Tissue fibrosis is the deposition of excessive extracellular matrix and can occur as part of the body's natural wound healing process upon injury, or as a consequence of diseases such as systemic sclerosis. Skin fibrosis contributes to significant morbidity due to the prevalence of injuries resulting from trauma and burn. Fibroblasts, the principal cells of the dermis, synthesize extracellular matrix to maintain the skin during homeostasis and also play a pivotal role in all stages of wound healing. Although it was previously believed that fibroblasts are homogeneous and mostly quiescent cells, it has become increasingly recognized that numerous fibroblast subtypes with unique functions and morphologies exist. This Review provides an overview of fibroblast heterogeneity in the mammalian dermis. We explain how fibroblast identity relates to their developmental origin, anatomical site and precise location within the skin tissue architecture in both human and mouse dermis. We discuss current evidence for the varied functionality of fibroblasts within the dermis and the relationships between fibroblast subtypes, and explain the current understanding of how fibroblast subpopulations may be controlled through transcriptional regulatory networks and paracrine communications. We consider how fibroblast heterogeneity can influence wound healing and fibrosis, and how insight into fibroblast heterogeneity could lead to novel therapeutic developments and targets for skin fibrosis. Finally, we contemplate how future studies should be shaped to implement knowledge of fibroblast heterogeneity into clinical practice in order to lessen the burden of skin fibrosis. Summary: This Review discusses the multifaceted aspects of fibroblast heterogeneity and the different roles of fibroblast subpopulations to help overcome skin scarring and fibrosis. Fibrosis is the replacement of functional connective tissue with excessive collagen-rich extracellular matrix (ECM). This results in the formation of fibrotic scars, which are the inevitable consequence of the body's repair process following tissue damage . FibrosiThe global impact of skin fibrosis is significant, with over 100 million people affected every year in the developed world . ScarrinSkin repair involves restoring tissue integrity through a complex and tightly controlled process that consists of the following overlapping stages: homeostasis, inflammation, proliferation and maturation . SeveralThis Review aims to discuss the current knowledge of the role of fibroblast heterogeneity in wound healing and fibrosis. We highlight the differences in fibroblast heterogeneity observed in human and mouse dermis, and summarize current insight into the understanding of cell-cell and cell-matrix communications that may shape fibroblast heterogeneity. Lastly, we contemplate how fibroblast heterogeneity could lead to the development of more-effective therapeutic modalities for wound healing.Skin is composed of two organized layers: a more superficial epidermal layer, mainly composed of keratinocytes, and a deeper dermal layer, primarily consisting of fibroblasts . There aDermato-myotome: the origin of the dorsal dermis.HOX code: describes a number of rules regarding the expression of homeobox (HOX) genes and their effects on segment identity.Mesoderm: one of the three primary germ layers that are sandwiched between the two other germ layers known as ectoderm and endoderm.Myofibroblast: differentiated fibroblasts that express α-SMA.Neural crest: a transient embryonic structure in vertebrates that gives rise to the peripheral nervous system and to several non-neural cell types.Stroma: the supportive tissue of an epithelial origin.Wound contraction: healing response following skin wounding that reduces the size of the tissue defect to decrease the amount of tissue that needs repair.Cell fate-mapping experiments have demonstrated that the differences in the embryological origin of dermal fibroblasts in human and mice depend on the anatomical location of the body . FibroblSeveral putative ‘pan-fibroblast’ markers have been well studied in the mouse, including platelet-derived growth factor receptor alpha (PDGFRA) . Using lEn1)-positive (En1+) and En1-negative (En1−) fibroblasts, and paired-related homeobox 1 (Prrx1)-positive (Prrx1+) and Prrx1-negative (Prrx1−) fibroblasts  code’  and flavin-containing dimethylaniline monooxygenase1 (FMO1)  . The twoetention .In murine skin, two additional fibroblast subpopulations have been identified in close association with hair follicle structures such as the hair follicle dermal papilla and the dermal sheath . These f+) CD90−  cells are enriched in the papillary dermis. Furthermore, papillary fibroblasts have increased proliferative potential, express podoplanin (PDPN) and netrin-1 (NTN1) and do not differentiate into adipocytes , matrix glial protein (MGP), peroxisome proliferator-activated receptor gamma (PPARG) and CD36, and readily undergo adipogenic differentiation, a hallmark of reticular fibroblasts. The differences in the papillary and reticular fibroblast behavior observed parallel those of murine skin (KANK4), aggrecan (ACAN), pregnancy-specific beta 1-glycoprotein (PSG1) and collagen 11 alpha 1  were upregulated in aged fibroblasts  CD26− as well as high expression of collagen chains such as collagen 6 alpha 5 (COL6A5), and primarily localized in the upper dermis. The second population defined by CD90+CD36+ surface expression, was abundant in the lower dermis and included pre-adipocytes. A third population had high expression of known pericyte markers, and two further populations were identified as CD90+CD39+CD26+ and Lin–CD90+CD39– regulator of G protein signaling 5 (RGS5)–. The fibroblast subpopulation markers were rapidly lost in culture, despite the cells usually maintaining their fibroblast function, highlighting the need to work with cells freshly isolated from human skin. The response of cultured cells to interferon gamma (IFN-γ), however, was retained. The upper dermal fibroblasts demonstrated an anti-inflammatory phenotype in response to IFN-γ stimulation and were able to support epidermal reconstruction when introduced into a decellularized dermis (ex vivo expansion of specific fibroblast subpopulations could herald the development of specific skin-based substitutes. The existing studies on the varied fibroblast cell surface markers in human unwounded skin underscore the extent of the future work needed to understand fibroblast heterogeneity in skin fibrosis.In contrast, a recent study identified at least five functionally distinct human dermal fibroblast subpopulations, and papillary and reticular dermal cells had distinct gene expression profiles . The fird dermis . These fSeveral signaling pathways have been implicated in the regulation of fibroblasts in fibrosis, the most studied among these being the Wnt pathway . Wnt sigAlthough it is clear that fibroblasts are under the control of regulatory signaling pathways, different fibroblast types also respond to distinct paracrine signals . LichtenOne of the hallmarks of wound healing is wound contraction (see Glossary, + pericytes and/or perivascular progenitor cells are the predominant myofibroblast source and can promote fibrosis in the skin, kidney and liver (Although originally considered a final differentiation state of fibroblasts, myofibroblasts are now recognized as a heterogeneous population of cells that derive from several progenitors. Specific subpopulations of dermal fibroblasts have greater myofibroblast potential . In animnd liver . Residennd liver , but endnd liver . Thus, mAn emerging concept in organ fibrosis is myofibroblast plasticity . AdipocyRecent evidence has highlighted that fibroblast heterogeneity is particularly crucial during wound healing . Identifin vivo models, in which splinted wounds are made on the mouse dorsum, which allows fibroblasts to be isolated following scar formation (in vitro models of skin scarring exist (in vivo wound model:Excisional  the most common wound repair in vivo model is the excisional in vivo wound model. Splinted full thickness cutaneous wounds are usually created on the dorsum of the rodent. Histology and macroscopic analysis is then used to analyze the skin wounds, to study the fibroblast and keratinocyte response to skin wounding (wounding .Hydrogel models: fibroblasts and keratinocytes are embedded in collagen hydrogels, with keratinocytes seeded on top of the hydrogel to provide a 3D environment as opposed to a 2D environment. Hydrogel models can be used in vitro and in vivo. Such hydrogel systems are easy to produce and can mimic a dermo-epidermal equivalent. However, there can be problems with mechanical stability of the gel, introducing variability (iability .Monolayer models: fibroblasts and keratinocytes are grown in monolayers in vitro. This allows for the study of the cellular microenvironment of keratinocytes and fibroblasts individually. When studying keratinocytes, monolayers can form a differentiated epidermis. However, monolayer models do not assess the cellular interactions of skin or reconstitute the complex 3D environment of the cell-cell and cell-matrix interaction of the skin (the skin .Skin-on-chip models: skin-on-chip systems culture skin tissue within a microfluidic system, which can control many physical and biochemical parameters, including medium flow, mechanical force and biochemical gradients. These in vitro systems have the ability to study the 3D skin environment interactions to a greater depth than monolayer and hydrogel models but are very complex and require great expertise (xpertise .Many studies of fibroblast heterogeneity utilize ormation . Howeverng exist  (Lotz etng exist . Traditing exist  (Kwak etng exist . Future Fibroblast heterogeneity could also hold promise for disease diagnosis as well as disease severity. For example, a study analyzing skin biopsies from 61 patients with scleroderma showed that expression profiles of their skin exhibited significant heterogeneity, and this was important for disease severity stratification and predicting the response to immunosuppressive treatments . UnderstSeveral groups have examined fibroblast heterogeneity in mouse embryonic mesenchymal cells using single-cell analysis and lineage tracing. Although there are numerous similarities between mouse and human skin, there are also a number of important differences. To date, most of the fibroblast subtypes have been identified in unwounded mouse skin; however, further work on unwounded human skin will show whether similar subtypes are conserved in human skin, and thus illuminate potential therapeutic avenues. The regulatory system underpinning fibroblast heterogeneity in human and murine skin remains unknown, with current studies focusing on the Wnt signaling pathway . Future As discussed here, fibroblasts are not a uniform cell population. Their heterogeneity has been extensively studied in murine skin, but further work is needed in human skin. Emerging work has identified multiple subsets of myofibroblasts, the key cellular drivers of fibrosis; however, these specific populations and their distinct contributions to fibrosis have yet to be fully elucidated. Identifying the regulatory signals of specific fibroblast subpopulations will aid in the development of new therapies to prevent scarring and other fibroses and to improve wound repair. Thus, future studies unraveling the heterogeneity of human fibroblast subpopulations have the potential to unveil new directions for fibroblast cellular therapy within the field of regenerative medicine.


Depression is a multidimensional construct consists of different symptom clusters. This paper aims to investigate if the impact of a stepped-care model, JC JoyAge, differs for older adults with different baseline depressive symptom clusters. Data came from 702 older people aged 65 and over who completed the JoyAge program. Their depression , anxiety, loneliness, and cognition were assessed by social workers at baseline and 12-months follow-up. Among them 609 were at risk or with mild symptoms and received group-based preventive care (prevention group), and 93 had moderate or above symptoms and received intensive intervention (intervention group). Their responses to PHQ-9 were coded to indicate affective, cognitive, behavioral, and somatic symptom clusters. It was found that somatic complaints had the highest prevalence (91%), followed by affective (83%), behavioral (60%), and cognitive symptoms (41%). Logistic regressions were used to estimate the effects of the program. For the prevention group, the JoyAge preventive care is more effective among those who reported behavioral symptoms , but less so in those who had cognitive appraisal issues . For the intervention group, the JoyAge intervention was more effective in treatment among those who reported more affective symptoms . The benefits of JC JoyAge stepped-care are differentially associated with participants’ baseline profile. Participants’ overall depressive symptom severity and the presentation of symptom clusters need to be taken into consideration when delivering the services.


Oral Squamous Cell Carcinoma (OSCC) remains a cancer with poor prognosis and high recurrence rate. Even with multimodal treatment options available for OSCC, tumor drug resistance is still a persistent problem, leading to increased tumor invasiveness among OSCC patients. An emerging trend of thought proposes that extracellular vesicles (EVs) play a role in facilitating tumor progression and chemoresistance via signaling between tumor cells. In particular, exosomes and microvesicles are heavily implicated in this process by various studies. Where primary studies into a particular EV-mediated chemoresistance mechanism in OSCC are limited, similar studies on other cancer cell types will be used in the discussion below to provide ideas for a new line of investigation into OSCC chemoresistance. By understanding how EVs are or may be involved in OSCC chemoresistance, novel targeted therapies such as EV inhibition may be an effective alternative to current treatment options in the near future. In this review, the current understandings on OSCC drug mechanisms under the novel context of exosomes and microvesicles were reviewed, including shuttling of miRNA content, drug efflux, alteration of vesicular pH, anti-apoptotic signaling, modulation of DNA damage repair, immunomodulation, epithelial-to-mesenchymal transition and maintenance of tumor by cancer stem cells. Oral cancer, categorized under head and neck cancer, consists of cancers occurring in the oral cavity. Cancers originated from oral cavity, tongue, lip and mouth accumulatively represents the 8th most common cancer with more than 300,000 cases per annum . There wCurrent treatments for oral cancer include surgery, chemotherapy, radiotherapy and immunotherapy . DependiCurrent research on OSCC drug resistance centers around elimination and inactivation of drug from cancer cells, increased response to DNA damage, reduced apoptosis and increased migratory abilities of cancer cells .These drde novo drug resistance and acquired drug resistance. De novo drug resistance is present before drug exposure and selection for drug resistance, while acquired drug resistance, also known as adaptive drug resistance refers to resistance that is developed over time after prolonged exposure to chemotherapy drugs  . They ofPlatinum-based chemotherapy drugs which are commonly used in combination with 5-FU are still the usual first-line treatment for OSCC, but the results are far from satisfactory. In advanced OSCC cases, chemotherapy drugs such as methotrexate, paclitaxel and docetaxel are more commonly used either alone or in combination . DespiteExtracellular vesicles (EVs) are lipid-bilayer enclosed vesicles that are naturally released from cells. Over the last decade, intercellular communication mediated by EVs present a new paradigm in which chemoresistance mechanisms should be investigated. Unlike traditional cell-cell signaling via cytokines, chemokines and growth factors , EVs conEVs can be differentiated into three main categories: exosomes (30–100 nm), microvesicles (MVs) , and the more recently discovered ‘oncosomes’ (1–10 μm) . These cExosomes are the smallest subset of extracellular vesicles secreted by cells. Their sizes (30–100 nm) vary depending on the cellular source or sample isolation and preparation methods . They weExosomes originate from the early endosome which accumulates intraluminal vesicles (ILVs) formed through the inward budding of its membrane . The endThis entire process can either be driven by the endosomal sorting complexes required for transport (ESCRT) or by other ESCRT-independent mechanisms such as neutral sphingomyelinase-dependent ceramide formation. Exosomes secreted from one cell can fuse with the plasma membrane of another cell and can be internalized through endocytosis or pinocytosis, therefore, transferring bioactive molecules from one cell to another . The exoExosomes play many important roles in enabling drug resistance in cancer cells through multiple mechanisms such as shuttling of miRNA, drug efflux, anti-apoptotic signaling etc. In order to understand the effect of exosomes in both donor and recipient cells, it is imperative to investigate the exosomal cargo being shuttled between tumor cells.Due to the exosome’s ability to transfer diverse loads of proteins, lipids and nucleic acids between cells, they have been dubbed as ‘Trojan horses’ . ExosomeThere is a significant body of evidence supporting the theory that exosomes, in their capacity to act as agents of intercellular communication, disperse the characteristic of drug resistance between tumor cells by delivering miRNAs . MicroRNA particular miRNA which has been reported widely in modulating cancer drug resistance is miR-21. Although the precise mechanism of action of miR-21 in OSCC has yet to be fully described, miR-21 is regarded as a key oncogenic factor that is associated with poor cancer prognosis and are highly expressed in tissues and blood of OSCC patients when compared to healthy tissues and blood control . MiR-21 The mechanism of miR-21 in OSCC drug resistance may also be through the regulation of STAT3, a transcription factor activated by IL-6. MiR-21 is known to affect STAT3 to promote tumor cell survival, induce anti-apoptotic effect, and also aid in cell cycle progression and angiogenesis . The supMiR-24 is another highly upregulated miRNA in OSCC tissues. It regulates a myriad of cellular activities and was reported to induce cell survival and cisplatin resistance in tongue squamous cell carcinoma when up-regulated . FrequenThe discovery of miRNA involvement in chemoresistance is still novel and studies relating to OSCC are minimal at the moment. Moreover, one miRNA may target various tumor-related gene transcripts and the activity of one gene transcript may be regulated by multiple miRNAs. Thus, it is challenging to design therapeutic strategies which can efficiently eliminate drug resistance via a single miRNA . There aMultidrug resistance (MDR) is a phenotype in which cancer cells exhibit simultaneous resistance to multiple chemotherapeutic drugs. Overexpression of drug efflux transporters of the ATP binding cassette (ABC) transporter family such as P-glycoprotein (P-gp) and copper efflux transporters gives rise to the MDR phenotype in tumor cells . Althougtrans-Golgi network (TGN) and are involved in copper-stimulated trafficking  demonstrated that cisplatin resistant ovarian carcinoma cells have a lesser endo-lysosome volume and expression of lysosome-associated membrane proteins (LAMP1 and LAMP2), implying a marked increase in lysosomal exocytosis to expel cisplatin . V-ATPase is a multi-subunit ATP driven proton pump that regulates the intracellular and extracellular pH . Aside fThe regulation of cellular pH on drug resistance is further proven by another study which discovered that upregulated proton pump gene expression in cisplatin-resistant cell lines which resulted in intracellular alkalization and elevated intracellular pH in the cells. As DNA-cisplatin adducts are more easily formed under acidic conditions, the increase in the cellular pH could attenuate its cytotoxicity effect and induce drug resistance, and this was shown when proton pump inhibitor synergistically potentiated the cisplatin cytoxicity . WhetherApoptosis is one form of programmed cell death initiated by apoptotic proteins such as caspases, Bcl-2-associated proteins and cytochrome C. Malignant tumors are often marked with the overexpression of anti-apoptotic proteins or signals. Current molecular markers that hold prognostic and predictive values are still incapable of resolving the heterogeneity and complexity of OSCC . MoleculA study conducted a molecular profiling of 171 OSCC cases via immunohistochemical analysis and ordered them by hierarchical clustering and the expression profiles of 23 anti-apoptotic proteins were investigated. Despite being able to divide the OSCC cases into two groups—apoptotic and anti-apoptotic, there was no association between these groups with clinical and pathological characteristics such as the overall survival of patients. Thus, there is a knowledge gap in connecting apoptosis and cancer prognosis that needs to be filled. However, the analysis did showed a group of pro-apoptotic proteins that are more prominent and could be use as targeted therapies . The comNotwithstanding the current lack of understanding between apoptosis and cancer survival, apoptotic inhibition is still a chemoresistance trait that needs to be studied in depth. It has been suggested that exosomes can promote anti-apoptotic signals in both donor and recipient cells. For instance, Caspase-3, a protein known as an executioner caspase is responsible for cleaving cellular substrates that are vital to cell survival, leading to membrane blebbing and disruption of cytoskeletal functions. The release of caspase-3 containing membrane vesicles is thought to facilitate removal of apoptotic proteins in order to promote cell survival .Exosomes can transduce anti-apoptotic signals to recipient tumor cells via different mechanisms. They can stimulate recipient cells via signal transduction facilitated by surface receptors to activate anti-apoptotic pathways . ExosomeAs discussed above, miRNA plays an important role in mediating various signaling pathways between tumor cells as a major exosomal cargo. One of the signaling pathways include anti-apoptosis. Guo et al. showed that exosomes derived from cancer-associated fibroblasts (CAF) confers cisplatin resistance to head and neck squamous cell carcinoma (HNSCC) via exosomal miR-196a . HNSCC cAside from exosomal enrichment of miRNAs conferring chemoresistance traits, miRNAs conferring chemosensitivity will be downregulated in resistant OSCC cells . demonstThese experiments provide extensive evidence on the role of exosomes in propagating anti-apoptotic signals, be it from neighboring OSCC cells or from the tumor microenvironment. Based on the above studies, the main exosomal content carrying apoptotic signals are found to be miRNAs. However, the role of other exosomal contents in mediating anti-apoptotic signals should also be investigated. This is important as DNA damage repair (DDR) mechanisms play an essential role in anticancer drug resistance. Drugs such as cisplatin and carboplatin intercalate between DNA bases and introduce inter- and intra-strand linkages. These effects result in the inhibition of DNA synthesis, triggering DNA damage response and inducing apoptosis . The humCisplatin and many chemotherapeutic drugs are known for its ability to induce apoptosis via DDR . Akiko eInterestingly, cisplatin-induced DNA lesions that stall replication forks can result in the formation of DNA double-stranded breaks (DSBs), resulting in chromosomal DNA fragments . It can Although chemotherapy and radiotherapy are distinct from each other, both therapies induce DNA damage. Moreover, it has been demonstrated beforehand that exosomes from breast cancer cells can alter the phosphorylation status of DDR proteins . ExosomeDNA lesions resulting from damage induced by cisplatin and its analogues are repaired mainly via the nucleotide excision repair (NER) pathway . HoweverIn order to prove the role of exosomes in modulating DDR mechanisms in chemoresistant OSCC, more studies establishing a direct link are needed. To date, there is only evidence showing the involvement of OSCC exosomes in mediating DNA repair, but none in a chemoresistance context.Aside from modulating DDR mechanisms, exosomes have been shown to possess immunomodulatory effects . CellulaA well investigated pathway which mediates the critical changes characteristic of innate and adaptive immune responses is the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway . NF-κB iAside from monocytes, exosomes from OSCC can also reprogram macrophages. The epithelial-mesenchymal transition (EMT) is a conserved process marked by the loss of epithelial characteristics and gain of mesenchymal phenotype of a cell at a genetic, epigenetic and morphological level . It is cThere is increasing evidence that suggests EMT contributes to chemoresistance. Earlier studies have established the connection between transcription factors regulating expression of EMT genes and drug sensitivity . The stuEMT can be mediated by exosomes in OSCC cells undergoing hypoxic stress. Hypoxia has often been postulated to act as an environmental pressure resulting in malignant evolution of tumor cells . The metA recent study conducted by  further Besides exosomal miR-21 and miR-155, other EMT-modulating factors which may be potential cargoes of OSCC exosomes are epidermal growth factor (EGF), CD47, miR-222 and other EMT transcription factors (EMT-TFs) . A separin vitro and in vivo, thus conferring drug resistance (in vitro model of diffuse large B-cell lymphoma (DLBCL), exosomes containing Wingless-related integration site (Wnt) signaling factor from DLBCL can induce a CSC phenotype in recipient cells (The cancer stem cell (CSC) hypothesis postulates that CSCs are responsible for the maintenance and recurrence of tumors . This smsistance . In an int cells . CSCs arnt cells . These eCSC-like phenotype can be induced by microRNA such as miR-146a . RoychouMicrovesicles are heterogenous, membranous sacs that are shed directly from the plasma membrane of a cell . They arThe biogenesis of microvesicles involves the outward blebbing and pinching of the plasma membrane. The process is accompanied by localized changes in the plasma membrane protein and lipid components which are involved in modulating membrane curvature . SynergiWhile it has not been extensively studied, microvesicles have been shown to modulate drug resistance in cancer cells, making them another factor to consider in combating drug resistance. Microvesicles isolated from OSCC have been shown to be larger and enriched than normal control cells. Their cargo contains oncogenic miRNA such as miR-21, miR-27a, miR-27b, and miR-155 . One of The involvement of microvesicles (MVs) in chemoresistance properties are not as extensive as exosomes. However, they have been implicated in intercellular communication within the tumor microenvironment such as stromal and immune cells, transport of drug efflux proteins, drug sequestration and anti-apoptotic signaling . The patMVs isolated from A2780 ovarian cancer cells were shown to carry P-glycoprotein (P-gp), a protein overexpressed in multidrug resistance (MDR) phenotype . InterceWhether or not MVs of OSCC carry out similar functions is yet to be investigated. On the other hand, MVs from OSCC have already been proven to induce apoptosis of T lymphocytes . OSCC paThe involvement of MVs in facilitating intercellular communication, changes in tumor microenvironment and drug resistance in OSCC needs more investigation. Besides that, improvements and optimisation of isolation protocols and methodologies are of paramount importance. There is a need to achieve an EV subtype yield with the highest purity and integrity in order to avoid studying MVs and exosomes in the same context. Characterization methods of EVs need continual improvement in order to distinguish between MVs and exosomes. The distinguishing of MVs and exosomes from each other will avoid wrongly attributing EV cargoes and signaling pathways to an EV subtype.The involvement of EVs in mediating chemoresistance in OSCC still requires ample investigation. To date, only a handful of direct links implicating EV involvement in OSCC chemoresistance have been established. Nevertheless, these findings provide sufficient basis that both exosomes and microvesicles play an important role in several chemoresistance mechanisms. In this review, we have discussed several roles of EVs in the regulation of chemoresistance in OSCC including its exosomal content, drug efflux by EVs, alteration of vesicular pH, anti-apoptotic signaling transmitted by EVs, modulation of DDR mechanisms, immunomodulation by exosomes, transport of EMT promoting genes and lastly, microvesicle-mediated chemoresistance.In summation of the above findings related to exosomes, exosomal content such as miR-21 and miR-24 are recognized as key oncogenic factors mediating OSCC cell survival. Aside from that, drug efflux by EVs for instance cisplatin efflux mediated by copper transporters ATP7A and ATP7B remains an active field of investigation. Novel studies suggest that cisplatin may be transported out by ATP7B via lysosomal exocytosis, albeit similar studies done on OSCC are yet to be made. Alteration of vesicular pH by vacuolar ATPases residing on the endomembrane presents a viable mechanism of OSCC chemoresistance as cisplatin sensitivity depends on the acidity of intracellular environment.Cancer stem cells are known for self-renewal and low proliferation properties, thus making them harder targets for DNA-damaging drugs. Findings in relation to microvesicle-mediated chemoresistance in OSCC however, are limited. Nevertheless, notable findings of MV-mediated chemoresistance include apoptosis induction of T lymphocytes and reprogramming of monocytes by OSCC MVs. However, it is important that EV characterization methods be standardized across laboratories to distinguish MVs from exosomes.Besides that, exosomes from cancer-associated fibroblasts can deliver anti-apoptotic signaling miRNAs that alter expression of genes regulating apoptosis. In addition, exosomal release of chromosomal DNA may be a potential mechanism utilized by OSCC to reduce aberrant DDR activation and apoptosis. Furthermore, exosomes from OSCC have been shown to be capable in reprogramming monocytes via the NF-κB pathway and macrophages via miR-29a-3p, thus mediating immunosuppression of the tumor microenvironment. Aside from that, an emerging perspective suggests that epithelial-to-mesenchymal transition is a chemoresistant mechanism in addition to being a metastatic process. Exosomes from OSCC cells carrying EMT-promoting cargoes such as miR-21, miR-155  and EMT-transcription factors may confer chemoresistance phenotypes to recipient cells. Last but not least, exosomal miR-146a are implicated in enhancing stemness in OSCC cells. In addition to that, the various pathways of biogenesis and endocytic mechanisms of EVs need to be mapped in order for EV inhibition to be considered as a synergistic complement to chemotherapy. Current research has no guarantee that EV inhibition will fully cripple tumor progression. In addition, due to the heterogeneity of OSCC cases, there is no guarantee that a study done on a few OSCC cell lines are fully applicable to all OSCC cases. Current molecular diagnostics utilizing tumor biomarkers are unable to accurately predict tumor evolution and behavior due to intra- and inter-tumor heterogeneity . The com


Fatty acids are an important energy source during exercise. Training status and substrate availability are determinants of the relative and absolute contribution of fatty acids and glucose to total energy expenditure. Endurance-trained athletes have a high oxidative capacity, while, in insulin-resistant individuals, fat oxidation is compromised. Fatty acids that are oxidised during exercise originate from the circulation (white adipose tissue lipolysis), as well as from lipolysis of intramyocellular lipid droplets. Moreover, hepatic fat may contribute to fat oxidation during exercise. Nowadays, it is clear that myocellular lipid droplets are dynamic organelles and that number, size, subcellular distribution, lipid droplet coat proteins and mitochondrial tethering of lipid droplets are determinants of fat oxidation during exercise. This review summarises recent insights into exercise-mediated changes in lipid metabolism and insulin sensitivity in relation to lipid droplet characteristics in human liver and muscle.The online version of this article (10.1007/s00125-020-05170-z) contains a slideset of the figures for download, which is available to authorised users. Wmax) and then declining towards 75% Wmax. Tracer studies revealed that this comprises a drop in oxidation of NEFAs and triacylglycerol fat sources (intramyocellular lipid [IMCL] and lipoprotein-derived triacylglycerols) [During physical exercise, the increase in energy demand is fuelled by oxidation of glucose and fatty acids . The relycerols) .Other sources of exercise-fuelling fatty acids are the IMCLs, of which the majority is stored in triacylglycerol-rich lipid droplets dispersed throughout the muscle . The obsThe aim of this review is to summarise recent insights into exercise-mediated changes in lipid metabolism and insulin sensitivity in relation to lipid droplet characteristics in human liver and muscle.Stable isotope measurements in combination with muscle biopsies taken before and after exercise give insights in substrate use during exercise. Both, individuals with type 2 diabetes and obese control participants mainly rely on fatty acids originating from the circulation , 7. AddiMyocellular lipid droplets are viewed as dynamic organelles that store and release fatty acids upon changes in energy demand and supply . Lipid dThese studies provide novel and important insights on lipid droplet utilisation in relation to their location and protein decoration and give a better understanding of how lipid-droplet turnover is regulated during exercise in healthy individuals. This type of data, however, is lacking in individuals with type 2 diabetes. For full comprehension of why lipid droplet utilisation is compromised during endurance exercise in individuals with type 2 diabetes, a tracer study to make the distinction between whether plasma or lipid droplet-derived fatty acids are used for oxidation, along with lipid droplet-specific analysis of lipid droplet coat proteins and analysis of lipid droplet location, should be performed pre- and post-endurance exercise in individuals with type 2 diabetes.The more pronounced utilisation of intramyofibrillar lipid droplets during exercise may well be related to the observation that, in skeletal muscle, most lipid droplets  are in close proximity to mitochondria –19. At tData on changes in lipid droplet–mitochondria tethering during exercise are only available for endurance-trained athletes. In male elite cross-country skiers, lipid droplet–mitochondria interactions increase upon an acute exercise bout despite unaltered IMCL content . In enduCompromised mitochondrial respiratory capacity is frequently reported in type 2 diabetes –27 and oAs well as increasing mitochondrial capacity, endurance training also is an effective intervention to improve fat oxidation and modulate fat storage in the skeletal muscle of lean sedentary participants . SeveralTraining studies in healthy lean participants show that endurance training for 6 weeks promotes IMCL utilisation during exercise , 35, 37.In most of the studies discussed above, the timing of meal intake relative to the training sessions was not monitored strictly or intentionally timed so that participants trained fasted. Interestingly, training in the (overnight) fasted state has gained popularity to promote fat oxidative capacity. Upon fasting, adipose tissue lipolysis and plasma NEFA levels increase. The increase in NEFA drives myocellular uptake of fatty acids and, thus, can promote IMCL storage and oxidation of fatty acids. Indeed, fat oxidation rates during acute exercise in the fasted state are higher than in the fed state , 42. AlsUpon 6 weeks of endurance training, IMCL content drops during a single exercise bout in the fasted state. This drop in IMCL content upon acute exercise was similar if the training was performed in the carbohydrate-fed state vs that fasted state , 37. CurDiabetologia [Intrahepatic lipid (IHL) storage is associated with type 2 diabetes and cardiovascular diseases. The poor accessibility of the liver in healthy individuals means that most studies towards the effect of acute exercise and exercise training on IHLs and lipid metabolism in humans are based upon non-invasive techniques, such as MRI and tracer studies. These studies have revealed that diet and exercise both affect IHL content in obese patients with a non-alcoholic fatty liver and/or type 2 diabetes . Upon enetologia . Reductietologia , 48, howetologia


Callosobruchus maculatus. Using population-genomic approaches, we detected modest parallelism in allele frequency change across replicate lines during adaptation to lentil. Mapping populations derived from each lentil-adapted line revealed a polygenic basis for two host-specific performance traits (weight and development time), which had low to modest heritabilities. We found less evidence of parallelism in genotype-phenotype associations across these lines than in allele frequency changes during the experiments. Differential gene expression caused by differences in recent evolutionary history exceeded that caused by immediate rearing host. Together, the three genomic datasets suggest that genes affecting traits other than weight and development time are likely to be the main causes of parallel evolution and that detoxification genes  could be especially important for colonization of lentil by C. maculatus.Genes that affect adaptive traits have been identified, but our knowledge of the genetic basis of adaptation in a more general sense (across multiple traits) remains limited. We combined population-genomic analyses of evolve-and-resequence experiments, genome-wide association mapping of performance traits, and analyses of gene expression to fill this knowledge gap and shed light on the genomics of adaptation to a marginal host (lentil) by the seed beetle  Eda and Pitx-1) ) linkage .In contrast, correlations in model-averaged effects for either trait between mapping populations were low. Genetic correlations based on genomic-estimated breeding values were sometimes higher, e.g., the genetic correlation between breeding values in BC-L14 based on the BC-L1 versus BC-L14 genotype-phenotype map was 0.17 for adult weight, but they were also idiosyncratic, with negative correlations occurring as often as positive ones. These results suggest that mostly different causal variants were segregating in the three mapping populations, or that causal variants were mostly in LD with different subsets of the sequenced SNPs or with different alleles at the SNP loci (negative genetic correlations indicate the latter was likely true in at least some cases). Another possibility is that epistatic interactions between genetic variants affecting weight or development time are prevalent, such that different combinations of alleles are favored at some of the same loci by selection on lentil Population genomic analyses revealed considerable parallelism in patterns of genome-wide allele frequency change, and to a lesser extent in the specific SNPs exhibiting the highest rates of standardized allele frequency change, with notably higher parallelism between L1 and L14A than between either of these and L2. This is consistent with previous analyses of these experiments focused on estimating selection and testing for parallelism in selection ,40, and Lower parallelism in the genetic architecture of the performance traits than in patterns of evolutionary change would not generally be expected because selection should only cause repeated patterns of change if the same genes or alleles affect traits under selection  [C. maculatus  . Specifirolinase ).C. maculatus adapting to lentil. Comparisons with other systems, especially those where host adaptation does not involve high mortality and a severe bottleneck or where host shifts also include interactions with competitors, mutualists, or predators . The most notable exception was that genetic regions that showed exceptional changes in allele frequency during lentil adaptation in L1 overlapped more than expected by chance with genes showing plastic differential expression in L1 when reared in mung bean versus lentil. This is consistent with a role for the evolution of plasticity in host adaptation . Hos e.g., ,124,125),125C. ma


While muscle dysfunction in these settings involve reduction of muscle mass and disrupted fibers’ metabolism leading to suboptimal muscle work, mechanistic research in the field has been limited by the lack of adequate animal models. Over the last years, we have established a rodent model of COPD-induced skeletal muscle dysfunction that allowed a disaggregated interrogation of the cellular and physiological effects driven by COPD from the ones unique to hypercapnia. We found that while COPD and hypercapnia synergistically contribute to muscle atrophy, they are antagonistic processes regarding fibers respiratory capacity. We propose that AMP-activated protein kinase (AMPK) is a crucial regulator of CO2 signaling in hypercapnic muscles, which leads to both net protein catabolism and improved mitochondrial respiration to support a transition into a substrate-rich, fuel-efficient metabolic mode that allows muscle cells cope with the CO2 toxicity.Patients with chronic pulmonary conditions such as chronic obstructive pulmonary disease (COPD) often develop skeletal muscle dysfunction, which is strongly and independently associated with poor outcomes including higher mortality. Some of these patients also develop chronic CO Dysfunction of non-ventilatory skeletal muscles, which encompasses reduction of muscle mass and force-generation capacity, is a relevant comorbidity in patients with chronic pulmonary diseases including chronic obstructive pulmonary disease (COPD) , 2020. IIt is generally accepted that maximal force-generation capacity predominantly depends on muscle mass, and submaximal force, or endurance, on fibers metabolic properties . Both fi2 retention, or hypercapnia, occurs in many COPD patients, particularly in advanced stages of pulmonary disease .Although research focused on the combined effects of hypoxia and hypercapnia on skeletal muscle has been conducted , insight2 exposure  muscles weight, and force-generation capacity scored by the grip strength test . These pexposure . To gainercapnia . To invery cells . AMPK wary cells , which iry cells . Indeed,O3/MuRF1 .2-induced AMPK-mediated accelerated muscle catabolism is teleologically consistent with the fact that AMPK represents a cellular stress sensor and thus, protein catabolism activated by AMPK contributes proteolysis-derived substrate to support other relevant cellular processes in an energetically challenged environment  pathway has been reported to modulate ribosomal biogenesis and protein synthesis   . Moreovete (OCR) ; fatiguete (OCR) . We havete (OCR) , which fnic rats . Thus, t2 could selectively activate optimization of mitochondrial respiration in this setting. Indeed, a recent analysis of EDL muscles proteome from chronically hypercapnic mice shows that the bioenergetics-related “ATP binding” is the most significantly downregulated term compared to normocapnic controls  mice established the relevance of AMPK in maintaining skeletal muscle oxidative metabolism  receptor interaction (Our previous data supports the concept that muscle weakness in COPD is associated with a decreased absolute but not specific contractility ,b, suggeeraction . Intereseraction . We speceraction . Future COPD and hypercapnia are frequently associated entities, and both contribute to skeletal muscle dysfunction. However, their interaction has so far not been mechanistically explored. By using an animal model of COPD-induced muscle dysfunction, we made observations that support the concept that COPD and hypercapnia synergize regarding muscle atrophy but antagonize on their respiratory effects. We hypothesize that AMPK is a critical mediator of this process, which supports a cellular environment operating under metabolic stress. Future research with AMPK loss-of-function analyses can elucidate the implications of these observations, which could be consequential to improve the management of hypercapnic COPD patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Albany Medical College Institutional Animal Care and Use Committee.JB conducted the experiments leading to the presented figures. AJ wrote the manuscript. All authors edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hepatitis B virus (HBV) is a globally-distributed pathogen and is a major cause of liver disease. HBV  can integrate into the host genome, but (unlike retroviruses) this integrated form is replication-defective. The specific role(s) of the integrated HBV DNA has been a long-standing topic of debate. Novel in vitro models of HBV infection combined with sensitive molecular assays now enable researchers to investigate this under-characterised phenomenon with greater ease and precision. This review covers the contributions these systems have made to understanding how HBV DNA integration induces liver cancer and facilitates viral persistence. We summarise the current findings into a working model of chronic HBV infection and discuss the clinical implications of this hypothetical framework on the upcoming therapeutic strategies used to curb HBV-associated pathogenesis. Chronic infection with the human hepatitis B virus (HBV) is one of the major drivers of liver disease and is the most common cause of liver cancer worldwide. Approximately one third of the world’s population has been exposed to the virus, and ~257 million people currently live with a chronic HBV infection ,2. ChronChronic HBV infections are generally asymptomatic for long periods (up to decades) as the virus replicates without triggering an antiviral immune response . DecadesSurveillance and ongoing care for chronic HBV infection is complicated by psychosocial factors, including limited access to or distrust of the health care system, financial burdens, social stigma, and systemic discrimination . A poor cis-mediated mechanisms  or trans-mediated mechanisms . This review focuses on how in vitro models have helped in furthering our understanding of how integrated HBV DNA contributes to virus persistence and pathogenesis.The mechanisms by which HBV causes liver cancer are still not well understood and are under intense investigation. One of the major factors thought to be involved in hepatocarcinogenesis is the integration of the viral DNA genome into cellular chromosomes. This reportedly induces pro-oncogenic pathways through several means , inHBV is the prototypical member of the Hepadnaviridae family, which are small enveloped, hepatotropic DNA viruses (~3.2 kbp) replicating via reverse transcription. The double-stranded DNA genome of HBV can take two forms : relaxedHBV replication starts with rcDNA-containing HBV particles attaching to heparan sulphate proteoglycans on the surface of hepatocytes ,13,14 F. This atThe HBV rcDNA genome is released into the nucleus, where it is repaired into the covalently closed circular DNA (cccDNA) form by multiple host DNA repair enzymes ,29,30,31While the majority of intra-capsid reverse transcription events result in the formation of rcDNA, a minority of virions contain HBV dslDNA (though this ratio can fluctuate in patients in different stages of infection ). The prThe role of integrated HBV DNA is currently unclear: unlike retroviruses, the integrated form of HBV is replication-deficient because of rearrangements that abrogate the expression of capsid, polymerase and functional pgRNA . FirstlyVarious functions have been ascribed to the integrated HBV DNA form . In vitro models  have enaIn vitro models vary in their ease of use (or reproducibility) in integration studies and inversely how closely they resemble integrations from a true infection in people with HBV. Tumour-derived cell lines from HBV patients contain integrations formed in the native setting but cannot be used to understand underlying mechanisms as they do not generate new integrations. Conversely, nuclear introduction of large numbers of HBV DNA molecules (via over-expressing constructs or transfection) can drive relatively high integration rates, allowing easy study of the integration process. However, this does not reflect a true infection; nuclear import of new HBV virions occurs relatively inefficiently in HBV infection ,49,50,51Integrated HBV DNA was initially described in liver cancers (or cell lines derived from these) and regenerative hepatic nodules in cirrhotic liver tissue ,73,74,75A fuller picture of HBV integration was revealed when tissues were analysed earlier in infection and with specifically designed PCR-based assays with higher sensitivity (methods for detecting HBV DNA integrations have been reviewed previously ). HBV inde novo HBV replication [While stable cell lines that overexpress HBV can force new DNA integrations ,53, intelication  nor viralication . TogetheSceI restriction enzyme into a chicken hepatoma cell line containing a single I-SceI recognition site [In vitro models have shown that it is the dslDNA form of HBV that integrates into the host genome. In a foundational study of an in vitro duck HBV model, Bill and Summers introduced a transgene coding for the I-ion site . They foThis study was also pivotal in showing that hepadnaviral DNA integration occurs at double-stranded DNA breaks in the cellular genome. These results are consistent in human HBV models: increased integrations were observed in HepG2.2.15 cells after treatment with Poly(ADP-Ribosyl)ation inhibitors (which slow down the repair of DNA breaks) or hydrogen peroxide (which increase oxidative DNA damage) ,53. Analysis of the termini of the integrated virus genomes have found frequent short deletions, suggesting the involvement the error-prone non-homologous end joining (NHEJ) DNA repair pathways. The majority of virus-host junctions observed in patient tissues and in vitro infection models show consistent results ,48. MoreSupporting this case in a true infection model, integration can also occur in terminally-differentiated HepaRG-based cells as well as non-dividing primary human hepatocytes . As NHEJAnother strong indicator of NHEJ pathway involvement is the largely random distribution of integrations throughout the host genome. Unlike many retroviruses that preferentially integrate into transcriptionally active regions in the host genome, HBV does not appear to have any major favoured sites of integration with respect to any cellular structures or functional motifs . Our stBy contrast, the HBV DNA integration sites in HCC tumour tissue are enriched in particular genetic regions (particularly cancer associated genes), though these likely arise due to selection towards integration events that induce cancer progression (as explained in the next section).There is a clear association between integrated HBV DNA with liver cancer; the frequency of HBV integrations in HBV-associated cancers  is muchIntegrated HBV DNA forms have viral promoters on their 3′ ends (the HBc and HBx promoters) that can drive downstream cellular transcription, which can in turn drive carcinogenesis. Indeed, this appears to be the case in some HBV-associated HCCs, in which integrations repeatedly occur close to the promoter of the human telomerase reverse transcriptase (hTERT), or within mixed-lineage leukaemia 4 (MLL4) or cyclin e1 (CCNE1) genes ,89,90,91However, these represent a minority of HCCs, with most having integrations in sites not associated with cancer. Moreover, insertions at these sites are not likely to be sufficient to initiate cancer. In vitro cell models have shown that while over-expression of hTERT can immortalise some human hepatocytes, these cells are unable to induce cancer formation even when injected into immune-deficient mice ,93,94. TAnother reported mechanism for HBV-associated HCC development is transformation by viral antigens. However, HBV proteins in themselves do not appear to be carcinogenic; transgenic mice expressing HBV antigens from native promoters do not show any liver pathohistology ,96. MoreA possible mechanism could be the altered expression of viral proteins (either wild-type or mutated) from the integrated HBV genomes. Expression of wild-type HBx protein alone leads to an increased risk of HCC development in older mice , but doeThe integrated HBV DNA leads to C-terminal truncation of the HBx ORF, which may be involved in carcinogenesis. A recent report has described the C-truncated HBx variants in HCC and found that they retain the ability to interact with the E3 Cul4A/DDB1 complex (previously shown to be important in modulating of transcription of HBV and likely host genes ), while Integrated HBV DNA genomes can also contain mutations in the HBsAg region. The PLC/PRF/5 hepatoma-derived cell line contain some of the few fully-sequenced integrated HBV DNA genomes; some of these show mutations, though it is unclear how representative these are for integrated forms in non-tumour tissue. One of these integrations includes a deletion in the HBsAg ORF . MutatioClearance of HBs is considered an end-point for HBV therapy . In this state, HCC risk is significantly reduced, and liver fibrosis is halted and can even reverse. Moreover, high levels of circulating HBsAg have been linked to virus-specific tolerance ,116 and Notably, integrated HBV DNA can be a source of HBsAg. While the episomal replication-competent cccDNA form of HBV can code for HBsAg, early studies of the hepatoma-derived cell lines Hep3Bs ,47 and PThe virus infects the whole liver following the initial infection. In this initial phase, the majority of HBsAg is derived from cccDNA, as integrated HBV DNA is rare  are the main antigens that are targeted .cccDNA-containing hepatocytes are targeted for cell death and replaced by mitosis of neighbouring hepatocytes. This facilitates the clonal expansion of hepatocytes containing integrations .In parallel, replicative space increases due to loss of cells expressing HBsAg from cccDNA. cccDNA is lost with mitosis ,50,123 aThe ratio of cccDNA to integrated HBV DNA frequency therefore decreases. Correspondingly, the ratio of both HBV e antigen and serum HBV DNA to HBsAg decreases (as observed in patient sera ).A new equilibrium emerges where the level of immune suppression by HBsAg  counteraLong-term exposure to the stably high levels of HBsAg can deplete HBV-targeting T-cells . In thisUsing the discoveries described in this review, we have developed a hypothetical model of HBV integration dynamics and how it can act as a stable source of HBsAg throughout chronic infection . While mThere are several aspects to this theoretical model that require further experimental support. Mathematical modelling would be ideal in determining if emergent behaviour from these initial assumptions and multiple interacting factors reflects clinical observations of HBV patients. To provide realistic ranges for parameters, precise experiments must be carried out to define the effect sizes of each of these factors . This may require a firmer understanding of anti-HBV immunity than the field currently has. Even relatively simple parameters, such as cccDNA per hepatocyte, remain poorly defined due to the technical difficulties in specific quantification and sampling of patient liver tissue in all phases of chronic HBV infection and various states . We believe that work in these fundamental areas of HBV research will be important to gain a broader understanding of chronic HBV infection and allow us to treat it more effectively. Such a model has implications on the development of therapeutic strategy to cure HBV . Firstly, while it is difficult to clear integrations that are established early, preventing new infections  at the appropriate points during chronic infection could limit new integrations. These therapies could also act to limit inflammation, preventing ongoing clonal expansion and amplification of existing integrations. If integrations indeed are causative in HCC (as many suspect), earlier interventions could limit cancer initiation by reducing new integration events and limiting clonal expansion of cell already with existing integrations. Indeed, exciting data presented at the 2020 Virtual Meeting for the American Association for the Study of Liver Diseases support these hypotheses ide analogue therapy reduced the extent of HBV DNA integration in chronic hepatitis B patients, presented by N. Chow). The efficacy of such strategies for HCC prevention is dependent on the relative contributions to tumorigenesis from (1) sudden clonal expansion of hepatocytes from new integrations that occur later in infections; or (2) the ongoing clonal expansion of cells with integrations established early on in infection. Therapies will be much more effective if the former is the case. Our model also suggests that some caution should be taken for any therapies that could induce long-lived flares . We would suggest that measurement of integration frequency be incorporated into the trial design when testing such agents. Finally, therapeutic modulation of HBsAg expression could alter these dynamics dramatically, given the multifaceted nature of HBsAg in this model. The aforementioned computer simulations or mathematical modelling would be ideal in identifying and designing future therapeutic strategies. In summary, the contributions of integrated HBV DNA towards HBV persistence and pathogenesis have been partially clarified and continue to be the topic of intense interest, particularly given its impact on current and upcoming therapies. Newly-developed highly controllable in vitro systems will allow the field to further dissect the roles of HBV DNA integration with greater precision in future studies.


Chronic obstructive pulmonary disease (COPD) is a complex chronic disease in which T cell-mediated pulmonary inflammation has been shown to play a key role. Accumulating evidence shows that COPD has many of the characteristics of an autoimmune response. An adaptive immune response directed against lung self-antigens, which are released during the initial innate inflammatory response and are triggered by constant exposure to cigarette smoke and epithelial injury, drives the persistent inflammatory response found in smokers. The development and severity of adaptive inflammation depend on the level of tolerance to self-antigens. For these reasons, the effect of regulatory T (Treg) cells on adaptive immunity in COPD patients is of particular interest and could be targeted therapeutically. The disturbance in immune homeostasis caused by changes in the number or function of Treg cells, which is related to cigarette smoke exposure, may be of importance in understanding the development and progression of COPD. The question arises whether quantitative and/or functional defects of Treg cells are linked to the development of COPD.Chronic obstructive pulmonary disease (COPD) presents as a persistent limitation in airflow that usually becomes progressively worse and is associated with exacerbated inflammatory responses of the lungs to irritating gases or particles . CigaretIn the present review, we will briefly summarize both current understanding of the functions of Treg cells and evidence for their involvement in the pathogenesis of COPD.in vitro or chronic antigen stimulation in vivo, particularly at sites of inflammation and environmental interfaces, such as the gut  syndrome  cell rely primarily on glycolytic metabolic pathways for proliferation. In contrast, established Treg cells rely more on mitochondrial oxidation pathways for their suppressive function. Both pathways actually represent two metabolism modulating mode. Glycolytic metabolism allows inflammation, whereas oxidative metabolism suppress inflammation. It has been shown that inhibiting glycolysis results in increased expression of Foxp3 during iTregs development , 17. ThoTreg cells provide essential protection to the body against an overactivated immune response. Reduced numbers and/or functional impairment of Treg cells is found in a number of immune-related diseases . It is, +Foxp3+ Treg cells can suppress dendritic cell and T cell functions through IL-10 and TGF-β, respectively . More recently, the mechanisms of action of Tregs have been expanded by several studies on their interactions with dendritic cells. Akkaya et al. . demonstIn addition to classical immunosuppressive functions, several studies have revealed much broader and multilevel mechanisms by which Treg cells regulate immunity and maintain tissue homeostasis. For example, recruitment of Treg cells to injured skeletal muscle to promote tissue repair was found to be mediated by the growth factor amphiregulin and regulated by IL-18 and IL-33 , suggestIdentifying specific markers that define Treg cells and distinguish them from activated effector T cells is important to completely understand the function of these cells. There is, however, still no consensus on reliable specific markers for identifying Treg cells.+CD25+ Tregs  represents an advance in this area . Tregs eOver the years, people have gradually realized that Treg cells are a heterogeneous cell population. This is a milestone in optimizing the efficacy of human Treg immunotherapy by selecting functionally different subpopulations of Tregs and identifying the ideal source of Tregs. Thus far, > 40 Treg subpopulation divisions defined by different surface molecules have been reported , 52.+Foxp3low resting cells  and CD45RA−Foxp3hi activated cells , both of which are suppressive in vitro, and pro-inflammatory cytokine-secreting CD45RA−Foxp3l° cells (fraction III), which are non-suppressive. CD45RA+ rTreg cells are bona fide suppressive Treg cells in which the TSDR within the Foxp3 enhancer is demethylated. This subpopulation predominates in cord blood and decreases with age because of thymic involution. Once activated, rTreg cells enhance Foxp3 expression, then actively proliferate, and finally convert to aTreg cells. As the main effectors of suppression, aTreg cells have the highest expression of Foxp3 and CD25 among the three subpopulations of Treg cells, show high capacity for immunosuppression and have a demethylated TSDR. Interestingly, rTreg and aTreg cells use different inhibitory mechanisms, associated with production of different anti-inflammatory cytokines, including IL-10 and TGF-β. In contrast, cells in fraction III shows low levels of TSDR demethylation, secrete pro-inflammatory cytokines and show decreased suppressive activity. More importantly, these cells express both Foxp3 and RORγt, have the potential to differentiate into Th17 cells and produce IL-17, IL-2, and TNF-α under inflammatory conditions  have potent immuno-suppressive function with limited proliferative capacity, whereas HLA-DR− memory Tregs (DR–mTreg) are less suppressive, but more proliferative. In contrast to mTreg subpopulations, naïve Tregs exhibit the least suppressive capacity, but the greatest proliferative capacity   fluids from smokers with normal lung function have markedly upregulated CD4patients . Similarpatients . In linepatients . Smokerspatients  and Chia second) . In cont second) . Increas second) , suggest+Foxp3+ T cells have been found in pulmonary lymphocyte follicles of moderate COPD patients, but not in lung parenchyma  and pro-inflammatory subpopulations in smokers, suggesting that inflammatory and anti-inflammatory responses coexist. Compared with healthy smokers, COPD patients show a higher proportion of pro-inflammatory subpopulations and a lower proportion of inhibitory subpopulations. This could reflect the progression of inflammation and the exhaustion of anti-inflammatory cells during the development of COPD .+ T cells, but also within different functional subsets of Treg cells  family members play crucial roles in maintaining the lineage integrity of T cells, including Tregs . Since p+), which appear to lack the energy to proliferate when exposed to bacteria (Contrary to our understanding that a deficiency of Treg cells may predispose to autoimmunity and inflammation , Treg cebacteria . Chronicbacteria . Both PDbacteria . To restbacteria .Another study has shown that Treg cells in mouse model indeed promote pulmonary fibrosis by producing the potent profibrotic cytokines TGF-β and PDGF-B . Since pIn summary, current data suggest that Treg cells have two functions in COPD. One is the beneficial maintenance of peripheral immune tolerance and the other is a deleterious reduction in antibacterial immunity, with a potential profibrotic effect. Because of the methodological complexity in defining Treg cells and the use of different sampling sites, it is not surprising that contrasting results occur in this field. Additional studies are needed to understand the involvement of Treg cells in the different phenotypes or stages of COPD before they can be considered a valid therapeutic target.JH was responsible for analysis of data and manuscript preparation. YS contributed with scientific expertise and manuscript preparation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Roux-en-Y gastric bypass (RYGB) is considered the gold standard for weight loss surgery and is an effective, safe treatment for morbid obesity and associated metabolic derangements. Complications such as small bowel obstruction are rare with a reported incidence of 5%. Obstruction caused by hiatal herniation of the gastric pouch and alimentary limb occurs even less frequently. Prompt recognition and treatment are imperative as delayed intervention may result in significant morbidity. At the time of this manuscript there have only been four reported cases in the literature highlighting a paucity of clinical guidance for the recognition and management of this complication. Here we present a case of acute small bowel obstruction secondary to hiatal herniation of the gastric pouch and proximal Roux limb. Furthermore, we review the literature and discuss the key aspects for the management of this complication. Roux-en-Y gastric bypass (RYGB) is effective in the treatment of morbid obesity and its associated metabolic derangements ,2. HowevA 68-year-old female presented with a one-year history of progressive nausea, emesis, and sharp epigastric abdominal pain leading to eventual acute intolerance of oral intake for four days. She previously underwent a retro-colic Roux-en-Y gastric bypass (RYGB) surgery 12 years prior for treatment of her morbid obesity. Eight weeks prior to presentation, she underwent magnetic resonance cholangiopancreatography for evaluation of her chronic symptoms. Imaging revealed a large hiatal hernia containing the gastric fundus and the absence of any additional acute processes Figure  and B. GAn optical trocar was utilized to gain entry into the abdominal cavity. Exploration of the esophageal hiatus was performed. A type IV hiatal hernia involving the entire gastric pouch and proximal Roux limb was identified. The bowel was distended and edematous but viable. The pars flaccida was opened with a laparoscopic bipolar vessel sealer, which demonstrated the left crus being densely adherent to the Roux limb. After careful dissection, the hernia sac and contents including gastric pouch and proximal Roux limb were reduced out of the hiatus. The gastric pouch and Roux limb were closely inspected, both of which appeared viable. The anterior and posterior hernia sacs were separated from the bilateral crus and mediastinal structures until the contents were reduced and adequate esophageal length was obtained. A posterior cruroplasty was performed with 2-0 Ethibond suture. Intraoperative upper endoscopy confirmed smooth passage along the length of the esophagus, a normal appearing gastric pouch, patent gastrojejunostomy free from stricture and ulceration with a dilated yet viable Roux limb. Upper gastrointestinal study on post-operative day one demonstrated no leak or obstruction of contrast passage. She was discharged on post-operative day three with resolution of her symptoms except mild nausea.At one-, nine-, and 27-month post-operative follow-up visits, the patient reported complete resolution of symptoms, improved tolerance of liquids and solids, and regular bowel function.According to the American Society for Metabolic and Bariatric Surgery, 252,000 metabolic operations were performed in 2018 for obesity and its associated metabolic derangement, with RYGB accounting for 17% of the total operations . UtiliziHiatal hernia occurrence after RYGB can occur quite frequently after gastric bypass . The relA review of the literature identified four reported cases making our patient only the fifth reported case of bowel obstruction from a hiatal hernia after gastric bypass Table  4-7]. O-7. O4-7]In the presented case, a diagnosis of obstruction was suspected based on the patient’s history of protracted, intermittent abdominal pain, nausea, vomiting, and constipation. Although a large hiatal hernia containing gastric pouch was identified on MRI, the Roux limb was not within the hernia, suggesting spontaneous herniation of the alimentary limb leading to the patient’s acute-on-chronic symptoms. This would explain the patient’s prior intermittent symptoms, which worsened upon Roux limb incarceration a few days prior to presentation. This phenomenon of acute-on-chronic symptoms has also been observed with internal hernias leading to acute presentation requiring operative intervention ,14.A laparoscopic approach to this patient was undertaken for the demonstrated benefits of laparoscopic versus open paraesophageal hernia repair in the non-bariatric surgery patient . Key tenSmall bowel obstruction after RYGB often requires urgent surgical intervention as compared to small bowel obstruction in non-bariatric patients where a period of non-operative therapy can be trialed. Therefore, timely diagnosis is critical to provide timely operative intervention. In the absence of more common causes of bowel obstruction after RYGB, such as internal hernia, abdominal adhesions, or even small bowel intussusception, the treating physician must be aware of the possibility of hiatal herniation of the alimentary limb as a possible cause to avoid a delay in diagnosis and treatment.Bowel obstruction after RYGB is not uncommon. Obstruction from a hiatal hernia containing the gastric pouch and Roux limb is infrequent but carries the potential for significant morbidity. Treating physicians must remain cognizant of this possibility as prompt diagnosis and treatment are crucial. Laparoscopic repair is safe, effective, and can permit fast recovery. This case report demonstrates an appropriate course of management for patients to consider when treating this condition.


Interpreting connections between the multiple networks of cell metabolism is indispensable for understanding how cells maintain homeostasis or transform into the decontrolled proliferation phenotype of cancer. Situated at a critical metabolic intersection, citrate, derived via glycolysis, serves as either a combustible fuel for aerobic mitochondrial bioenergetics or as a continuously replenished cytosolic carbon source for lipid biosynthesis, an essentially anaerobic process. Therein lies the paradox: under what conditions do cells control the metabolic route by which they process citrate? The Warburg effect exposes essentially the same dilemma—why do cancer cells, despite an abundance of oxygen needed for energy-generating mitochondrial respiration with citrate as fuel, avoid catabolizing mitochondrial citrate and instead rely upon accelerated glycolysis to support their energy requirements? This review details the genesis and consequences of the metabolic paradigm of a “truncated” Krebs/TCA cycle. Abundant data are presented for substrate utilization and membrane cholesterol enrichment in tumors that are consistent with criteria of the Warburg effect. From healthy cellular homeostasis to the uncontrolled proliferation of tumors, metabolic alterations center upon the loss of regulation of the cholesterol biosynthetic pathway. Deregulated tumor cholesterogenesis at the HMGR locus, generating enhanced carbon flux through the cholesterol synthesis pathway, is an absolute prerequisite for DNA synthesis and cell division. Therefore, expedited citrate efflux from cholesterol-enriched tumor mitochondria via the CTP/SLC25A1 citrate transporter is fundamental for sustaining the constant demand for cytosolic citrate that fuels the elevated flow of carbons from acetyl-CoA through the deregulated pathway of cholesterol biosynthesis. Since the 1970’s, sophisticated biochemical research tools and techniques have yielded a cornucopia of new detail on the molecular mechanisms of mitochondrial cellular bioenergetics, the individual pathway steps of intermediary metabolism, and the metabolic regulation of cell proliferation. The amassment of enzymological data on metabolic pathways, detailing complex enzymatic controls, spotlighted one of the fundamental and persistent metabolic controversies in the field of cell growth and proliferation—particularly the most clinically intractable obstacle: cancer.Thus, over 50 years ago the debate re-emerged over which metabolic profile initiates or dictates the cancer cell phenotype, known for nearly 100 years worldwide as the Warburg vs. the Crabtree effect .Warburg’s original hypothesis, based on his careful tissue slice respiration measurements with the manometer apparatus he invented, posits that cancer cells, unlike normal tissue, derive the bulk of their ATP by means of glycolysis, a less efficient ATP-generating pathway, despite an abundance of systemic oxygen and the presumptive capacity for high yields of ATP via mitochondrial aerobic oxidative phosphorylation. Warburg’s aerobic glycolysis concept for tumors proposed that the aberrant metabolic profile in tumor cells was based on malfunctioning mitochondria. Crabtree, offered a different interpretation of Warburg’s theory of tumor metabolism. Based on similarly careful tissue slice respiration measurements in a variety of tumors, he proposed that in order to supply their ATP requirements, the high rate of metabolic flux through glycolysis actually depresses the tumor’s capacity for normal mitochondrial oxidative respiration via feed-back regulatory processes. Crabtree wrote, “The tentative conclusion is that glycolytic activity exerts a significant checking effect on the capacity for respiration of tumour tissue.”-Crabtree-effect debate remains an open issue today. But, in Warburg’s era it wasn’t possible to detail any mitochondrial characteristics on a molecular level that might reveal their malfunction in cancer. And even with today’s more sophisticated techniques, if differences on the molecular structure-function level between normal and tumor mitochondria were indisputably documented, how could we determine whether such differences were a cause or consequence of the cancer phenotype?  played by an increased membrane cholesterol content, which together help perpetuate the unrestrained cell proliferation phenotype of cancer.Yet, in our view, despite the almost logarithmic increase in research publications on the Warburg effect and cancer over the last two decades the Warburg effect’s proposal of an aberrant respiratory pattern in tumors can be tightly linked with the long-held, well-documented, deregulated, and enhanced cholesterol synthesis ;(2)the tumor cell’s membranes become enriched with cholesterol as a result of the well-evidenced enhanced rate of cholesterol biosynthesis in tumors;(3)cholesterol enrichment of tumor mitochondrial membranes promotes and necessitates the continuous cytoplasmic supply of the precursor substrate (acetyl-CoA) for cholesterogenesis via the preferential export of citrate from mitochondria;(4)+-dependent citrate transporter  and the mitochondrial inner membrane citrate transport protein  might be critical to the proposed metabolic sequelae that largely define cell proliferation as the major phenotypic hallmark of cancer.implies, as others have, that communication or cross-talk between the plasma membrane NaMore than 30 years ago, inspired by an increasing abundance of exciting research implicating the pivotal role assumed by altered enzyme regulation of the cholesterol synthesis pathway in tumor cell proliferation, many laboratories (including ours) began to focus on the affect such a change in cholesterol biosynthesis would have on mitochondria as the cell’s main energy-generating machinery. As entry into the thrust of this review, the mitochondrial metabolomics involved in cholesterogenesis must be highlighted.The role of mitochondria is clearly of interest for two reasons. First, the stoichiometry of cholesterol biosynthesis requires 36 ATP per molecule, wherein mitochondrial oxidative phosphorylation would be the presumed major and most efficient ATP provider. Second, cytosolic acetyl-CoA is the initial precursor substrate of cholesterol (18 acetyl-CoA/cholesterol), whose multi-step synthesis is almost entirely localized on the endoplasmic reticulum (ER). In normal, oxygen-respiring cells amply supplied with glucose, the mitochondrion is a major cellular locus for glycolysis-derived acetyl-CoA, principally as a result of carbohydrate breakdown.SLC25A1 gene) in a 1:1 exchange for the electroneutral import of another TCA intermediate, malate  carbon source   . UltimatLC25A11) .But, wait! The operation of the conventional Krebs/TCA cycle  is usually appreciated first, without considering the interplay of the numerous metabolite and ion transport proteins embedded in the mitochondrial inner membrane that allow communication with the cytosol . ParticiFor example, are we to believe that all . Subsequent research leaves little doubt that inhibition of HMGR activity, and the resulting lack of cholesterol synthesis, suppresses cell division. Especially provocative are two findings  might become differentially cholesterol-enriched, in some cases incorporating enlarged lipid-raft domains. Such enrichment might alter the phase-transition properties of membrane lipids, thereby modifying the functional characteristics of integrated membrane-associated proteins . Would d2-requiring, Krebs/TCA cycle-linked bioenergetics enterprise, pregnenolone synthesis, and the operation of the more than 50 substrate transport protein complexes in at least 25 subfamilies embedded in the lipid bilayer, such as the CTP/SLC25A1 transport protein , or phosphoenolpyruvate . Using tcyto— > pyruvatemito— > acetyl-CoAmito— > citratemito—> [CTP]— > citratecyto) was accompanied by a negligibly small ADP-initiated O2 uptake  cholesterol-enriched hepatoma 3924A mitochondria relative to their normal liver counterparts. Both intra-mitochondrial and external-milieu citrate levels were assayed periodically on aliquots of actively respiring mitochondria from the rapid (hepatoma 3924A) and slow (hepatoma 16) growing tumors vs. their control livers over an extended incubation time-course . Strikin2 uptake . These f2 uptake .2 uptake occurred when supplied with either pyruvate or citrate, the cholesterol-rich tumor mitochondria respired almost indistinguishably as well as normal when fueled with substrates beyond citrate in the TCA cycle sequence: viz., isocitrate, α-ketoglutarate, succinate, and even with glutamate . That is, although virtually no ADP-stimulated Olutamate .2 uptake  reversed the nearly absent respiration of pyruvate-fed O2 uptake . These rIn this regard it is worth considering that early studies with isolated mitochondria from a variety of tumors  displayeExperimental approaches to the same or similar questions can often vary from one laboratory to another, especially as new technologies are applied. Explicitly presented methods, carefully followed and reproduced by different laboratories, become the “gold standard” by which the global veracity of experimental results are confirmed and established. However, deviation from one laboratory’s carefully specified methods, regardless of how seemingly inconsequential, will often yield results by the second that differ from the first. Thus, contradictory conclusions about fundamental mechanisms based upon data derived by different methods, and/or relying on a single tumor system, can be misleading, at best!Mindful of these considerations, the striking results seen on accelerated mitochondrial CTP-facilitated citrate transport in both slow- and rapidly growing rat hepatomas relative to normal liver organelles, encouraged consideration that there was a direct, positive correlation between the extent of mitochondrial cholesterol enrichment and citrate export, as described above. Would normal liver mitochondria, if purposefully enriched with cholesterol, mimic the behavior of tumor mitochondria with respect to their handling of citrate?Supplementary Material, for detailed methods; A variety of diverse methods have been employed over many years to alter the membrane cholesterol content of viable cells in experimental animals  includinThe results obtained after exogenous cholesterol enrichment of normal mitochondria, titrated to three increasing levels of cholesterol relative to control, mimicked remarkably both the re-routing pattern of CTP-promoted citrate export, and, in like manner, altered respiratory-linked oxidative phosphorylation observed with naturally occurring cholesterol-rich hepatoma mitochondria  Cholesterol Synthesis and DNA Replication in Synchronized Tumor Cells,” below, the BTC uptake rate was directly measured and was confirmed to rapidly enter proliferating, cultured murine lymphoma cells during ex vivo tissue slices, supports the following general conclusions:Comprehensive evaluation of these metabolic patterns from normal and tumor sources, employing both isolated mitochondria and viable (1)When mitochondria of hepatomas become enriched with cholesterol by means of accelerated cholesterogenesis due to loss of feedback regulation of HMGR, they manifest an altered metabolic profile that exhibits a preferential export of citrate.(2)max for the mitochondrial membrane CTP.Tumor mitochondria that are cholesterol enriched evince a significantly increased V(3)Preferential citrate efflux from the experimental tumor mitochondria deprives the conventional Krebs/TCA cycle of this critical respiratory substrate for oxidative phosphorylation-linked ATP generation regardless of ambient oxygen levels.(4)The specific CTP inhibitor, BTC, blocks the preferential efflux of citrate from cholesterol-rich tumor mitochondria, thereby restoring citrate-fueled respiration and re-establishing the Krebs/TCA cycle respiration-linked oxidative phosphorylation process of normal mitochondria.(5)The ability to support respiration-linked oxidative phosphorylation fueled by alternative substrates joining the Krebs/TCA cycle beyond the formation of citrate appears unaffected by the enhanced rate of citrate export from cholesterol-rich tumor mitochondria.(6)The ratio of mitochondrial citrate to cytosolic citrate in the cholesterol-rich tumor mitochondria may be a critical expression of cell proliferation in cancer.Although the regulation of cholesterol biosynthesis in normal cells is effected by means of several highly complex mechanisms, including feedback inhibition of HMGR by mevalonate-fueled pathway intermediates, hydroxysterols and a diverse host of other cellular metabolites , one cou14C]-citrate over a 4-hr time course for both normal and hepatoma 3924A was clearly established. In addition to [14C]-citrate, we also used [14C]-acetate as substrate for the PMS, bypassing the ATP-citrate lyase conversion of citrate to acetyl-CoA, the immediate carbon source for cholesterogenesis. Both substrates yielded near identical results  between acetate and mevalonate. Alternatively, the tumor PMS showed no difference in the 9-fold accelerated rate of cholesterol synthesis when fueled by either acetate or mevalonate, indicating that with the tumor system, regardless of whether the initial cholesterol synthesis substrate was supplied before (acetate) or after  the HMGR rate-limiting step, carbon flux was unchanged.Data reduction on a per-cell basis indicated that the tumor PMS had the capacity to synthesize cholesterol from either citrate or acetate more than 9-fold faster than did normal PMS. When normal liver PMS was supplied with -pyruvate incorporation into cholesterol, but concomitantly inhibited [3H]-thymidine incorporation into DNA, thereby arresting cell proliferation  is shared by BTC. Attempts to even partially overcome the BTC inhibition of DNA replication by the addition of 0.5 mM mevalonate to the culture required inclusion of the plasma membrane cation ionophore A23187 to the incubation. This permitted cytosolic Ca2+/Mg2+ to become replenished from the culture medium (∼0.5 mM), compensating, partially, for the chelating effects of BTC, and illuminating the mevalonate pathway’s well-established promotion of DNA replication . Experiments with isolated nuclear preparations from the 70Z/3 murine lymphoma affirmed that DNA synthesis was not significantly inhibited in BTC-treated, mevalonate-supplied incubations, but could be completely restored merely by addition of 5 mM Mg1, limiting the supply of cytosolic citrate and thus acetyl-CoA, and thereby ultimately mevalonate, arrests further progression through S-phase, abruptly halting cell proliferation.Analysis of further experimental permutations with this tumor system  gave autIn terms of the generally protracted time-line of tumor cell growth, therefore, accumulated data accentuate these tightly linked metabolic hallmarks of uncontrolled cell proliferation: deregulation of cholesterogenesis at the HMGR metabolic locus; mitochondrial membrane enrichment with cholesterol; preferential export of pyruvate-generated (glycolysis-generated) citrate from cholesterol-enriched mitochondria; and the requirement of a continual supply of mevalonate for ensuing DNA synthesis and cell replication. Collectively, these metabolomic profiles coincide to help endorse our proposed “truncated Krebs/TCA cycle” model .Nearly a decade before the introduction of the collective term “exosomes” to characterize exfoliated vesicles from normal and tumor cells , researcDespite recent decades employing sophisticated membrane cholesterol imaging and other analytical techniques that have elucidated the roles and evolution of shed vesicles (whether exosomes or ectosomes) in intercellular trafficking, and their potential involvement in immune surveillance , cholestMitochondrial membrane cholesterol enrichment in the graded growth series of Morris hepatomas 16 and 3924A , 1986 prAlthough no longer the case , at the +/K+-ATPase. Significantly, when the ascites culture was fed 54 μg mevalonate/106 cells for 3 h, the microvesicles exfoliated into the CFAF were cholesterol enriched 81% more than the shed vesicles from cultures minus added mevalonate. The data emphasize three important features that uphold the preferential carbon-flow concept of mitochondrially exported citrate that feeds deregulated, enhanced cholesterogenesis in tumors: (1) exfoliated vesicles contain higher amounts of cholesterol than the tumor plasma membranes from which they were shed; (2) supplying mevalonate to ascites tumor cultures dramatically yields an even further increase in the amount of cholesterol in the exfoliated microvesicles; and (3) the process of cholesterol-enriched microvesicle shedding in these tumors not only occurs both in vivo and in vitro, but is continuous and, in vitro, rapid (within 3 h), highlighting the tumor’s accelerated carbon flux through cholesterol synthesis anabolism.These studies  showed tin vivo [prostate cancer is a prime example— was reported to dramatically inhibit lipogenesis, enhance apoptosis and induce cell cycle arrest  the ubiquitously observed loss-of-control of HMGR activity promoting an elevated cholesterol biosynthesis rate, the operation of which is mandatory for DNA synthesis and cell division, and fueled by cytosolic citrate; (2) preferential metabolic routes, involving critical participation of the mitochondrial CTP and, potentially, the plasma membrane PMCT, through which an augmented supply of cytosolic citrate is sustained. Herein, we have enlarged the scope of tumor cell metabolomics by considering not only the mitochondrial bioenergetic carbon flux patterns in tumors and their adherence to the Warburg effect, but by emphasizing the paramount role assumed by cytoplasmic citrate, we have delineated consequences for tumor cell proliferation when accelerated cholesterogenesis prevails. Thus:1.The molecular basis for the absence or reduced control of citrate-fueled cholesterogenesis in tumors has been traced to a combination of defective feedback control elements and/or proteolytic degradation failures at the rate-controlling HMGR step of the synthetic pathway, in concert with a constitutively elevated expression of the HMGR enzyme. Our own research documented a 10-fold increase in the HMGR protein/cell in a rapidly proliferating heptoma, and a corresponding 9-fold increase in the rate of citrate-fueled cholesterol synthesis, compared with the normal liver system. Notwithstanding the cause, continuously escalated tumor cholesterogenesis requires a commensurately elevated and continuous supply of citrate as its fundamental biosynthetic substrate.2.In situ, the extent of such cholesterol enrichment appears to correlate with the proliferative rate of the particular tumor and the heterogeneity of its tissue environment.Membranes of tumor cell mitochondria, as well as their plasma membranes, have been long known to possess more cholesterol than those of their normal tissue counterparts. 3.2 consumption. Exogenous enrichment of normal liver mitochondria with titratable increased levels of cholesterol mimics this abnormal curtailment of early substrate Krebs/TCA cycle respiration in tumor mitochondria, proportional to the level of cholesterol enrichment. By blocking citrate export, the specific CTP inhibitor BTC completely reverses the decrease in pyruvate- and citrate-fueled respiration associated with aberrant membrane cholesterol enrichment. Yet, under either tumor or exogenously cholesterol-enriched-normal mitochondrial experimental conditions, fueling mitochondrial respiration-linked oxidative phosphorylation with substrates that enter the Krebs/TCA cycle beyond the aconitate hydratase step yields normal rates of O2 consumption. Thus, mitochondrial membrane cholesterol enrichment occurring during the progression from normal-homeostatic to tumorigenic-cell proliferative states directly effects increased CTP activity and drives accelerated export of mitochondrial citrate into the cytosol .Mitochondria from an increasingly graded growth series of rat hepatomas display corresponding enrichment with cholesterol, with directly correlating increased rates of citrate export, and proportionate curtailment of pyruvate- or citrate-fueled O4.ex vivo hepatoma tissue slice incubations fueled with pyruvate or citrate. Furthermore, following the preferential utilization of either substrate enabled the tracking of the carbon flow either through the tumor’s deregulated cholesterogenesis pathway to cholesterol, or into the CO2 end-product of normal tissue respiration. As with the isolated mitochondrial experiments, BTC reverses the pattern by preventing export of mitochondrially generated citrate to the cytosol, thus depriving the cholesterogenesis pathway of its precursor source of cytosolic acetyl-CoA . Considered together, these results are congruent with our proposed “truncated” Krebs/TCA cycle (2 level, satisfies the deduced mitochondrial dysfunction originally proposed by the Warburg effect.These aberrant tumor respiratory patterns with isolated hepatoma mitochondria are replicated in CA cycle  portrayi5.The critical importance of supplying cytoplasmic citrate from mitochondria as prerequisite for cell cycle progression is well documented. We demonstrated that BTC, the classic CTP inhibitor, rapidly enters exponentially growing ascites lymphoma cell cultures and arrests DNA synthesis , 9. BTC 6.in vivo and in vitro, revealed the rapid and continuous exfoliation of microvesicles, heavily enriched with cholesterol, from the tumor’s plasma membranes into the cell-free ascites fluid. Addition of mevalonate to the ascites culture medium dramatically increased the cholesterol content of the already-cholesterol-enriched exfoliated microvesicles. It is clearly recognized that microvesicle shedding from tumor plasma membranes commensurate with deregulated cholesterogenesis has provocative implications regarding the role of these processes in cancer immunosurveillance.Together with the continuous accelerated citrate export from the hepatoma’s cholesterol-enriched mitochondria, the loss of regulatory control at the HMGR locus of the cholesterogenesis pathway forecasts a potential accumulation of cholesterol, with consequential cholesterol enrichment of plasma membrane lipid rafts. Our exploratory studies with murine ascites hepatomas, 7.Finally, speculations are raised about potential regulated coordination between the CTP and PMCT in tumors.All original research documented in this review was performed in the laboratory of PSC by research associates accredited in the references herein, in the Laboratory of Biochemistry, Department of Biology, New York University. RAP and many others performed the research. PSC and RAP contributed equally to the writing of this review, and both approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.