in-depth knowledge about the available computational tools. Understanding the intricacies of these tools is crucial for effectively applying them to analyze protein aggregation. SAP  is a technology used to identify regions within therapeutic antibodies that are prone to aggregation. By employing full antibody atomistic molecular dynamics simulations, researchers can pinpoint these aggregation-prone regions. This knowledge allows them to perform targeted mutations and engineer antibodies with improved stability. The application of SAP has significantly enhanced in the stability of therapeutic antibodies compared to the wild type. This technology has the potential to be integrated into the screening and discovery phase of antibody development, enabling the development of more stable and effective antibodies for different diseases.et al., a comprehensive analysis was performed on 611 protein sequences and their respective lifetimes using the aggregation predictor known as TANGO. The results of the study revealed an interesting correlation between protein lifetime and aggregation propensity. It was observed that proteins with shorter lifetimes exhibited a higher tolerance for aggregation.166 Phenomenological algorithms utilize experimental data to understand the factors that contribute to protein aggregation and establish empirical aggregation scales. These algorithms, such as AGGRESCAN167 and Zyggregator,168 are based on the rationalization of experimentally determined factors that influence protein aggregation. Incorporating these factors allows the algorithms, to predict the protein aggregation, more effectively.169In a study by De Baets 166 PASTA 2.0,170 Amyloid Mutants,171 FoldAmyloid172 and Waltz173 analyze various characteristics of protein sequences. These include the propensity of a sequence to adopt a defined β-enriched conformation, the packing density of proteins, the composition and patterning of residues and the ability to adopt topologically restricted conformations commonly observed in amyloid-like states. By considering these sequence properties, these algorithms offer insights into the potential for protein aggregation, enabling researchers to predict and understand aggregation tendencies more effectively.145The second class of computational approaches is centered around the theoretical assessment of specific sequence properties associated with protein aggregation. Algorithms within this category such as TANGO,174 Algorithms such as APPNN,175 FISH Amyloid176 and netCSSP177 are noteworthy examples of these machine learning-based approaches. These algorithms effectively utilize the power of neural networks to accurately identify and predict protein aggregation, offering researchers valuable tools for studying and understanding this important phenomenon.Studies of protein aggregation are increasingly relying on the development of machine learning methods. These methods harness the capabilities of artificial neural networks to detect sequential features that exhibit a strong correlation with protein aggregation. Remarkably, machine learning approaches have achieved comparable, and in some cases even superior, performance compared to the traditional predictors.7.The stability of protein-based drugs is a crucial factor in ensuring their functional integrity for therapeutic applications. Challenges related to proper conditions during manufacturing, storage and transportation as well as immune responses, proteases and physiological conditions in the human body, significantly influence protein stabilities. Protein aggregation poses a substantial risk to therapeutic efficacy as it can induce immunogenicity and diminish the effectiveness of protein products. It is important to note that the choice of stabilization strategies may depend on the specific characteristics of the protein-based drugs, including its size, complexity and intended application. Each protein may require a tailored approach to optimize stability and ensure its therapeutic efficacy and safety. The integration of multiple computational and experimental methods is necessary to address the various challenges in protein-based therapeutic development. In this context, computational methodologies provide value by accelerating experimental processes for design and optimization of therapeutic proteins. Furthermore, this integration of computational and experimental methods provides a more efficient way of reaching a deeper understanding of drug discovery processes and their Overall, the combined efforts of computational modeling and experimental approaches hold promise in advancing therapeutic protein design and implementation in clinical settings.The manuscript's evolution was a collaborative effort among the authors. Mahdie Rahban initially drafted the manuscript based on insights received from Ali Akbar Saboury. Faizan Ahmad meticulously reviewed the first draft, offering insightful comments for improvement. Subsequently, Mieczyslaw A. Piatyszek, Thomas Haertlé, and Luciano Saso independently reviewed the manuscript, providing valuable corrections and comments. Finally, Mahdie Rahban and Ali Akbar Saboury amalgamated all the corrections and comments from the co-authors, shaping the final manuscript. This version underwent confirmation and approval by all authors before submission.There are no conflicts to declare.


Plasmacytomas are localized monoclonal plasma cell lesions with no evidence of systemic involvement which are divided into solitary bone plasmacytoma (SBP) and extra-medullary plasmacytoma (EMP). The diagnosis of plasmacytomas (PCM) in the oral regions is challenging given the atypical clinical manifestations and low frequency. Here, we report an extremely rare case of plasmacytoma in an elderly male which initially appeared to be arising from the left buccal mucosa on clinical examination but after radiological imaging and intra-operative findings, the epicentre was found to be in the left infratemporal fossa (ITF). The patient underwent en-bloc compartment resection with high clearance of the ITF which proved to be an effective management strategy. It is crucial for the head and neck surgeon to be aware of the solitary bone plasmacytoma in the oral and maxillofacial region in order to identify it early and provide these patients with the best care possible before complications arise. Plasma cell tumours are classified into multiple myeloma and plasmacytomas. Plasmacytomas are localized monoclonal plasma cell lesions with no evidence of systemic involvement which are divided into solitary bone plasmacytoma (SBP) and extra-medullary plasmacytoma (EMP) . Most coThe diagnosis of plasmacytomas (PCM) in the oral regions is challenging given the atypical clinical manifestations and low frequency. Therefore, identification of the clinical, radiographic, and histopathological features of this neoplasm is of immense importance to head and neck surgeons to make an appropriate diagnosis. Here, we report an extremely rare case of plasmacytoma which initially appeared to be arising from the left buccal mucosa on clinical examination but after radiological imaging and intra-operative findings, the epicentre was found to be in the left infratemporal region.A 65-year-old male presented to our hospital with complaint of swelling over the left cheek with swelling under the left eye since the past 6 months. The swelling was associated with pain since the last one month which was intermittent in nature and pricking in character which aggravated with jaw movements and relieved with oral painkiller. On examination of the oral cavity, a 4*3 cm submucosal growth was palpable in the left buccal mucosa extending anteriorly 2 cm short of oral commissure, posteriorly involving the retromolar trigone with upper and lower gingivo-buccal sulcus free of the lesion. The lesion had a smooth surface, well-defined margins, non-tender, non-mobile and there was no evidence of cervical lymphadenopathy.Biopsy of the growth was done on out-patient basis which was reported as normal study. Later, tru-cut biopsy of the lesion reported it as a salivary gland tumour. Contrast enhanced Computed Tomography (CT) scan showed an evidence of lobulated infiltrative soft tissue attenuation mass, possibly malignancy, approximately 3.7*3.4*5.5 cm epicentered in the left infratemporal region invading into the left masticator space, pterygopalatine fossa, with associated erosion of pterygoid plates. Fig.  MediallyThe patient underwent En-block compartment resection with high clearance of ITF. In this case, ITF clearance was done while preserving the mandibular condyles as they were not involved. Fig.  The specPlasmacytoma was first described by Schridde, in 1905, as an uncommon solitary growth of neoplastic monoclonal plasma cells . The IntThe aetiology of oral cavity plasmacytoma is unknown, but risk factors include exposure to chemicals, radiation, viral infection (Epstein-Barr virus in immunocompromised patients) chronic inflammation, and genetic disorders , 6]..6].Clinical presentation of plasmacytomas may be asymptomatic or non-specific like pain and swelling near the affected bone (SPB). EMPs most frequently arise from upper respiratory tract mucosa, causing symptoms such as epistaxis, rhinorrhoea, and nasal obstruction but they can arise in any soft tissue, including oral cavity mucosa . It is iSystemic plasma cell disorders such as multiple myeloma have increased blood calcium, deranged renal function, anaemia and multiple bone lesions (CRAB syndrome) which is not seen in true plasmacytoma . HoweverThe present case report describes a 65-year-old male patient with a suspected salivary gland tumour arising from the left infratemporal fossa (ITF). Based on CECT and tru-cut biopsy, the patient was planned for Compartment resection with ITF clearance. Since mouth opening was adequate on examination, mandible not involved on imaging, and appeared free of tumour intra-operatively, the mandible condyles were spared. As routine blood investigations including renal function tests were normal. Serum protein electrophoresis and urine analysis for Bence Jones protein was done post-operatively and showed normal study.On histology, these tumours show sheets of plasma cells with varying differentiation of atypical plasma cells (i.e. abundant cytoplasm with eccentric located nuclei), inclusion bodies and cartwheel chromatin pattern . CD138 iThe ITF is a complex anatomical region containing vital structures such as the maxillary artery, pterygoid muscles, and trigeminal nerve branches. Tumours in this region are rare and account for less than 1% of all head and neck malignancies. The surgical approach to ITF tumours is challenging due to the complex anatomy and proximity to vital structures. ITF tumours require a multidisciplinary approach involving otolaryngologists, maxillofacial surgeons, and neurosurgeons. Different surgical approaches include open or endoscopic, transcervical, transmandibular, or transmaxillary routes and high ITF approaches . The choImaging plays a crucial role in diagnosing and managing Contrast-enhanced computed tomography (CECT), and magnetic resonance imaging (MRI) is the chosen imaging modality. CECT is preferred for initial evaluation due to its wider availability, better resolution of bony structures, and ability to detect calcifications. MRI is preferred for evaluating soft tissue and neural involvement and planning the surgical approach .Metastatic lesions, osteoblastomas, Ewing sarcomas, rhabdomyosarcomas, and histiocytomas are a few differential diagnoses for SBP. Osteolytic osteoblastic metastatic lesions have localised radiolucency that resembles SBP.Plasmacytoma is a radiosensitive tumour and hence, radiotherapy is the main choice of treatment for both SPB and extramedullary plasmacytoma. Used with curative intent, and local control rates of > 80% can be achieved , 13. FraNearly 80% of SBP patients do not experience any recurrences even after five years, while 50–80% of individuals survive SBP for ten years . The likSolitary bone plasmacytoma is a lymphoproliferative disorder that involves clonal proliferation of plasma cells. Our case reports an extremely rare case of plasmacytoma presenting as a submucosal swelling in the oral cavity but epicentered in the left ITF. Compartment resection with high ITF clearance is an effective surgical approach for such lesions.Although the clinical and radiographic signs of SBP might not be non-specific, clinicians should always keep them in mind when making a differential diagnosis for oral cavity lesions. It is crucial for the head and neck surgeon to be aware of the solitary bone plasmacytoma in the oral and maxillofacial region in order to identify it early and provide these patients with the best care possible before complications arise.


Experimental studies in animal models of aging such as nematodes, fruit flies or mice have observed that decreased levels of insulin or insulin signaling promotes longevity. In humans, hyperinsulinemia and concomitant insulin resistance are associated with an elevated risk of age-related diseases suggestive of a shortened healthspan. Age-related disorders include neurodegenerative diseases, hypertension, cardiovascular disease, and type 2 diabetes. High ambient insulin concentrations promote increased lipogenesis and fat storage, heightened protein synthesis and accumulation of non-functional polypeptides due to limited turnover capacity. Moreover, there is impaired autophagy activity, and less endothelial NO synthase activity. These changes are associated with mitochondrial dysfunction and oxidative stress. The cellular stress induced by anabolic activity of insulin initiates an adaptive response aiming at maintaining homeostasis, characterized by activation of the transcription factor Nrf2, of AMP activated kinase, and an unfolded protein response. This protective response is more potent in the long-lived human species than in short-lived models of aging research resulting in a stronger pro-aging impact of insulin in nematodes and fruit flies. In humans, resistance to insulin-induced cell stress decreases with age, because of an increase of insulin and insulin resistance levels but less Nrf2 activation. These detrimental changes might be contained by adopting a lifestyle that promotes low insulin/insulin resistance levels and enhances an adaptive response to cellular stress, as observed with dietary restriction or exercise. Humans and most animal species exhibit the phenomenon of aging prior to dying a natural death. There is an age-dependent increase of physical damage to cellular constituents and changes in cellular and organ function.At the cellular level, age associated damage includes the accumulation of defective macromolecules such as oxidized lipids, proteins and deoxyribonucleic acid (DNA), as well as the formation of protein aggregates. There is increased production of free radicals and less adenosine triphosphate (ATP) from dysfunctional mitochondria in the context of lower availability of nicotinamide adenine dinucleotide (NAD+) and altered nutrient sensing. Cell repair and turnover mechanisms are impaired as evident from impaired proteostasis, decreased autophagy and lower stem cell activity –4. In moCan a natural course of aging be defined? Is there a primary lesion which kicks off a cascade of defects, and what is the role of insulin in this process? First of all, there is a genetic basis to the duration of life, otherwise the strikingly different lifespans between species such as between mice and humans or frogs and turtles could not be explained , 13. HowIn support of the network concept is the experience from anti-aging trials. The DrugAge database of aging-related drugs lists several hundred compounds for which significant extension of the lifespan in at least one model has been reported. Drug targets include many different cell functions ranging from glutathione metabolism to synaptic transmission which argues against a dominant role of defects in only one cellular compartment . SimilarWe conclude that cell, organ and organismal physiology has several “weak spots” with low resistance towards metabolic, inflammatory, toxic or other types of stress. For instance, depending on genetic background, environment, lifestyle or developmental stage, there may be accumulation of DNA damage in excess of DNA repair capacity, protein aggregation during periods of high peptide synthesis overburdening protein turnover or disaggregation mechanism, high levels of oxygen radical formation in the context of intense mitochondrial activity and failing radical scavenging responses, or accumulation of oxidized lipids because of deficient autophagy Figure 1Caenorhabditis elegans , more than doubles the natural lifespan of the nematode  elegans –23. Lifelifespan .In mice or humans, the regulation of the insulin/IGF-1 signal transduction pathway is more complex because of an additional upstream anabolic hormone, growth hormone. This pituitary hormone promotes IGF-1 production from the liver and other tissues, but the two hormones have partly opposite effects. For instance, growth hormone induces insulin resistance but promotes insulin production whereas IGF-1 promotes insulin sensitivity and reduces insulin secretion . TherefoMany studies have observed an extended lifespan in mice if growth hormone expression, or binding to its receptor are impaired. Longevity is increased in both sexes of Ames or other dwarf mice with deficient production of growth hormone together with prolactin and thyroid stimulating hormone or with isolated growth hormone deficiency , 30. Micvia knocking out the Ins1 gene and one of two Ins2 alleles in female mice appeared to increase maximum lifespan (p < 0.059)  .FOXO3A . For instance, small-breed domestic dogs exhibit a higher mass-specific metabolic and growth rate than large dogs, and therefore oxidative damage of lipids is seen. Nevertheless, small-breed dogs live significantly longer , 47. In In humans, epidemiological studies suggest a pro-aging effect of insulin. Insulin resistance increases with aging, but centenarians usually preserve normal glucose tolerance, low levels of fasting insulin and higher insulin sensitivity, when compared with adults > 75 years of age –54. The In adults with normal glucose tolerance, there is a parallel increase of fasting insulin levels and insulin resistance with aging, and this is associated with central obesity , 57. HypAnother approach of studying the health impact of hyperinsulinemia is to determine the insulinemic potential of the diet as assessed by food frequency questionnaires evaluated by measuring circulating C-peptide concentrations. Analyses of the prospective Nurses’ Health Study and the Health Professionals Follow-up Study  showed that a higher insulinemic potential of diet was associated with increased risk of all-cause, cardiovascular and cancer mortality . Of noteInsulin is a potent anabolic hormone. Just doubling fasting insulin levels is enough for suppression of lipolysis by approximately 50% and promotion of lipogenesis in adipocytes while hepatic gluconeogenesis is not yet inhibited (reviewed in ). A Mendvia the insulin receptor, causing the phenomenon of insulin resistance. This may involve decreased insulin receptor expression, but the major reason is impaired signal transduction because of diminished tyrosine autophosphorylation of the receptor, removal of bound phosphate residues by phosphatases and suboptimal downstream signaling along the insulin receptor substrate (IRS) – (PI3K) – protein kinase B (PKB/AKT) pathway  and subsequent activation of the mitogen-activated kinase protein kinase kinase (MEK) - extracellular signal-regulated kinase (ERK) is not affected by insulin resistance and contributes to these effects of hyperinsulinemia  activity, the latter resulting in increased protein synthesis and impaired autophagy –95. Incrulinemia Figure 2ulinemia , 102.in vitro elicits a senescent cell phenotype, characterized by cell cycle arrest and adoption of a senescence-associated secretory phenotype which includes the secretion of proinflammatory mediators, microRNAs and vesicles  vesicles . The provesicles . Increas in vivo . High am in vivo .Another age-associated marker is DNA damage. Prolonged incubation of animal or human cells with 0.5 nmol/l insulin caused DNA damage in the context of increased radical oxygen species production from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondria . WhetherAs reviewed above, high insulin concentrations cause cell stress because of excess anabolic activity which include (i), increased lipogenesis and fat storage also in non-adipocytes, (ii), increased protein synthesis and accumulation of non-functional polypeptides because of limited turnover capacity, (iii), impaired autophagy activity, (iv) increased progression of stressed cells towards a senescent stage. These changes are associated with mitochondrial dysfunction and increased levels of radical oxygen species , 118. HyTaken together, hyperinsulinemia in the context of insulin resistance appears to exhibit a pro-aging role. Whether these effects become clinically relevant probably depends on the body’s ability to mount an appropriate defense response for containing the detrimental consequences of hyperinsulinemia and insulin resistance. One well documented health risk associated with increased insulin levels is type 2 diabetes. We have previously argued that the progression to overt type 2 diabetes is prevented if there is a persistent protective/adaptive response which includes an anti-inflammatory defense response to nutrient-induced inflammation, increased neutralization of free radicals and improved mitochondrial function for the reduction of oxidative stress, and an upregulated ability to lessen endoplasmic reticulum stress by an unfolded protein response and autophagy . We suggOxygen radicals initiate a cell protective response by activation of nuclear factor erythroid 2 – related factor 2 (Nrf2), a key transcriptional factor for the expression of more than 250 genes involved in cytoprotective processes such as redox regulation, xenobiotic metabolism, DNA repair, and protein homeostasis including the unfolded protein response –126. TheLoss of proteostasis because of excessive protein synthesis is a major consequence of an acute rise of insulin levels, but this is apparently contained by the unfolded protein response of the endoplasmic reticulum . MisfoldA third important signal of cell stress is a decrease of ATP levels versus adenosine diphosphate (ADP) and adenosine monophosphate (AMP) concentrations, which results from increased consumption and deficient production of ATP. Low ATP levels lead to the activation of AMP-activated protein kinases. This group of kinases modulates the activity of many metabolic enzymes, histones and transcription factors by phosphorylation and by promoting their acetylation. One important consequence is the restoration of mitochondrial homeostasis –136.As mentioned, several lifestyle factors have been observed to lower levels of fasting and postprandial insulin as well as of insulin resistance. These factors include dietary restriction and exercise , 137. Invia the mitogen-activated kinase pathway and less production of NO by endothelial NO synthase. These potentially aging-promoting effects are contained by an adaptive cellular activity characterized by anti-oxidative, anti-inflammatory, protein chaperone, DNA repair and overall turnover process which is more potent in the long-lived human species than in short-lived models of aging research (The anabolic hormone insulin induces cell stress because of increased biosynthetic activity and reduced clearance/repair of damaged cellular components. Insulin resistance is a potentiating factor because of increased signaling research –147. Thevia Nrf2 becomes less potent but possibly not in centenarians (Of note, lifestyle factors that are considered to improve healthspan and possibly lifespan in humans modify both sides of the balance. Dietary restriction and exercise have been found to lower levels of insulin and insulin resistance. Concomitantly, dietary restriction, dietary phytochemicals and exercise activate the Nrf2-dependent cellular stress response and modify microbiota composition and function in a favorable way. During aging, the cell stress response enarians –151, andenarians , 152. BoHK: Writing – original draft. KK: Writing – review & editing. SM: Writing – review & editing.